Alcaligenes

J Microbiol, 2004 Dec, 42(4), 346 - 52
Enzymatic and Non-enzymatic Degradation of Poly (3-Hydroxybutyrate-co-3-Hydroxyvalerate) Copolyesters Produced by Alcaligenes sp . MT-16; Choi GG et al.; Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters with a variety of 3HV contents (ranging from 17 to 60 mol%) were produced by Alcaligenes sp . MT-16 grown on a medium containing glucose and levulinic acid in various ratios, and the effects of hydrophilicity and crystallinity on the degradability of the copolyesters were evaluated . Measurements of thermo-mechanical properties and Fourier-transform infrared spectroscopy in the attenuated total reflectance revealed that the hydrophilicity and crystallinity of poly(3HB-co-3HV) copolyesters decreased as 3HV content in the copolyester increased . When the prepared copolyester film samples were non-enzymatically hydrolysed in 0.01 N NaOH solution, the weights of all samples were found to have undergone no changes over a period of 20 weeks . In contrast, the copolyester film samples were degraded by the action of extracellular polyhydroxybutyrate depolymerase from Emericellopsis minima W2 . The overall rate of weight loss was higher in the films containing higher amounts of 3HV, suggesting that the enzymatic degradation of the copolyester is more dependent on the crystallinity of the copolyester than on its hydrophilicity . Our results suggest that the degradability characteristics of poly(3HB-co-3HV) copolyesters, as well as their thermo-mechanical properties, are greatly influenced by the 3HV content in the copolyesters.

J Synchrotron Radiat, 2005 Jan 1, 12(Pt 1), 13 - 8 Epub 2004 Dec 23.
Heterologous metalloprotein biosynthesis in Escherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin from Alcaligenes xylosoxidans; Harris RL; This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli . The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre . Both proteins were processed and exported to the periplasm of E . coli, which is consistent with their periplasmic location in their native host A . xylosoxidans . NiR could be readily purified from the periplasmic fraction of E . coli but the enzyme as isolated possessed only type-1 Cu centres . The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification . Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis . Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A . xylosoxidans . The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.

Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 74 - 8
Site-directed mutagenesis alters DnaK-dependent folding process; Yoshimune K et al.; The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE) . When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine . These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK . This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 285 - 93
Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10; Alemayehu D et al.; A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo . The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level . Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans . The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound . The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

Can J Microbiol, 2004 Aug, 50(8), 633 - 44
A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems; Gilbride KA et al.; Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques . 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system . Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters . Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities . There was little overlap in the composition of the culturable community between mills at the genus level . Furthermore, RSGP variation was almost as high within a mill as between mills . Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species . Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum . These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.

J Cyst Fibros, 2004 Aug, 3(3), 151 - 7
Antibiograms of resistant Gram-negative bacteria from Scottish CF patients; Mackenzie FM et al.; Background: Over a 19-month pilot phase, 93 multiply resistant Gram-negative isolates from Scottish cystic fibrosis patients were sent to a referral laboratory for further investigation . Methods: In common with the referring diagnostic laboratories, disc diffusion testing was carried out . Antibiotic susceptibility testing was also established by MIC methodology . NCCLS methods were used throughout . Twenty antibiotics were tested . Results: Comparing disc diffusion results against MIC results, there were 167 (14%) major errors . By MIC, Pseudomonas aeruginosa (n=59), Stenotrophomonas maltophilia (n=16), Burkholderia cepacia (n=10) and Alcaligenes xylosoxidans (n=7) were susceptible to 18%, 11%, 4% and 35% of the antibiotics tested, respectively . Colistin and tobramycin were the most active agents against P . aeruginosa with 60% and 49%, respectively, testing susceptible . Minocycline and gentamicin were most active against S . maltophilia with 58% and 18%, respectively, testing susceptible . B . cepacia were most susceptible to co-trimoxazole (10%) and ciprofloxacin (10%) . Five and six of the seven A . xylosoxidans isolates were susceptible to piperacillin and imipenem, respectively . Conclusions: Improved methods for susceptibility testing of such clinical isolates need to be employed in routine diagnostic laboratories . Levels of resistance in referred isolates were very high and similar to those described in the USA.

Biosci Biotechnol Biochem, 2004 Sep, 68(9), 1921 - 8
Purification and characterization of aromatic amine dehydrogenase from Alcaligenes xylosoxidans; Kondo T et al.; Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on beta-phenylethylamine . The molecular mass of the enzyme was 95.5 kDa . The enzyme consisted of heterotetrameric subunits (alpha2beta2) with two different molecular masses of 42.3 kDa and 15.2 kDa . The N-terminal amino acid sequences of the alpha-subunit (42.3-kDa subunit) and the beta-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively . The enzyme had a quinone cofactor in the beta-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form . The pH optima of the enzyme activity for histamine, tyramine, and beta-phenylethylamine were the same at 8.0 . The enzyme retained full activity after incubation at 70 degrees C for 40 min . It readily oxidized various aromatic amines as well as some aliphatic amines . The Michaelis constants for phenazine methosulfate, beta-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 microM respectively . The enzyme activity was strongly inhibited by carbonyl reagents . The enzyme could be stored without appreciable loss of enzyme activity at 4 degrees C for one month at least in phosphate buffer (pH 7.0).

Indian J Exp Biol, 2003 Dec, 41(12), 1469 - 72
Biological control of Fusarium wilt of pigeonpea Cajanus cajan (L.) Millsp with chitinolytic Alcaligenes xylosoxydans; Vaidya RJ et al.; Alcaligenes xylosoxydans protected pigeonpea from Fusarium wilt in a pot experiment and field trials . When seeds of pigeonpea (C . cajan) were treated with A . xylosoxydans and sown in soil infested with Fusarium, the incidence of wilt was reduced by 43.5% and resulted in 58% higher grain yield . The antifungal activity of A . xylosoxydans was based on chitinase production and was comparable in efficacy to commercial antifungal agents such as benlate, monitor WP, thiram and bavistin.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 65 - 72
Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp . MMRR7; Rajeshkumar K et al.; Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand {COD}: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number {MPN} >1100) . The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey's manual of systematic bacteriology . Among the five selected bacterial strains, the strain designated as MMRR7 and identified as Alcaligenes sp . was found to give a maximum reduction in COD (62%) in 5 d of incubation . Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent . Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp . MMRR7 gave a maximum COD reduction of 91% in 120 h . This study clearly indicates the possibility of using Alcaligenes sp . MMRR7 for the effective treatment of dairy wastewater.

Appl Biochem Biotechnol, 1999 Spring, 77-79, 445 - 54
Conversion of industrial food wastes by alcaligenes latus into polyhydroxyalkanoates; Yu PH et al.; Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties . As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism . Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs . The final dried cell mass and specific polymer production of A . latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, respectively . These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.

Acta Crystallogr D Biol Crystallogr, 1997 Jul, 53(Pt 4), 406 - 18
Structures of a blue-copper nitrite reductase and its substrate-bound complex; Dodd FE; Copper-containing nitrite reductases (NiR's) have been conveniently subdivided into blue and green NiR's which are thought to be redox partners of azurins and pseudo-azurins, respectively . Crystal structures of two green NiR's have recently been determined . Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively . The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented . The Cu-Smet distance is the same as those in the green NiR's . Thus, the length of this interaction is unlikely to be responsible for differences in colour . Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise 'classical' blue Cu centre . Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination . These results are discussed in terms of enzyme mechanism.

Acta Crystallogr D Biol Crystallogr, 1996 Jul, 52(Pt 4), 858 - 63
A re-evaluation of the crystal structure of chloromuconate cycloisomerase; Kleywegt GJ; It is shown here that the reported 3 A crystal structure of chloromuconate cycloisomerase from Alcaligenes eutrophus {Hoier, Schlomann, Hammer, Glusker, Carrell, Goldman, Stezowski & Heinemann (1994) . Acta Cryst . D50, 75-84} was refined in the incorrect space group I4 . In addition, a stretch of about 25 residues near the N-terminus is out-of-register with the density in the original structure . From the coordinates and structure factors deposited in the Protein Data Bank (PDB), it was possible to determine the correct space group to be I422 . The structure was then re-refined, using the original data reduced to I422, to a crystallographic free R factor of 0.264 at 3 A resolution (conventional R factor 0.189) . With conservative refinement and rebuilding methods, the errors in the chain tracing could be identified and remedied . Since the two molecules per asymmetric unit in the original structure are actually related by crystallographic symmetry, the observed differences between them are artefacts . In particular, the differences between, and peculiarities of the metal-binding sites are unreal . This case shows the dangers of crystallographic refinement in cases with unfavourable data-to-parameter ratios, and the importance of reducing the number of parameters in such cases to prevent gross errors (for instance, by using NCS constraints) . It also demonstrates how the evaluation and monitoring of model quality during the entire refinement and rebuilding process can be used to detect and remedy serious errors . Finally, it presents a strong case in favour of depositing not only model coordinates, but also experimental data (preferably, both merged and unmerged data).

Acta Crystallogr D Biol Crystallogr, 1996 Sep, 52(Pt 5), 937 - 41
Direct-Method Structure Determination of the Native Azurin II Protein Using One-Wavelength Anomalous Scattering Data; Xiao-Feng Z; The one-wavelength anomalous scattering (OAS) X-ray diffraction data of azurin II, a copper-containing protein from Alcaligenes xylosoxidans were collected at the Photon Factory, Japan at a 'routine' wavelength of 0.97 A . The structure had been originally solved by the molecular-replacement method {Dodd, Hasnain, Abraham, Eady & Smith (1995) . Acta Cryst . D51, 1052-1064} . As a technique of ab initio structure determination, the direct method {Fan, Hao, Gu, Qian, Zheng & Ke (1990) . Acta Cryst . A46, 935-939} was attempted to break the phase ambiguity intrinsic to OAS data . The phases were then improved using the solvent-flattening method . The final electron-density map clearly shows most Calpha positions and many side chains and it is traceable without prior knowledge of the structure . It is concluded that the direct method is capable of phasing anomalous scattering data collected at one wavelength from moderate-sized native proteins (M(w) approximately 20 kDa) which contain copper or atoms with a similar scattering power.

Acta Crystallogr D Biol Crystallogr, 1994 Jan, 50(Pt 1), 75 - 84
Crystal structure of chloromuconate cycloisomerase from Alcaligenes eutrophus JMP134 (pJP4) at 3 A resolution; Hoier H; Chloromuconate cycloisomerase (E.C . 5.5.1.7) is an enzyme involved in the 2,4-dichlorophenoxyacetate degradation pathway of Alcaligenes eutrophus JMP134 (pJP4) . The crystal structure of this protein was determined at 3 A resolution by molecular-replacement techniques using atomic coordinates from the reported crystal structure of the homologous muconate cycloisomerase (E.C . 5.5.1.1) from Pseudomonas putida as the search model (42% identical positions in the sequences) . Structure refinement by simulated-annealing and restrained least-squares techniques converged at R = 0.195 . In the crystals studied, space group I4, the protein is present as two octamers per unit cell with two subunits per asymmetric unit . Each subunit consists of two globular domains, one of which forms an alpha/beta-barrel . Comparison of this structure with that of muconate cycloisomerase reveals the reasons for the altered substrate specificity of chloromuconate cycloisomerase . Marked differences are observed in polarity, accessibility and hydrogen-bonding potential in the channel leading into the active site as well as in the active center itself.

Rev Port Pneumol, 2003 Nov, IX(5 Suppl 1), 35 - 36
{EPIDEMIOLOGICAL SURVEY OF BACTERIA ISOLATED FROM THE RESPIRATORY TRACT OF CYSTIC FIBROSIS PATIENTS}; Quintas S et al.; With the aim of characterizing the evolution of the epidemiological profile of respiratory bacterial infections of patients having Cystic Fibrosis (CF), the authors conducted a retrospective analysis about it's incidence and prevalence in 78 CF patients followed at the CF Specialized Centre, Paediatric Department, Santa Maria Hospital, Lisbon, during a 5 years period (1995-1999) . Pseudomonas aeruginosa was the most frequently isolated bacteria during the first three years of the study (60-73%), being surpassed by Staphylococcus aureus . However, Pseudomonas aeruginosa always remained the principal agent of chronic colonization (44-59%), with a peak of beginning between 0 and 5 years (34%) . A significative increase of the prevalence of intermitent and chronic colonization with Staphylococcus aureus was verified during this five years (48% to 83% and 32% to 54%) . The prevalence of isolations of Meticillin Resistant Staphylococcus aureus and of Burkholderia cepacia almost duplicated during this period . The taxes of isolation and chronic colonization with Alcaligenes xylosoxidans raised sharply beyond 1997 (from 3% and 0% in 1996 to 7% and 5% in 1997 and 10% and 7% in 1999) . Chronic colonization with Haemophilus influenzae kept a median prevalence of 22%, in spite of an increase in isolations (from 42% to 61%) . Fifty five per cent of the patients were chronically colonized by two or more agents . In view of these results, the authors discuss the therapeutic schemes and the measures to limit cross-infection which have been being advocated for CF patients in our centre.

Biochemistry, 2004 Jul 13, 43(27), 8670 - 9
Crystal structure of 4-chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states; Gulick AM et al.; 4-Chlorobenzoate:CoA ligase (CBAL) is a member of a family of adenylate-forming enzymes that catalyze two-step adenylation and thioester-forming reactions . In previous studies, we have provided structural evidence that members of this enzyme family (exemplified by acetyl-CoA synthetase) use a large domain rotation to catalyze the respective partial reactions {A . M . Gulick, V . J . Starai, A . R . Horswill, K . M . Homick, and J . C . Escalante-Semerena, (2003) Biochemistry 42, 2866-2873} . CBAL catalyzes the synthesis of 4-chlorobenzoyl-CoA, the first step in the 4-chlorobenzoate degredation pathway in PCB-degrading bacteria . We have solved the 2.0 A crystal structure of the CBAL enzyme from Alcaligenes sp . AL3007 using multiwavelength anomalous dispersion . The results demonstrate that in the absence of any ligands, or bound to the aryl substrate 4-chlorobenzoate, the enzyme adopts the conformation poised for catalysis of the adenylate-forming half-reaction . We hypothesize that coenzyme A binding is required for stabilization of the alternate conformation, which catalyzes the 4-CBA-CoA thioester-forming reaction . We have also determined the structure of the enzyme bound to the aryl substrate 4-chlorobenzoate . The aryl binding pocket is composed of Phe184, His207, Val208, Val209, Phe249, Ala280, Ile303, Gly305, Met310, and Asn311 . The structure of the 4-chlorobenzoate binding site is discussed in the context of the binding sites of other family members to gain insight into substrate specificity and evolution of new function.

Biodegradation, 2004 Jun, 15(3), 205 - 12
Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp . strain ZL5; Liu Y et al.; A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China . The isolate was identified as a Sphingomonas sp . strain on the basis of 16S ribosomal DNA analysis . Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities . This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp . strain KP7, also capable of growing on phenanthrene but not naphthalene . A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5 . Curing the plasmid with mitomycin C and transferring the plasmid to E . coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation . The C23O gene located on plasmid pZL was cloned and overexpressed in E . coli JM109(DE3) . The ring-fission activity of the purified C23O from the recombinant E . coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol >> 3,4-dihydroxyphenanthrene > 3-chlorocatechol.

Proteomics, 2004 Jul, 4(7), 2028 - 36
Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867; Zhao B et al.; Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway . Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions . One set of the enzymes is constitutive whereas the other is strictly inducible . To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized . A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate . The proteome profiles of P . alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis . Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing . Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms . The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp . U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P . alcaligenes . In addition, the induction of stress proteins and other adaptation phenomena were also observed.

J Biol Chem, 2004 Sep 3, 279(36), 37250 - 60 Epub 2004 Jun 25.
Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans; Hintner JP et al.; The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12 . The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp . KP7 . The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum . The enzyme from P . salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant . The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate . The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified . For comparison, the putative gentisate 1,2-dioxygenase from C . glutamicum was functionally expressed in E . coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1353 - 6
Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase; Iwakiri R et al.; We engineered biphenyl-degrading Alcaligenes sp . strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707) . Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination . The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions . The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions . Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

Biotechnol Lett, 2004 Apr, 26(8), 617 - 22
Transcription level of granule-associated phaP and phaR genes and granular morphogenesis of poly-beta-hydroxyalkanoate granules in Ralstonia eutropha; Seo MC et al.; The transcription levels of the granule-associated phaP and phaR genes in Ralstonia eutropha were regulated through the transformation of the phbC genes from R . eutropha and Alcaligenes latus into the poly-beta-hydroxybutyrate synthase-negative mutant . The granular morphogenesis of short chain length, poly-beta-hydroxyalkanoate (scl-PHA) was closely associated with the mRNA transcription levels of the phaP and phaR genes, especially with the ratio of phaP/phaR genes . The phasin protein encoded by the phaP gene increased the number of granules, while the PhaR protein of the phaR gene enlarged the size of the scl-PHA granules in R . eutropha.

Rev Argent Microbiol, 2004 Jan-Mar, 36(1), 47 - 51
Microbial dehalogenation of polychlorinated biphenyls in aerobic conditions; Araoz B et al.; From soils contaminated with polychlorinated biphenyls (PCBs) a strain of Alcaligenes sp . able to grow in a mineral medium with a commercial mixture of PCBs as carbon source was isolated . This strain consumed up to 200 ppm in seven days in laboratory conditions . After 24 h of incubation, some new congeners of PCBs could be recognized by mass spectrometry . Through the identification of these compounds it was possible to postulate examples of possible transformations by dechlorinations of penta- and tetra-chlorinated congeners into tri-chlorinated ones . The properties of the isolated strain are appropriate for bioremediation of contaminated areas and also for using in bioreactors in order to remove the xenobiotic chemical.

Pneumologie, 2004 May, 58(5), 309 - 15
{Isolation measurements for cystic fibrosis patients}; Vonberg R et al.; BACKGROUND: Respiratory tract infections significantly contribute to morbidity and mortality of cystic fibrosis patients . METHODS: We conducted a systematic literature review (Pubmed 01/1966 up to 09/2003) in order to present recommendations for the isolation of CF patients colonized with Burkholderia cepacia spp., Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Alcaligenes spp . Evidence and quality of 64 publications dealing with pathogen transmission or isolation measurements of colonized patients were evaluated . RESULTS: B . cepacia spp . was dealt most often with and 35 of 36 authors recommended the isolation of patients colonized with this pathogen . Isolation of patients colonized with P . aeruginosa was proposed by 21 of 25 authors . Only 5 studies concerned S . maltophilia or Alcaligenes spp . CONCLUSIONS: A) B . cepacia spp . colonized patients need to get a single room for their own . B) P . aeruginosa colonized CF patients should be separated from non-colonized CF patients . C) Patients harbouring even multi drug resistant P . aeruginosa, S . maltophilia or Alcaligenes spp . may not share their room with immunocompromised patients and should also be isolated when treated in intensive care units.

J Ind Microbiol Biotechnol, 2004 Mar, 31(3), 137 - 47 Epub 2004 Apr 27.
Antibacterial effects of knotwood extractives on paper mill bacteria; Lindberg LE et al.; Hydrophilic knotwood extracts from 18 wood species were assessed in disc diffusion and liquid culture tests for antibacterial effects against three species of paper mill bacteria . The Pinus sylvestris, P . resinosa, P . contorta, and P . banksiana extracts decreased or inhibited bacterial growth . The susceptibility order was P . sylvestris > P . resinosa > P . contorta > P . banksiana, correlating with the concentrations of pinosylvin and pinosylvin monomethyl ether in these wood species . Also, Pseudotsuga menziesii and Thuja occidentalis extracts had a small inhibitory effect . The Gram-positive Bacillus coagulans was more susceptible to the extracts than the Gram-negative Burkholderia multivorans and Alcaligenes xylosoxydans . The main components in the Pinus knotwood extracts were pinosylvin monomethyl ether and pinosylvin, suggesting these to be the active components . Therefore, pure pinosylvin, pinosylvin monomethyl ether, and dihydro-pinosylvin monomethyl ether were also tested . All compounds showed antibacterial effects . However, higher concentrations were needed for these pure compounds than for the knotwood extracts . Pinosylvin had stronger antibacterial effects than pinosylvin monomethyl ether . This work shows that knotwood extracts, especially from Pinus species, have a potential for use as natural biocides in papermaking.

Environ Pollut, 1993, 81(1), 47 - 50
Evaluation of Acacia nilotica (L.) del . and Casuarina equisetifolia forst . for tolerance and growth on microbially treated dyestuff wastewater; Kanekar P et al.; Two tree species, Acacia nilotica (L.) Del . and Casuarina equisetifolia Forst., were tested for their tolerance and growth on dyestuff wastewater containing phenol, aniline, and methyl violet . The wastewater was treated microbially by using a culture of Pseudomonas alcaligenes in a fixed-film bioreactor . The plants were watered with untreated and treated wastewater and tap water (control) at the rate of 2 litre per plant per week . A . nilotica exhibited 100% survival with both untreated and treated wastewater, and growth (increase in height) was comparable to that of control plants . However, growth of C . equisetifolia was adversely affected . It exhibited 100% survival with treated wastewater but only 87% survival with untreated waste-water and the percentage increase in height was less with both treated and untreated wastewater . There was no effect on soil except for an increase in chloride content.

Extremophiles, 2004 Feb, 8(1), 45 - 51 Epub 2003 Oct 01.
Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses; Ma Y et al.; Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method . Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically . The library was dominated by 16S rDNAs of Gram-negative bacteria (24% alpha-Proteobacteria, 31% beta-Proteobacteria, 33% gamma-Proteobacteria, and 2% delta-Proteobacteria), with a lower percentage of clones corresponding to Gram-positive bacteria . Forty cloned sequences were similar to that of known bacterial isolates (> 97% sequence similarity), represented by the species of the genera Brevundimonas, Comamonas, Alcaligenes, Stenotrophomonas, and Klebsiella . Eighteen cloned sequences showed less affiliation with known taxa (< 97% sequence similarity) and may represent novel taxa.

J Bacteriol, 2004 Apr, 186(8), 2328 - 39
A new amidohydrolase from Bordetella or Alcaligenes strain FB188 with similarities to histone deacetylases; Hildmann C et al.; The full-length gene encoding the histone deacetylase (HDAC)-like amidohydrolase (HDAH) from Bordetella or Alcaligenes (Bordetella/Alcaligenes) strain FB188 (DSM 11172) was cloned using degenerate primer PCR combined with inverse-PCR techniques and ultimately expressed in Escherichia coli . The expressed enzyme was biochemically characterized and found to be similar to the native enzyme for all properties examined . Nucleotide sequence analysis revealed an open reading frame of 1,110 bp which encodes a polypeptide with a theoretical molecular mass of 39 kDa . Interestingly, peptide sequencing disclosed that the N-terminal methionine is lacking in the mature wild-type enzyme, presumably due to the action of methionyl aminopeptidase . Sequence database searches suggest that the new amidohydrolase belongs to the HDAC superfamily, with the closest homologs being found in the subfamily assigned acetylpolyamine amidohydrolases (APAH) . The APAH subfamily comprises enzymes or putative enzymes from such diverse microorganisms as Pseudomonas aeruginosa, Archaeoglobus fulgidus, and the actinomycete Mycoplana ramosa (formerly M . bullata) . The FB188 HDAH, however, is only moderately active in catalyzing the deacetylation of acetylpolyamines . In fact, FB188 HDAH exhibits significant activity in standard HDAC assays and is inhibited by known HDAC inhibitors such as trichostatin A and suberoylanilide hydroxamic acid (SAHA) . Several lines of evidence indicate that the FB188 HDAH is very similar to class 1 and 2 HDACs and contains a Zn(2+) ion in the active site which contributes significantly to catalytic activity . Initial biotechnological applications demonstrated the extensive substrate spectrum and broad optimum pH range to be excellent criteria for using the new HDAH from Bordetella/Alcaligenes strain FB188 as a biocatalyst in technical biotransformations, e.g., within the scope of human immunodeficiency virus reverse transcriptase inhibitor synthesis.

FEBS Lett, 2004 Mar 12, 561(1-3), 173 - 6
Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer; Farver O et al.; Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant . This mutation increases the reduction potential of the T1 copper center . Kinetic results suggest that the change in driving force has a dramatic influence on the reactivity: The T2Cu(II) is initially reduced followed by ET to T1Cu(II) . The activation parameters have been determined and are compared with those of the wild-type (WT) AxCuNiR . The reorganization energy of the T2 site in the latter enzyme was calculated to be 1.6+/-0.2 eV which is two-fold larger than that of the T1 copper center in the WT protein.

Biotechnol Appl Biochem, 2004 Dec, 40(Pt 3), 271 - 5
A novel synthetic peptide derivative from lactoferrin exhibiting antimicrobial activity; Ramesh CV et al.; A tetrapeptide derivative Boc-L-Lys(Boc)-L-Arg-L-Asp-L-Ser(Bu(t))-OBu(t) (PEP 1261; Boc is butoxycarbonyl, Bu(t) is t-butyl and OBu(t) is t-butyl ester), synthesized by a solution-phase strategy, exhibited antimicrobial activity against a broad spectrum of micro-organisms at an optimal concentration of 500 mug/ml . Whereas the tetrapeptide salt (L-Lys-L-Arg-L-Asp-L-Ser.HCl) was found to be fairly effective against bacterial cultures, it was not effective against fungal cultures . Comparative growth studies showed that PEP 1261 was equally as potent as the conventional antibiotics kanamycin, streptomycin and actidione for the Gram-negative bacteria Escherichia coli, Pseudomonas alcaligenes and the non-filamentous fungus Saccharomyces cerevisiae (baker's yeast), whereas 62 and 88.9% inhibition were observed for Gram-positive organisms such as Staphylococcus aureus and Bacillus thuringiensis respectively . PEP 1261 might exert its antimicrobial activity by permeabilizing the bacterial membrane, and this was confirmed by an increase in beta-galactosidase activity.

Environ Toxicol Chem, 2004 Feb, 23(2), 325 - 30
Biodegradation of 2-chlorophenol in forest soil: effect of inoculation with aerobic sewage sludge; Lallai A et al.; Decontamination of 2-monochlorophenol-containing forest soil was studied in laboratory experiments . We found that in sterile soil, sorption of chlorophenol can occur . Chlorophenol disappearance of approximately 55% was observed in native soil; both soil sorption and degradation by indigenous soil populations caused this disappearance . In native soil, however, the rate of chlorophenol disappearance was enhanced up to slightly more than 90% by inoculation with a sludge taken from the aeration tank of a municipal wastewater treatment plant . In this sludge, the presence of Alcaligenes and Pseudomonas spp . was observed . In other experiments, addition to the soil of a laboratory culture preacclimated to 2-monochlorophenol did not lead to a greater increase in chlorophenol disappearance . In contrast to native soil, inoculation of sterile soil had no effect on disappearance of the chlorophenol . A possible explanation for the lack of cometabolic degradation is that autoclaving of the soil destroys the organic substances within it.

J Basic Microbiol, 2004, 44(1), 59 - 65
Isolation and characterization of microorganisms capable of decolorizing various triphenylmethane dyes; Sharma DK et al.; Various soil and sludge samples collected from the vicinity of textile dyeing industries and waste disposal sites were used for enrichment of microbial population in the presence of triphenylmethane (TPM) dye Acid Violet-17 (AV-17) . Twenty-five (25) isolates were screened for their ability to decolorize AV-17 dye added at a rate of 10 mgl(-1) in mineral salts medium (MSM) agar plates . Five bacterial isolates belonging to Bacillus sp., Alcaligenes sp . and Aeromonas sp . were selected on the basis of their higher decolorization ability and were used to develop a bacterial consortium . The consortium was able to efficiently decolorize various TPM dyes viz . Acid Violet-17 (86%), Acid Blue-15 (85%), Crystal Violet (82%), Malachite Green (82%) and Brilliant Green (85%) . The consortium will be further used for designing efficient and cost effective treatment system for effluents of textile processing industries (TPI).

Arch Pediatr, 2003 Sep, 10 Suppl 2, 342s - 346s
{Pathogenic bacteria in cystic fibrosis}; Mariani-Kurkdjian P et al.; Since the CF gene identification in 1989 and despite the improvement of our knowledge in the physiopathology of the disease, bronchopulmonary infection determines the vital prognosis . Following Staphylococcus aureus infection, patients are colonized or colonized by Pseudomonas aeruginosa, greatly involved in the pulmonary deterioration . Other bacteria may be involved Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes sp . Intensive antibiotic treatment of primocolonisation helps to prevent or delay chronic colonisation . Chronic colonization needs a rational long term antibiotic strategy to prevent the occurrence of multiresistant germs; antibiotic cures are performed every 3 or 4 months before pulmonary exacerbation symptoms.

Ukr Biokhim Zh, 2003 Mar-Apr, 75(2), 90 - 3
{Obtaining bioluminescent bacterial strains for detection of thallium ions}; Gruzina TG et al.; The possibility of Alcaligenes eutrophus T1 luxphenotype creation by temperature induced mutagenesis has been shown . These mutants are able to emit light after induction by thallium ions . This biological method of thallium detection possesses high specificity and sensitivity (0.5 microM of metal) . Such microbial cells can be used to quantify bioavailability of thallium part in the environment.

Biotechnol Adv, 1986, 4(2), 245 - 59
Microbial polysaccharides with actual potential industrial applications; Paul F et al.; The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose . Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined . The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents . A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars . The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1487 - 97
Cybernetic model predictive control of a continuous bioreactor with cell recycle; Gadkar KG et al.; The control of poly-beta-hydroxybutyrate (PHB) productivity in a continuous bioreactor with cell recycle is studied by simulation . A cybernetic model of PHB synthesis in Alcaligenes eutrophus is developed . Model parameters are identified using experimental data, and simulation results are presented . The model is interfaced to a multirate model predictive control (MPC) algorithm . PHB productivity and concentration are controlled by manipulating dilution rate and recycle ratio . Unmeasured time varying disturbances are imposed to study regulatory control performance, including unreachable setpoints . With proper controller tuning, the nonlinear MPC algorithm can track productivity and concentration setpoints despite a change in the sign of PHB productivity gain with respect to dilution rate . It is shown that the nonlinear MPC algorithm is able to track the maximum achievable productivity for unreachable setpoints under significant process/model mismatch . The impact of model uncertainty upon controller performance is explored . The multirate MPC algorithm is tested using three controllers employing models that vary in complexity of regulation . It is shown that controller performance deteriorates as a function of decreasing biological complexity.

Microbiol Res, 2003, 158(3), 215 - 27
In silico analysis of a flavohemoglobin from Sinorhizobium meliloti strain 1021; Lira-Ruan V et al.; Hemoglobins (Hbs) have been characterized from a wide variety of eubacteria, but not from nitrogen-fixing rhizobia . Our search for Hb-like sequences in the Sinorhizobium meliloti genome revealed that a gene coding for a flavohemoglobin (fHb) exists in S . meliloti (SmfHb) . Computer analysis showed that SmfHb and Alcaligenes eutrophus fHb are highly similar and could fold into the same tertiary structure . A FNR-like box was detected upstream of the smfhb gene and mapping analysis revealed that the smfhb gene is flanked by nos and fix genes . These observations suggest that smjhb is regulated by the concentration of O2 and that SmfHb functions in some aspects of nitrogen metabolism.

Biotechnol Lett, 2003 Sep, 25(17), 1421 - 4
Removal and destruction of high concentrations of gaseous toluene in a two-phase partitioning bioreactor by Alcaligenes xylosoxidans; Daugulis AJ et al.; A two-phase bioreactor consisting of hexadecane dispersed in an aqueous, cell-containing medium (organic fraction = 0.33) was used to trap toluene vapours from an air stream . The affinity for toluene by the solvent resulted in high efficiency of removal and transfer to the aqueous phase based on equilibrium transfer . The system was readily able to handle a loading capacity of 748 mg l(-1) h(-1) at a toluene degradation efficiency of greater than 98%.

Biotechnol Lett, 2003 Jul, 25(14), 1203 - 7
Delivery of benzene to Alcaligenes xylosoxidans by solid polymers in a two-phase partitioning bioreactor; Daugulis AJ et al.; Toxic levels of benzene were decreased to sub-inhibitory levels in a bioreactor via absorption by polymer beads or cylinders (poly(ethylene-co-vinyl acetate) or poly(styrene-co-butadiene)) . After inoculation with Alcaligenes xylosoxidans, the remaining benzene in the aqueous phase as well as the benzene taken up by the polymers was degraded to completion . The capacity of these polymers for benzene, and benzene diffusivity within the polymers were also determined.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 337 - 41 Epub 2003 Feb 26.
Regioselective hydroxylation of quinolinic acid, lutidinic acid and isocinchomeronic acid by resting cells of pyridine dicarboxylic acid-degrading microorganisms; Uchida A et al.; Microorganisms aerobically degrading quinolinic acid, lutidinic acid or isocinchomeronic acid were isolated and the microbial regioselective hydroxylation of these pyridine dicarboxylic acids was studied . Alcaligenes sp . UK21 cells converted quinolinic acid into 6-hydroxypicolinic acid, suggesting the involvement of two enzyme reactions catalyzing hydroxylation at position C6 and decarboxylation at position C3 of quinolinic acid . Resting cells of Alcaligenes sp . UK21 accumulated 94.9 mM 6-hydroxypicolinic acid (13.2 g l(-1)), with a 96% molar conversion yield by 48 h incubation . Rhizobium sp . LA17 and Hydrogenophaga sp . IMA01 catalyzed the regioselective hydroxylation of lutidinic acid and isocinchomeronic acid into 6-hydroxylutidinic acid and 6-hydroxyisocinchomeronic acid, respectively . 6-Hydroxylutidinic acid accumulated up to 95.4 mM (17.5 g l(-1)) by 24 h incubation in the resting cells reaction, using Rhizobium sp . LA17, with a 99% molar conversion yield . Resting cells of Hydrogenophaga sp . IMA01 produced 88.7 mM 6-hydroxyisocinchomeronic acid (16.2 g l(-1)) by 24 h incubation, with a 81% molar conversion yield.

Gene, 2003 Jul 17, 312, 239 - 48
Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867; Yeo CC et al.; Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate . Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates . The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X . The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID . Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon . Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue . In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO . This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene . In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers . However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate . Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

Microbiol Res, 2003, 158(2), 195 - 9
Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909; Santiago MF et al.; The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated . The de novo pyrimidine biosynthetic enzymes were measured in extracts of P . alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity . Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present . Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound . Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes.

Biotechnol Bioeng, 2003 Sep 30, 83(7), 854 - 63
In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli; Hong SH et al.; The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E . coli and recombinant E . coli producing poly(3-hydroxybutyrate) {P(3HB)} . The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production . It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions . To prove the role of ED pathway on P(3HB) production, a mutant E . coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon . The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4) . The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E . coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E . coli as predicted by metabolic flux analysis .

Biotechnol Lett, 2003 Jan, 25(1), 83 - 7
Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions; Ueji S et al.; Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp . were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties . The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased . The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.

Biotechnol Lett, 2003 May, 25(9), 715 - 7
Purification and characterization of chitinase from Alcaligenes xylosoxydans; Vaidya R et al.; Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography . The molecular mass of chitinase was estimated to be 45 kDa and 44 kDa by SDS-PAGE and gel-filtration, respectively . The enzyme was optimally active at 50 degrees C (over 30 min) and pH 5 . Activity staining after PAGE showed a single band . The Km for chitin was 3 g l-1 . Cu2+ and Na+ at 5 mM inhibited chitinase activity to 25% while Ca2+, Mg2+ and Ba2+ had no effect at the same concentration . The purified enzyme degraded mycelia of Aspergillus niger.

Biotechnol Lett, 2003 May, 25(9), 665 - 70
Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high molar fractions of 3-hydroxyvalerate by a threonine-overproducing mutant of Alcaligenes sp . SH-69; Choi GG et al.; A threonine overproducing mutant of Alcaligenes sp . SH-69 was isolated and its ability to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), was investigated . The 3HV fraction in poly(3HB-co-3HV) produced from glucose as the sole carbon source exceeded 22 mol%, which is approximately six times higher than that achieved by the wild type under the same culture conditions . Furthermore, the addition of a relatively low concentration (10 mM) of propionic acid, valeric acid or levulinic acid to the glucose medium greatly increased the molar fraction of 3HV in the copolyester, to 38-77 mol% . The results suggest that metabolic engineering of the biosynthetic pathways supplying polyhydroxyalkanoate monomers, such as the threonine biosynthetic pathway, can lead to new poly(3HB-co-3HV)-producing strains.

Arch Microbiol, 2003 Aug, 180(2), 81 - 7 Epub 2003 Jul 03.
Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by Alcaligenes sp . strain UK21; Uchida A et al.; In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp . strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined . Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized . The enzyme catalyzed the incorporation of oxygen atoms of H(2)O into the hydroxyl group . The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively . Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase . 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized . The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits . The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors . The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.

FEMS Microbiol Rev, 2003 Jun, 27(2-3), 385 - 410
Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes; Mergeay M et al.; Ralstonia metallidurans, formerly known as Alcaligenes eutrophus and thereafter as Ralstonia eutropha, is a beta-Proteobacterium colonizing industrial sediments, soils or wastes with a high content of heavy metals . The type strain CH34 carries two large plasmids (pMOL28 and pMOL30) bearing a variety of genes for metal resistance . A chronological overview describes the progress made in the knowledge of the plasmid-borne metal resistance mechanisms, the genetics of R . metallidurans CH34 and its taxonomy, and the applications of this strain in the fields of environmental remediation and microbial ecology . Recently, the sequence draft of the genome of R . metallidurans has become available . This allowed a comparison of these preliminary data with the published genome data of the plant pathogen Ralstonia solanacearum, which harbors a megaplasmid (of 2.1 Mb) carrying some metal resistance genes that are similar to those found in R . metallidurans CH34 . In addition, a first inventory of metal resistance genes and operons across these two organisms could be made . This inventory, which partly relied on the use of proteomic approaches, revealed the presence of numerous loci not only on the large plasmids pMOL28 and pMOL30 but also on the chromosome . It suggests that metal-resistant Ralstonia, through evolution, are particularly well adapted to the harsh environments typically created by extreme anthropogenic situations or biotopes.

J Appl Microbiol, 2003, 95(1), 160 - 6
Development of a Gram-negative selective agar (GNSA) for the detection of Gram-negative microflora in sputa in patients with cystic fibrosis; Moore JE et al.; AIMS: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF) . METHODS AND RESULTS: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30 g), bacteriological agar no.1 (10 g), yeast extract (5 g), crystal violet (2 mg), nisin (48 mg), novobiocin (5 mg), cycloheximide (100 mg), amphotericin (2 mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF . GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined . Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1.50 x 102 CFU ml-1 sputum Pseudomonas aeruginosa, 2.38 x 102 CFU ml-1 sputum Burkholderia cepacia genomovar IIIb and 6.70 x 103 CFU ml-1 sputum Stenotrophomonas maltophilia . A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection . CONCLUSIONS: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged . SIGNIFICANCE AND IMPACT OF THE STUDY: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.

Infect Immun, 2003 Jun, 71(6), 3196 - 205
Modulation of virulence by two acidified nitrite-responsive loci of Salmonella enterica serovar Typhimurium; Kim CC et al.; Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen . The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively . Maximal induction of the promoters by nitrite was dependent on pH . The nipAB promoter was regulated by oxygen in an Fnr-dependent manner . The nipC promoter was also regulated by oxygen but in an Fnr-independent manner . The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS . Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD(50)s) than did the wild type in C57BL/6J mouse infections . The lower LD(50)s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria . We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

Biotechnol Bioeng, 2003 Jul 5, 83(1), 104 - 11
Evaluation of spectrofluorometry as a tool for estimation in fed-batch fermentations; Hagedorn A et al.; Native culture fluorescence was investigated as an additional source of information for predicting biomass and glucose concentrations in a fed-batch fermentation of Alcaligenes eutrophus . Partial least squares (PLS) regression and a feed forward neural network (FFNN) coupled with principle component analysis (PCA) were each used to model the kinetics of the fermentation . Data from three fermentations was combined to form a training set for model calibration and data from a fourth fermentation was used as the testing set . The fluorescent soft-sensors were compared with a previously developed feed forward neural network soft-sensor model which used oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), aeration rate, feed rate, and fermentor volume to estimate biomass and glucose concentrations . The best model performance for predicting both biomass and glucose concentrations was achieved using the native fluorescence-based models . Real data predictions of the biomass concentration in the testing set were obtained using both the PLS and FFNN PCA modeling utilizing fluorescence measurements plus the rate of change of the fluorescence measurements . Accurate predictions of the glucose concentration in the testing set were obtained using the FFNN PCA modeling technique utilizing the rate of change of the fluorescence measurements . Substrate exhaustion was indicated qualitatively by a first-order PLS model utilizing the rate of change of fluorescence measurements . These results indicate that native culture fluorescence shows promise for providing additional valuable information to enhance predictive modeling which cannot be extracted from other easily acquired measurements .

Int Microbiol, 2003 Mar, 6(1), 57 - 64 Epub 2003 Mar 13.
Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5; Park JE et al.; Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+) . The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca . Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+) . A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E . coli . The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+) . By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance . Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H . alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.

J Food Prot, 2003 Apr, 66(4), 592 - 8
Comparison of sodium hypochlorite-based foam and peroxyacetic acid-based fog sanitizing procedures in a salmon smokehouse: survival of the general microflora and Listeria monocytogenes; Bagge-Ravn D et al.; The effects of fog sanitization with peroxyacetic acid (hydrogen peroxide, peracetic acid, and acetic acid in combination) on general hygiene (aerobic plate count) and on Listeria monocytogenes were assessed in a slicing area at a salmon smokehouse and compared with the effects of foam sanitization with sodium hypochlorite (routinely performed at the smokehouse) . Two hundred twenty-three environmental samples were collected with sponges and swabs after each of the sanitization procedures, and 68 samples were collected during production . The total culturable aerobic plate count was determined for each sample, and a total of 288 bacterial strains were randomly isolated and tentatively identified to genus level by physiological and biochemical tests . The microflora was dominated by Neisseriaceae, Enterobacteriaceae, and lactic acid bacteria during production . Foam sanitization caused a change in the composition of the flora, with Pseudomonas spp . and Alcaligenes spp . being the dominant gram-negative bacteria and Kurthia spp . and Bacillus spp . being the surviving gram-positive bacteria . Bacteria were very sensitive to fog sanitization, and yeasts accounted for almost half of the surviving flora . By a selective isolation method, strains of L . monocytogenes were isolated and subsequently characterized by random amplified polymorphic DNA (RAPD) typing . Following foam sanitization, 14 to 42% of the samples contained <10 CFU per site, whereas 29 to 78% of the samples collected after fog sanitization contained this level of bacteria . The prevalence of L . monocytogenes was unchanged, but L . monocytogenes was found only in poorly cleaned areas such as drains . The RAPD types for all positive samples were identical to the type that had persisted in the smokehouse since 1995, indicating the importance of drains as a niche.

J Mol Biol, 2003 Apr 25, 328(2), 429 - 38
Atomic resolution structures of native copper nitrite reductase from Alcaligenes xylosoxidans and the active site mutant Asp92Glu; Ellis MJ et al.; We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E) . The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis . Despite much effort and technological progress, still very few structures are known at such resolution . For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A . Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively . These structures provide the basis from which to build a detailed mechanism of this important enzyme .

J Basic Microbiol, 2003, 43(1), 75 - 9
Comparison of aspartate transcarbamoylase regulation in Pseudomonas alcaligenes and Pseudomonas mendocina; Santiago MF et al.; The regulation of aspartate transcarbamoylase activity in cell extracts of Pseudomonas alcaligenes ATCC 14909 and Pseudomonas mendocina ATCC 25411 was compared . Under saturating substrate concentrations, pyrophosphate, CTP, UDP and ADP were highly inhibitory of the P . alcaligenes transcarbamoylase activity while pyrophosphate, UDP, ADP, ATP and GTP were the most effective inhibitors of the P . mendocina transcarbamoylase . By examining transcarbamoylase inhibition by ribonucleotide triphosphates, it was possible to differentiate these species assigned to different DNA homology groups and such an analysis might prove useful in the reclassification of Pseudomonas species.

Lett Appl Microbiol, 2003, 36(3), 129 - 34
The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans; Vaidya RJ et al.; AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria . METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar . Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation . This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans . The mutant Alc . xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type . CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc . xylosoxydans . SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.

Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 153 - 62
{Cloming, sequence analysis of imidase gene from Alcaligenes eatrophus and its expression in E . coli}; Wang Y et al.; A hydantoin-cleaving microorganism 112R4 is screened and identified to be Alcaligenes eutrophus . The resting cell of Alcaligenes eutrophus 112R4 can catalyze the hydrolysis of hydantoin, dihydropyrimidine and succinimide effectively, but not function to 5-monosubstituted hydantoins or 5,5'-disubstituted hydantoins . The microorganism can utilize succinimide as a sole carbon source and nitrogen source, which indicates the presence of a complete transformation pathway of succinimide, and a hydantoin-cleaving enzyme, imidase, is suggested to be contained in this metabolic pathway . A 6 kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R4 is shown to be correlative with the transformation of succinimide . A 2 kb DNA fragment containing the gene of imidase is subcloned and sequenced . Deletion analysis verifies that one open reading frame of 876 nucleotides, which encodes a peptide of 291 amino acids, with a calculated molecular weight of 33688, is responsible for the encoding of imidase . This is the first report of the nucleotide and amino acid sequences of imidase (GenBank accession number: AF373287) . A homology search performed in protein database reveals an identity of 14% with polysaccharide deacetylase conserved domain, an identity of 60% with N-terminal 20 amino acids of Blastobacter sp . A17p-4, but no apparent similarity with all known cyclic-amide-cleaving enzymes . This result suggested that the imidase should be classified as a new member of cyclic amidases . Under the control of lac promoter and IPTG induction, the imidase activity of transformed E . coli reached 3200 U/L, which is about 7-fold higher than that of gene donor strain.

Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 247 - 54
{Optimization of fermentation conditions for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Alcaligenes eutrophus}; Du G et al.; A close relationship, between the initial addition time and concentration of propionate and HV fraction, was observed in shaking culture of Alcaligenes eutrophus for the production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) . The optimal initial addition time of propionate was determined at the onset of PHBV formation period . Although relatively high HV unit could be obtained under high propionate concentration, the growth and product synthetic activities were inhibited obviously . Different ratios of glucose to propionate were fed to stimulate the formation of HV unit and the results were compared . The optimizing feeding strategy of propionate was proposed based on the consideration of several fermentation index . The final cell dry weight, PHB concentration, PHB content and HV fraction in PHBV reached 52.1 g/L, 40.8 g/L, 78.3% and 16.2 mol%, respectively, Yield coefficient of HV unit to propionate and PHBV productivity were obtained to be 0.5 g/g and 0.74 g/(L/h), respectively.

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 160 - 3
{Studies on the parasites of Paris polyphylla var . yunnanensis}; Wang S et al.; Two bacteria and three fungi were isolated from the hypogeal stems of Paris polyphylla var . yunnanensis . The bacteria were identified as Bacillus cereus and Pseudomonas alcaligenes, and the fungi were identified as Periconia sp., Pachnocybe albida and Hormomyces paridiphilus . The results on liquid culture of B . cereus, P . alcaligenes and H . paridiphilus indicated that the colloidization and polysaccharide content increasing in hypogeal stems of P . polyphylla var . yunnanensis were due to the extracellular polysaccharide secretion of the parasitical fungus H . paridiphilus.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 52 - 6
{Studies on microbial factor on color change of Dunhuang mural . I . Classification of microbes on color-changed mural and property of some typical species}; Feng Q et al.; Microbial strains were isolated from 51 typical color changed samples of mural in 6 grottos of Dunhuang Mogao Gretto . After identification, it is proved that belong to 6 genera of bacteria and 5 genera of mould . Bacillus, Alcaligenes and Penicillium were dominant species in all of them . In the imitative experiment, it is found that Cladosporium sp., A . niger and two strains of bacteria have a great effect on the color change of red pigment of mural and aging of sizing agent.

Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 579 - 85
{Cloning of a new catechol 1,2-dioxygenase gene (tfd C) from Plesiomonas and its expression in the E . coli}; Ma Z et al.; A new catechol-1,2-dioxygenase gene (tfd C) was cloned from the Plesiomonas using the PCR method . Primers were designed according to the reported sequence of Catechol-1,2-dioxygenase (C120) gene from Alcaligenes eutroplus . The amplified fragment contained a 765 bp open reading frame (ORF), encoding a protein of 255 amino acids . The new tfd C gene shared a high homology with the one cloned from Alcaligenes eutroplus, showing only one base difference at 693 site (C-->A) and consequently one amino acid difference at 228 site (P-->T) . The ORF was cloned to the plasmid pBluescriptII KS, which was transferred to E . coli JM109 and a positive clone, pBt2G, was then selected . A significant activity of C120 was detected in the positive clone . When the ORF was cloned to the plasmid pET-30a, which was transferred to E . coli BL21(DE3) plysS, the expected 33 kD protein was detected from a positive clone, pET30A, by SDS-PAGE . The C120 is a key enzyme in degrading aromatic pollutants in the environment . In order to use plants to degrade aromatic pollutants, the gene will be introduced into the turfgrass . To express the gene properly in plants, its translation initiation codon was modified from GTG to ATG . A similar activity of C120 was obtained following the modification.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 290 - 5
{Effects of nitrogen feeding on the accumulation of poly-beta-hydroxybutyrate with Alcaligenes eutrophus}; Du G et al.; On the basis of analysis of PHB fermentation processes, the effects of ammonium sulfate feeding rate at PHB formation period on the PHB accumulation by Alcaligenes eutrophus were investigated . It was shown that the complete absence of nitrogen source at PHB formation phase would lead to the decline of PHB synthetic activity, and the obvious influences of different nitrogen feeding rate on PHB synthesis were observed . Higher PHB content, but relative lower cell dry weight, PHB concentration and PHB productivity could be obtained at slower nitrogen feeding rate . The excessive nitrogen feeding rate resulted in the drop of PHB content, which led to the decrease of PHB concentration and PHB productivity . The better results could be achieved when the ammonium sulfate feeding rate was set at around 0.5 g/h.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 26 - 31
{The construction of a recombined E . coli strain with stable and high production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)}; Tian J et al.; Plasmid pJMC2 was constructed by cloning the parDE fragment of RK2 into pTZ18U-PHB which harbored phaCAB from Alcaligenes eutrophus and was transferred into E . coli HMS174 and E . coli JM107 separately . It is very stable in its hosts cultured in medium without ampicillin . E . coli HMS 174(pTZ18U-PHB) and E . coli JM107(pTZ18U-PHB) produced P(3HB-co-3HB) in a low phosphate concentration medium(18 mmol/L) . The proportion of 3-hydroxyvalerate(3HV) in the polymer was 5%-8% . A fed-batch culture of E . coli HMS174(pJMC2) was conduct in a 5 L automatically controlled fermentor, the final dry cell weight, P(3HB-co-3HV) content, and th 3HV proportion were 42.5 g/L, 70% and 4.9% respectively.

J Clin Microbiol, 2003 Jan, 41(1), 492 - 4
Evaluation of MicroScan Autoscan for identification of Pseudomonas aeruginosa isolates from cystic fibrosis patients; Saiman L et al.; Accurate identification of gram-negative bacilli from cystic fibrosis (CF) patients is essential . Only 57% (108 of 189) of nonmucoid strains and 40% (24 of 60) of mucoid strains were definitively identified as Pseudomonas aeruginosa with MicroScan Autoscan . Most common misidentifications were Pseudomonas fluorescens-Pseudomonas putida (i.e., the strain was either P . fluorescens or P . putida, but the system did not make the distinction and yielded the result P . fluorescens/putida) and Alcaligenes spp . Extending the incubation to 48 h improved identification, but 15% of isolates remained misidentified . The MicroScan Autoscan system cannot be recommended for the identification of P . aeruginosa isolates from CF patients.

J Gen Appl Microbiol, 1998 Aug, 44(4), 269 - 274
Purification and characterization of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp . XY-234; Araki T et al.; A beta-1,3-xylanase-producing bacterium, Alcaligenes sp . XY-234, was isolated from the marine environment . The organism produced endo-1,3-beta-xylanase at a high level in the culture fluid . The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies . The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pI was 4.0 . The enzyme hydrolyzed beta-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-beta-D-xyloside, and beta-1,4-xylan . The Km of the enzyme was 4.0 mg/ml . Optimal pH and temperature were 7.5 and 40 degrees C, respectively . It was stable from pH 6.0 to 10 and at a temperature of less than 40 degrees C . The enzyme was strongly inhibited by 1 mM HgCl(2)., AlCl(3), CuCl(2), FeCl(3), HgCl(2), Pb(CH(3)COO) (2), and N-bromosuccinimide.

J Environ Sci (China), 2002 Oct, 14(4), 445 - 50
A reactor system combining reductive dechlorination with co-metabolic oxidation for complete degradation of tetrachloroentylene; Lee TH et al.; A laboratory sequential anaerobic-aerobic bioreactor system, which consisted of an anaerobic fixed film reactor and two aerobic chemostats, was set up to degrade tetrachloroethylene (PCE) without accumulating highly toxic degradation intermediates . A soil enrichment culture, which could reductively dechlorinate 900 microM (ca . 150 mg/L) of PCE stoichiometrically into cis-1,2-dichloroethylene (cis-DCE), was attached to ceramic media in the anaerobic fixed film reactor . A phenol degrading strain, Alcaligenes sp . R5, which can efficiently degrade cis-DCE by co-metabolic oxidation, was used as inoculum for the aerobic chemostats consisted of a transformation reactor and a growth reactor . The anaerobic fixed film bioreactor showed more than 99% of PCE transformation into cis-DCE in the range of influent PCE concentration from 5 microM to 35 microM at hydraulic retention time of 48 h . On the other hand, efficient degradation of the resultant cis-DCE by strain R5 in the following aerobic system could not be achieved due to oxygen limitation . However, 54% of the maximum cis-DCE degradation was obtained when 10 mumol of hydrogen peroxide (H2O2) was supplemented to the transformation reactor as an additional oxygen source . Further studies are needed to achieve more efficient co-metabolic degradation of cis-DCE in the aerobic reactor.

Res Microbiol, 2002 Nov, 153(9), 579 - 84
Molecular characterization of a salt-tolerant bacterial community in the rice rhizosphere; Tripathi AK et al.; The diversity of salt-tolerant bacteria present in the rhizosphere of Oryza sativa was investigated . Fourteen bacterial strains, isolated after enrichment in nitrogen-free, semi-solid medium and showing tolerance to 3% NaCl, were analyzed by restriction patterns produced by amplified DNA coding for 16S rDNA (ARDRA) with enzymes Sau3AI, AluI and RsaI which showed that they were represented by 4 ARDRA types . Biodiversity among the 14 strains was also analyzed by the random amplified polymorphic DNA (RAPD) technique with a 10-mer primer . Partial nucleotide sequence of 16S rDNA assigned these clusters to Serratia marcescens, Pseudomonas aeruginosa, Alcaligenes xylosoxidans and Ochrobactrum anthropi . Notably, all four bacterial species are potential human pathogens that infect immunocompromised patients.

Appl Environ Microbiol, 2002 Dec, 68(12), 5925 - 32
A novel high-cell-density protein expression system based on Ralstonia eutropha; Srinivasan S et al.; We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124 . This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression . Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively . By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E . coli.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Jul-Aug, (4), 60 - 2
{Persistent properties of protozoa-associated bacteria}; Plotnikov AO et al.; The species structure and persistent properties (antilysozyme and antihistone activity) of bacteria forming associations with protozoa is revealed . Among them, 68.9% of the isolates were enterobacteria, the remaining organisms belonged to the families Aeromonas, Alcaligenes, Pseudomonas, Vibrio, etc . Within the family Enterobacteriaceae bacteria of the Escherichia group prevailed . 50.4% of the isolates were found to have antilysozyme activity and 97%--antihistone activity . The level of persistent properties in the representatives of allochthonous microflora was higher than that in the representatives of autochthonous microflora . In addition to antilysozyme activity antihistone activity was noted in protozoa-associated bacteria, which could be of importance for the formation of symbiotic links in natural associations . These data may be used in sanitary and hygienic practice for microecological monitoring of the environment.

Biodegradation, 2002, 13(2), 141 - 7
Degradation of aliphatic polyester films by commercially available lipases with special reference to rapid and complete degradation of poly(L-lactide) film by lipase PL derived from Alcaligenes sp; Hoshino A et al.; Commercial lipases were examined for their degradation efficiency of aliphatic polyester films . In 100 days immersion of polyester films in lipase solutions at 37 degrees C at pH 7.0, Lipase Asahi derived from Chromobacterium viscosum degraded polybutylene succinate-co-adipate (PBSA), poly (e-caprolactone) (PCL) and polybutylene succinate (PBS), and Lipase F derived from Rhizopus niveus degraded PBSA and PCL during 4-17 days . Lipase F-AP15 derived from Rhizopus orizae could degrade PBSA in 22 days . In these cases, PBS and PBSA were mainly degraded to dimers, whereas PCL was mainly degraded to monomers . Only poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) and poly (L-lactide) (PLA) were not degraded in the experiments . However, PLA degraded completely at 55 degrees C, pH 8.5 with Lipase PL during 20 days . This result could be explained with the sequential reactions of the chemical hydrolysis of the polymer to oligomers at higher pH and temperature, and the succeeding enzymatic hydrolysis of oligomers to the monomers.

Environ Pollut, 2002, 120(3), 691 - 700
Runoff rates, chemical speciation and bioavailability of copper released from naturally patinated copper; Karlen C et al.; The release of copper, induced by atmospheric corrosion, from naturally patinated copper of varying age (0 and 30 years) has been investigated together with its potential ecotoxic effect . Results were generated in an interdisciplinary research effort in which corrosion science and ecotoxicology aspects were combined . The aim of the investigation was to elucidate the situation when copper-containing rainwater leaves a roof in terms of runoff rate, chemical speciation, bioavailability and ecotoxicity effects . Data have been collected during a three-year field exposure conducted in the urban environment of Stockholm, Sweden . The potential environmental effects have been evaluated using a combination of a copper specific biosensor test with the bacterium Alcaligenes eutrophus and the conventional 72-h growth inhibition test with the green alga Raphidocelis subcapitata . The results show annual runoff rates between 1.0 and 1.5 g/m2 year for naturally patinated copper of varying age . The runoff rate increased slightly with patina age, which mainly is attributed to the enhanced first flush effect observed on thicker patina layers . The total copper concentration in investigated runoff samplings ranged from 0.9 to 9.7 mg/l . Both computer modeling and experimental studies revealed that the majority (60-100%) of released copper was present as the free hydrated cupric ion, Cu(H2O)6(2+), the most bioavailable copper species . However, other copper species in the runoff water, such as, e.g . Cu(OH)+ and Cu2(OH)2(2+), were also bioavailable . The copper-containing runoff water, sampled directly after release from the roof, caused significant reduction in growth rate of the green alga . It should be emphasized that the results describe the runoff situation immediately after release from the copper roof and not the real environmental ecotoxicity . Therefore the data should only be used as an initial assessment of the potential environmental effect of copper runoff from building applications . Future risk assessments should also consider dilution effects of copper, changes in its chemical speciation and bioavailability during environmental entry, and type and sensitivity of the receiving ecosystem.

Eur J Biochem, 2002 Oct, 269(19), 4868 - 78
Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production; Lin PH et al.; An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine . The bacterium was classified as Variovorax paradoxus by phylogenetic analysis . The gene was cloned and sequenced . The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids . The V . paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity) . After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography . The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography . A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained . After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase . The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively . After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained . The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine . Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives . The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+ . Thus, the N-D-AAase from V . paradoxus can be considered a chiral specific and metal-dependent enzyme.

Syst Appl Microbiol, 2002 Aug, 25(2), 183 - 8
Accumulation of poly(3-hydroxybutyrate) from octanoate in different pseudomonas belonging to the rRNA homology group I; Diard S et al.; It is admitted that one of the characteristics of pseudomonads is their inability to accumulate poly(3-hydroxybutyrate) . In this paper, we show that poly(3-hydroxyoctanoate) synthesis is restricted to Pseudomonas rRNA homology group I, which includes both fluorescent and nonfluorescent species . However, within the genus Pseudomonas, the P . aeruginosa complex can be subdivided into two groups: the "P . aeruginosa group", which includes P . aeruginosa, P . alcaligenes, P . citronellolis, P . mendocina, produce poly(3-hydroxyoctanoate) from octanoate and the "P . oleovorans group" which includes the type strain of P . oleovorans, P . pseudoalcaligenes and two Pseudomonas sp., produce poly(3-hydroxybutyrate) during cultivation on octanoate . Strain GPo1 (ATCC 29347) formely identified as P . oleovorans and known to produce various medium-side-chain PHAs such as poly(3-hydroxyoctanoate) has been reclassified in the P . putida complex.

Prikl Biokhim Mikrobiol, 2002 Jul-Aug, 38(4), 401 - 4
{Use of maleic acid by mixed cultures of microorganisms}; Safronova IIu et al.; In a mixed batch culture, Alcaligenes xylosoxidans subsp . xylosoxidans 260 transformed maleic acid into malic acid . Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid . Both bacterial cultures dissociated with the formation of R, S, and M forms . At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A . xylosoxidans and Bacillus subtilis . At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively . Association of bacteria A . xylosoxidans and B . subtilis was not stable under flow conditions water.

J AOAC Int, 2002 Jul-Aug, 85(4), 917 - 24
Characterization of copolymer hydroxybutyrate/hydroxyvalerate from saponified vernonia, soybean, and "spent" frying oils; Saeed KA et al.; Poly(beta-hydroxyalkanoate)s (PHAs) were biosynthesized by Ralstonia eutropha (formerly known as Alcaligenes eutrophus) by using saponified soybean, vernonia, and "spent" frying oils . These PHAs were isolated and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), gas chromatography/mass spectrometry (GC/MS), proton nuclear magnetic resonance spectrometry (1H NMR), and 2-dimensional homonuclear (1H-1H) correlation spectroscopy (COSY) . The analytical results revealed that the PHAs produced from saponified vernonia and soybean oils were copolymers of hydroxybutyrate (HB) and hydroxyvalerate (HV), that is, P(HB/HV)s, whereas the saponified "spent" frying oil produced only poly(beta- hydroxybutyrate) (PHB) homopolymer . MALDI-MS, GC/MS, and NMR independently confirmed the composition of the PHAs . Saponified soybean oil and vernonia oil PHAs contained approximately 4 and 1% HV units, respectively . For comparison, commercial PHB and P(HB/HV), produced by R . eutropha by using glucose and a cosubstrate of glucose and propionic acid, respectively, as carbon sources, were similarly characterized.

Zh Mikrobiol Epidemiol Immunobiol, 2002 May-Jun, (3), 60 - 3
{Intestinal microflora in rats under the conditions of a chronic toxic lesion of the liver}; Sozinov AS et al.; Intestinal microflora in healthy rats and its changes under the conditions of experimental chronic toxic hepatitis were studied . The study revealed that in intact animals the microflora of the small intestine was represented by bacteria of the genera Escherichia, Enterobacter, Moraxella, Alcaligenes, Staphylococcus, Streptococcus . Bacteria of the genera Escherichia, Enterobacter, Moraxella, Alcaligenes, Staphylococcus, Corynebacterium and Clostridium were isolated from the large intestine . No bacteria were found in the systemic blood, the contents of the portal vein, as well as in the liver parenchyma and the mesenterial lymph nodes . As the result of dysbiosis induced by the introduction of kanamycin and in chronic hepatitis caused by carbon tetrachloride the sharp decrease in the species composition of microbial communities (up to 2-3 species) in the small intestine and was observed along with penetration of bacteria into the blood stream, the mesenterial lymph nodes and the liver parenchyma . The tendency towards the restoration of the quantitative and qualitative microflora composition was noted following administration into experimental animals of bactisubtil and amixin--an inductor of interferonogenesis.

Chemosphere, 2002 Jun, 47(10), 1073 - 80
Bioavailability of zinc in runoff water from roofing materials; Heijerick DG et al.; Corrosion and runoff from zinc-coated materials and outdoor structures is an important source for the dispersion of zinc in the environment . Being part of a large inter-disciplinary research project, this study presents the bioavailability of zinc in runoff water immediately after release from the surface of 15 different commercially available zinc-based materials exposed to the urban environment of Stockholm, Sweden . Runoff water was analysed chemically and evaluated for its possible environmental impact, using both a biosensor test with the bacteria Alcaligenes eutrophus (Biomet) and the conventional 72 h growth inhibition test with the green alga Raphidocelis subcapitata . Chemical speciation modelling revealed that most zinc (94.3-99.9%) was present as the free Zn ion, the most bioavailable speciation form . These findings were confirmed by the results of the biosensor test (Biomet) which indicated that all zinc was indeed bioavailable . Analysis of the ecotoxicity data also suggested that the observed toxic effects were due to the presence of Zn2+ ions . Finally, regression analysis showed that, for this type of runoff samples, the rapid screening biosensor was capable of predicting (a) the total amount of zinc present in the runoff samples (R2 of 0.93-0.98; p < 0.05) and (b) the observed 72 h-EbC50s (R2 of 0.69-0.97; p < 0.05).

Pediatr Pulmonol, 2002 Aug, 34(2), 101 - 4
Alcaligenes infection in cystic fibrosis; Tan K et al.; The aim of this study was to investigate the effect of chronic Alcaligenes species infection of the respiratory tract on the clinical status of patients with cystic fibrosis . We conducted a retrospective case-controlled study . The microbiological records of all patients attending the Leeds Regional Pediatric and Adult Cystic Fibrosis Units from 1992-1999 were examined . Chronic Alcaligenes infection was defined as a positive sputum culture on at least three occasions over a 6-month period . These patients were compared with controls matched for age, gender, respiratory function, and Pseudomonas aeruginosa infection status . Respiratory function tests, anthropometric data, Shwachman-Kulczycki score, Northern chest x-ray score, intravenous and nebulized antibiotic treatment, and corticosteroid treatment were compared from 2 years before to 2 years after Alcaligenes infection.From a clinic population of 557, 13 (2.3%) fulfilled the criteria for chronic infection . The median age at acquisition of infection was 17.2 years (range, 6.5-33.6) . There was no significant difference in the changes of percentage predicted values for FEV(1), FVC, FEF(25-75), or Shwachman-Kulczycki and Northern chest x-ray scores, or in weight, height, and body mass index z-scores between Alcaligenes-infected cases and controls . There was also no significant difference in the use of antibiotics (intravenous and nebulized) or corticosteroids (inhaled and oral).We conclude that in our clinic, chronic infection with Alcaligenes species was uncommon . Chronically infected patients showed no excess deterioration in clinical or pulmonary function status from 2 years before to 2 years after primary acquisition .

Aviakosm Ekolog Med, 2002, 36(2), 28 - 32
{Species composition and microbial contamination of atmospheric humidity condensate and potable water from the Mir water supply systems during Mir main missions 4 through 27}; Skuratov VM et al.; Analyzed were quantitative and qualitative characteristics, and processes of formation and transformation of microflora in the atmospheric humidity condensate (AHC) and potable water (PW) regenerated from the condensate by the long-operating system (SRV-K) aboard space station Mir . The paper presents data on species composition of microflora of AHC, and PW regenerated by SRV-K in the period from 1989 through to 1999 . The following microbial species were identified in AHC: Alcaligenes (14.8%), Bacillus (3.7%), Citrobacter (7.4%), Clostridium (11.1%), Enterobacter (7.4%), Proteus (7.4%), Pseudomonas (18.5%), and Staphylococcus (100%) . There were two PW cocks in SRV-K--"hot" and "cold" . "Hot" PW microflora included Alcaligenes (15%), Bacillus (5%), Citrobacter (20%), Clostridium (5%), Enterobacter (15%), Proteus (10%), Pseudomonas (15%), and Staphylococcus (75%) . "Cold" PW microflora included Aeromonas (14.3%), Alcaligenes (21.4%), Bacillus (7.1%), Citrobacter (7.1%), Clostridium (7.1%), Enterobacter (7.1%), Moraxella (7.1%), Pseudomonas (7.1%), and Staphylococcus (78.5%) . Microbial content averaged 8.9 x 10(2) +/- 1.3 (0.01-4) x 10(3) CFU/ml in condensate and 1.5 +/- 0.1 (0-3.0) CFU/ml in regenerated potable water.

Folia Microbiol (Praha), 2002, 47(3), 247 - 54
Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia; Totevova S et al.; Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contaminated by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106) . Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strains Pseudomonas alcaligenes KP2 and P . fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103 . DNA-DNA hybridization was used to compare both strains with well-known PCB-degraders Burkholderia cepacia strain LB400 and Ralstonia eutropha strain H850 . The strain KP12 employs the same meta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degrader Pseudomonas cepacia P166 . Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes . We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.

Biodegradation, 2001, 12(6), 393 - 400
Fluoranthene degradation in Pseudomonas alcaligenes PA-10; Gordon L et al.; Pseudomonas alcaligenes strain PA-10 degrades the four-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically . HPLC analysis of the growth medium identified four intermediates, 9-fluorenone-1-carboxylic acid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formed during fluoranthene degradation . Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acid and 9-hydroxy-1-fluorene-carboxylic acid resulted in increases in fluoranthene removal, while pre-exposure to 9-fluorenone and 9-fluorenol resulted in a decrease in fluoranthene degradation . The rate of indole transformation was similarly affected by pre-exposure to these metabolic intermediates, indicating a link between fluoranthene degradation and indigo formation in this strain.

Microb Ecol, 2002 Mar, 43(2), 280 - 9 Epub 2002 Feb 20.
Diversity of Pseudomonas spp . isolated from rice rhizosphere populations grown along a salinity gradient; Rangarajan S et al.; Along the coastline of Tamil Nadu, five sites were chosen to assess the diversity of Pseudomonas populations isolated from rice (Oryza sativa) cultivated along a salinity gradient . One of these sites was under organic farming while the other four were under inorganic farming . A total of 256 Pseudomonas strains isolated from these five sites were analyzed using both phenotypic (substrate utilization patterns and antibiotic resistance assay) and genotypic (PCR-RFLP of 16S rDNA) characteristics . The results derived from this study indicate that soil salinity affects rhizosphere Pseudomonas populations . It was observed that increasing salinity led to decreasing diversity . Fluorescent pseudomonads were the dominant species found in the non-saline site, while in the saline sites they were replaced by salt-tolerant species, in particular Pseudomonas alcaligenes and P . pseudoalcaligenes . An interesting observation was the increase in diversity found in the saline site under organic farming . Organic farming was found to be capable of mitigating the harmful effects of saline stress to a large extent, and restoring the Pseudomonas diversity, thereby making it comparable with the diversity encountered in the non-saline site.

Microb Ecol, 2002 Mar, 43(2), 199 - 216 Epub 2002 Jan 22.
Autecological properties of 3-chlorobenzoate-degrading bacteria and their population dynamics when introduced into sediments; Bott TL et al.; Ecologically significant properties of wild-type and genetically engineered bacteria capable of degrading 3-chlorobenzoate (3-CB) were compared in the laboratory, and isolates were introduced into streambed sediments in microcosms to observe their population dynamics . 3-CB metabolism, growth on algal extract, temperature optima, and ingestion by protozoa were ecological properties considered relevant to the persistence of these bacteria if introduced into nature . Cell-specific Vmax for 3-CB metabolism and cell-specific mineralization rates each spanned approximately 2 orders of magnitude, but isolates did not rank consistently . The Ks for 3-CB metabolism for Alcaligenes sp . BR60 was approximately 40-fold lower than the mean value for the other isolates, which differed only approximately 4-fold among themselves . All isolates grew on an algal extract nearly as well as on tryptone-yeast extract, implying potential for survival on natural metabolic substrates in situ . Most isolates had temperature optima that were 3-15 degrees C higher than maximum stream water temperature (22 degrees C) . Ciliates preferentially ingested P . acidovorans M3GY, and either P . putida RC-4(pSI30) or its parent strain were least preferred, but microflagellates did not exhibit consistent preferences . Fluorescent antibodies were prepared against isolates to permit detection of target cells in natural communities . In three different microcosm experiments the cell densities of introduced isolates declined over a period of days . In one experiment, 3-CB additions (100 mg/L) led to increases of P . alcaligenes C-0 and P . acidovorans M3GY cell densities within 1 day, although P . putida RC-4(pSI30) took 4 days . In a second experiment, the persistence of P . putida RC-4(pSI30) and its parent strain P . putida RC-4 were compared and rates of initial population decline were not statistically different . 3-CB addition stimulated the growth of other organisms while densities of the P . putida strains further declined . In a third experiment exposure to 100 mg 3-CB/L slowed the rate of decline of P . acidovorans M3GY densities compared to a 10 mg/L concentration or unamended control . Competition with the native flora was a significant factor affecting the persistence of introduced 3-CB degraders.

Antimicrob Agents Chemother, 2002 Jun, 46(6), 2014 - 6
Detection of a variant metallo-beta-lactamase, IMP-10, from two unrelated strains of Pseudomonas aeruginosa and an alcaligenes xylosoxidans strain; Iyobe S et al.; The gene bla(IMP-10) of a variant metallo-beta-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1 . Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.

Can J Microbiol, 2002 Mar, 48(3), 189 - 99
Response of spring rape (Brassica napus var . oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant; Belimov AA et al.; Responses of rape (Brassica napus var . oleifera L.) to inoculation with plant growth promoting rhizobacteria, Pseudomonas putida Am2, Pseudomonas putida Bm3, Alcaligenes xylosoxidans Cm4, and Pseudomonas sp . Dp2, containing 1-aminocyclopropane-l-carboxylate (ACC) deaminase were studied using growth pouch and soil cultures . In growth pouch culture, the bacteria significantly increased root elongation of phosphorus-sufficient seedlings, whereas root elongation of phosphorus-deficient seedlings was not affected or was even inhibited by the bacteria . Bacterial stimulation of root elongation of phosphorus-sufficient seedlings was eliminated in the presence of a high ammonia concentration (1 mM) in the nutrient solution . Bacterial effects on root elongation of potassium-deficient and potassium-sufficient seedlings were similar . The bacteria also decreased inorganic phosphate content in shoots of potassium- and phosphorus-sufficient seedlings, reduced ethylene production by phosphorus-sufficient seedlings, and inhibited development of root hairs . The effects of treatment with Ag+, a chemical inhibitor of plant ethylene production, on root elongation, ethylene evolution, and root hair formation were similar to bacterial treatments . The number of bacteria on the roots of phosphorus-deficient seedlings was not limited by phosphorus deficiency . In pot experiments with soil culture, inoculation of seeds with bacteria and treatment with aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis in plants, increased root and (or) shoot biomass of rape plants . Stimulation of plant growth caused by the bacteria was often associated with a decrease in the content of nutrients, such as P, K, S, Mo, and Ba, in shoots, depending on the strain used . The results obtained show that the growth-promoting effects of ACC-utilizing rhizobacteria depend significantly on the nutrient status of the plant.

J Biol Chem, 2002 Jun 28, 277(26), 23725 - 32 Epub 2002 Apr 18.
The X-ray structure of ferric Escherichia coli flavohemoglobin reveals an unexpected geometry of the distal heme pocket; Ilari A et al.; The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 A and refined to an R-factor of 19% . The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region . An inspection of the heme environment in E . coli flavohemoglobin reveals an unexpected architecture of the distal pocket . In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species . Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide . Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.

J Bacteriol, 2002 May, 184(9), 2399 - 403
Novel carbohydrate-binding module of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp . strain XY-234; Okazaki F et al.; A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp . strain XY-234, has been cloned and sequenced . txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da . The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus . The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan . TxyA was capable of binding specifically to beta-1,3-xylan . The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469 . Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively . Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.

Eur J Clin Microbiol Infect Dis, 2002 Feb, 21(2), 108 - 13
Investigation of an outbreak due to Alcaligenes xylosoxydans subspecies xylosoxydans by random amplified polymorphic DNA analysis; Lehours P et al.; In 1999, over a 3-week period, Alcaligenes xylosoxydans subsp . xylosoxydans was isolated from five blood cultures and one cerebrospinal fluid specimen from five children hospitalized in a pediatric hematology ward as well as from two respiratory therapy devices of two children hospitalized in an intensive care unit . The infection control unit of the hospital conducted an epidemiological investigation and identified a detergent-disinfectant solution as the source of contamination . Conventional biochemical tests, antimicrobial susceptibility tests and random amplified polymorphic DNA (RAPD) fingerprinting were used to compare clinical and environmental isolates . RAPD analysis proved to be more discriminant than biotyping or antibiotyping in this context and identified the common source of the outbreak.

Biotechnol Prog, 2002 Mar-Apr, 18(2), 252 - 6
Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10; Garcia-Ruiz V et al.; A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor . The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through {(2-hydroxyethyl)thio}acetic acid and thiodiacetic acid as intermediates . Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion . Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels . Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose . Oxidation to {(2-hydroxyethyl)thio}acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity . Inhibition of TDG transformation in the presence of several alcohols is also shown.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 255 - 62
The hydrogen-sensing apparatus in Ralstonia eutropha; Lenz O et al.; Molecular hydrogen is widely used by microorganisms as a source of energy . One of the best studied aerobic hydrogen oxidizers, the beta-proteobacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), harbors two distinct {NiFe}-hydrogenases which catalyze the heterolytic cleavage of H2 into 2H+ and 2e- . The genes encoding the hydrogenase subunits are arranged in two large operons together with accessory and regulatory genes involved in hydrogenase biosynthesis . Both operons are transcribed from strong sigma54-dependent promoters . Transcription requires the activation by the HoxA protein, a member of the NtrC family of response regulators . HoxA is only active when H2 is present in the environment . H2 recognition is mediated by a signal transduction complex consisting of the soluble histidine protein kinase HoxJ and a regulatory {NiFe}-hydrogenase which acts as an H2 receptor . Biochemical and genetic data suggest that signal transduction between the RH and HoxJ involves an electron transport process . According to our current model the histidine protein kinase HoxJ inactivates HoxA by phosphorylation in the absence H2 . This property of the HoxJ-HoxA regulator pair is quite different from the behaviour of common two-component regulatory systems . Phosphorylation of HoxA is blocked in the presence of H2 provided the RH can contact HoxJ and transmit the signal to the kinase . Furthermore, hydrogenase gene expression is subject to a global regulatory network in response to the carbon and energy source . HoxA is a major component of this epistatic control the molecular mechanism of which is not yet understood.

Mikrobiologiia, 2002 Jan-Feb, 71(1), 59 - 65
{Nonlinearity in the growth of bacterial colonies: conditions and causes}; Panikov NS et al.; The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size . The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density . In this work, Pirt's model was tested on two bacteria--Alcaligenes sp . and Pseudomonas fluorescens--having markedly distinct cultural properties and grown on agarized medium with pyruvate . The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (10(5)-10(6) cells), and for different depths of the agar layer, determining the amount of available substrate . A linear growth mode was observed only with P . fluorescens and only in the case of growth from a microdroplet . When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of Kr (the rate of colony radius increase) occurring after 2-3 days of growth . The growth of P . fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp . turned out to be directly related to the medium layer thickness . The departure from linearity is consistently explained by a new kinetic chart stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center . The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by age of cells . The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.

J Nat Prod, 2002 Mar, 65(3), 395 - 8
Halogenated metabolites from the new Okinawan red alga Laurencia yonaguniensis; Takahashi Y et al.; A novel brominated diterpene based on the rare neoirieane skeleton, named neoirietetraol (1), has been isolated along with a halogenated C15 acetogenin, (3Z)-laurenyne (2), from a new Laurencia species, L.yonaguniensis Masuda et Abe, species inedita, collected at Yonaguni Island, Okinawa Prefecture, Japan . The structures of these metabolites were elucidated by spectroscopic data (IR, 1H NMR, 13C NMR, 2D NMR, and MS) . Neoirietetraol (1) was toxic to the brine shrimp (Altemia salina; LC50, 40.1 microM) and also showed weak antibacterial activities against two marine bacteria, Alcaligenes aquamarinus and Escherichia coli.

Biomacromolecules, 2002 Mar-Apr, 3(2), 291 - 6
Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19; Kim do Y et al.; An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150 . The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa . The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC . The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol . It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100 . The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide) . Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents . It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms . The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units . The results demonstrated in this study suggest that the MCL-PHA depolymerase from P . alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.

Biochemistry, 2002 Mar 12, 41(10), 3430 - 8
Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center; Prudencio M et al.; Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers . The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer . The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed . Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers . On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site . As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV) . Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers . The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm . The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme . This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site . However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur . When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer . The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased . The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.

Biochemistry, 2002 Feb 19, 41(7), 2353 - 60
Six- to five-coordinate heme-nitrosyl conversion in cytochrome c' and its relevance to guanylate cyclase; Andrew CR et al.; The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step . Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.4 +/- 0.5) x 10(4) M(-1) x s(-1), which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket . Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm(-1), suggesting a distorted Fe-N-O coordination geometry . The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k(6-5) = (8.1 +/- 0.7) x 10(3) M(-1) x s(-1), implying that an additional molecule of NO is required to form the 5c-NO adduct . Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c' binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position . The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c' and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts.

Hybrid Hybridomics, 2001, 20(5-6), 325 - 32
Rapid identification of Ralstonia eutropha strain CH34 using monoclonal antibodies; Melchior F et al.; Ralstonia eutropha strain CH34 (formerly Alcaligenes eutrophus CH34) is an aerobic Gram-negative and facultative chemolithotrophic bacteria with plasmid-bound resistance to heavy metals . The presence of Ralstonia eutropha strain CH34 is an indication of environmental heavy metals pollution . The major purpose of this work was to produce monoclonal antibodies (MAbs) against the metal transport outer membrane proteins . In this way, bacteria outer membranes, grown with or without iron, were purified . The electrophoretic pattern of the outer membrane revealed that, in iron starvation conditions, at least four proteins were overexpressed . These outer membranes were used to immunize mice to produce MAbs . About 200 hybridomas were tested by enzyme-linked immunoadsorbent assay (ELISA) . Most of these hybridomas exhibited cross reactions with Escherichia coli and Klebsiella aerogenes . Two hybridomas, AE5/7 and AE5/9, produced MAbs that detected specifically Ralstonia eutropha strain CH34 . Analysis by Western blotting showed that these MAbs recognized a protein with a molecular weight of about 41 kDa . Moreover, the presence of the two megaplasmids was required for the full expression of the 41-kDa protein, as demonstrated by screening of the derivatives strains by ELISA . These MAbs could be used for a specific and rapid detection of Ralstonia eutropha strain CH34, using direct immunological methods.

J Mol Biol, 2002 Feb 8, 316(1), 51 - 64
Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism; Ellis MJ et al.; Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO) . Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites . Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite . At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site . We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254 . The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite . Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme . For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated . The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively . The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme . Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons . The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water . Therefore, the ligand is no longer able to perform proton abstraction . Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel . The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed . The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme .

Biodegradation, 2001, 12(4), 209 - 17
Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp . JS867 under oxygen limited conditions; Smets BF et al.; Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 under varying degrees of oxygen limitation was examined . Complete 2,4-DNT removal was observed under oxygen excess with near stoichiometric release (83%) of nitrite . Average kinetic parameters were estimated based on a dual-Monod biokinetic model with 2,4-DNT and O2 as growth limiting substrates . The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO2- . Under aerobic conditions, mumax, KsDNT, and KiNO were 0.058 (0.004)hr(-1), 3.3(+/-1.3) mg 2,4-DNT/L, and 1.2(+/-0.2)hr(-1), respectively . At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced . JS867 was able to use NO2- as a terminal electron acceptor when grown on glucose or succinate under anaerobic conditions . However, during growth on 2,4-DNT and under O2-limited conditions, JS867 did not use released nitrite as electron acceptor . The nearly constant molar ratios of DNT removed over NO2- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued . No evidence of nitroreduction was obtained under the examined oligotrophic conditions . JS867 displayed a high affinity for oxygen consumption with K(SO2) value of 0.285(+/-0.198) mg O2/L . Our results indicate that under oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.

Am J Nephrol, 2001 Nov-Dec, 21(6), 502 - 6
CAPD-associated peritonitis caused by Alcaligenes xylosoxidans sp . xylosoxidans; Tang S et al.; Alcaligenes xylosoxidans is an uncommon cause of peritonitis in patients on maintenance continuous ambulatory peritoneal dialysis (CAPD) . Peritonitis caused by A . xylosoxidans usually carries a poor prognosis because of the pathogen's virulence and its universal resistance to most antimicrobial agents . Even after early Tenckhoff catheter removal, the transport property of the peritoneum is often irreversibly damaged, leading to permanent technique failure . We report 2 patients with CAPD-associated peritonitis due to A . xylosoxidans sp . xylosoxidans who were successfully cured with a combination of piperacillin and tazobactam . One of them subsequently returned uneventfully to CAPD .

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 510 - 4
{High-level production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by feb-batch culture of Alcaligenes eutrophus}; Cai YB et al.; Fermentation strategies for production P (3HB-co-3HV) from glucose and propionic (or valeric) acid by Alcaligenes eutrophus were studied . During the culture, we controlled pH of the broth by feeding precusors of 3HV- propionic or valeric acid after Ammonia feeding stopped . When propionic acid were used as the precusor, for 50 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 149.9 g/L, 124.9 g/L, 83.3% and 12.4 mol%, respectively, with a PHA productivity of 2.50 g h-1 L-1 . When valeric acid were used as the precusor, for 45 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 160.2 g/L, 119.0 g/L, 74.2% and 17.7 mol%, respectively, with a PHA productivity of 2.64 g h-1 L-1 . Prior to this study, it hasn't been reported to obtain such high level productivity and 3HV fraction at the same time by Alcaligenes eutrophus.

Mikrobiologiia, 2001 Nov-Dec, 70(6), 745 - 52
{Autotrophic synthesis of polyalkanoates by Alcaligenes eutrophus in presence of carbon monoxide}; Volova TG et al.; The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophus was found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide . The gas mixtures that contained from 5 to 20 vol% CO did not inhibit the key enzymes of PHA synthesis--beta-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase . In the presence of CO, cells accumulated up to 70-75 wt% PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate . Chromatographic-mass spectrometric analysis showed that the PHA synthesized by A . eutrophus is a copolymer containing more than 99 mol% beta-hydroxybutyrate and trace amounts of beta-hydroxyvalerate . The PHA synthesized under the conditions described did not differ from that synthesized by A . eutrophus cells from electrolytic hydrogen.

Appl Environ Microbiol, 2002 Jan, 68(1), 152 - 60
Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb; Kaur R et al.; Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp . strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner . The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily . Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml) . The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex . To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R) . The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb . Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R . In addition, E . coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells . A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone . Overproduction of VHb-R in the hmp mutant of E . coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress . Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions . Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host . Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.

Appl Environ Microbiol, 2002 Jan, 68(1), 102 - 5
Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene; Carvalho MF et al.; Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals . A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site . A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization . Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species . Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively) . Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures . In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate . To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

J Clin Microbiol, 2001 Dec, 39(12), 4452 - 5
Identification of Pandoraea species by 16S ribosomal DNA-based PCR assays; Coenye T et al.; The recently described genus Pandoraea contains five named species (Pandoraea apista, Pandoraea pulmonicola, Pandoraea pnomenusa, Pandoraea sputorum, and Pandoraea norimbergensis) and four unnamed genomospecies . Pandoraea spp . have mainly been recovered from the respiratory tracts of cystic fibrosis (CF) patients . Accurate genus- and species-level identification by routine clinical microbiology methods is difficult, and differentiation from Burkholderia cepacia complex organisms may be especially problematic . This can have important consequences for the management of CF patients . On the basis of 16S ribosomal DNA sequences, PCR assays for the identification of Pandoraea spp . were developed . A first PCR assay was developed for the identification of Pandoraea isolates to the genus level . PCR assays for the identification of P . apista and P . pulmonicola as a group, P . pnomenusa, P . sputorum, and P . norimbergensis were also developed . All five assays were evaluated with a panel of 123 bacterial isolates that included 69 Pandoraea sp . strains, 24 B . cepacia complex strains, 6 Burkholderia gladioli strains, 9 Ralstonia sp . strains, 5 Alcaligenes xylosoxidans strains, 5 Stenotrophomonas maltophilia strains, and 5 Pseudomonas aeruginosa strains . The use of these PCR assays facilitates the identification of Pandoraea spp . and avoids the misidentification of a Pandoraea sp . as a B . cepacia complex isolate.

Mol Microbiol, 2001 Nov, 42(3), 587 - 601
Discovery and distribution of super-integrons among pseudomonads; Vaisvila R et al.; Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes . The discovery of 'super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed . Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors . Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044 . This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats . Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron . Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 6 - 12
Microbial production of poly-D-3-hydroxybutyrate from CO2; Ishizaki A et al.; This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha . Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R . eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system . Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding . Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9% . However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R . eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture . It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.

Mikrobiol Z, 2001 Jul-Aug, 63(4), 69 - 75
{Effect of introduced microorganisms on copper and cobalt mobility in soil}; Nikovskaia GN et al.; The paper deals with the change of copper and cobalt mobility in contaminated soil when activating microbiological processes by introduction of sources of carbon, nitrogen, phosphorus, metal-resistant microorganisms . When Bacillus cereus BKM 4368 growing in soil fertilized with glucose one can observe a 5-fold increase of cocentration of heavy-metal mobile forms as compared with sterile soil . The use of acetate as a carbon source and Alcaligenes euthrophus CH 34 culture as inoculum will provide a 3-fold decrease of concentration of copper and cobalt mobile forms in the process of microorganisms growth . Contribution of the soil microflora to the process of transformation of heavy metals under the investigated conditions was too low . The results obtained give a possibility to regulate the level of mobile forms of heavy metals in soil by means of choosing the adequate sources of carbon and microbial cultures.

J Clin Microbiol, 2001 Nov, 39(11), 3942 - 5
Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis; Saiman L et al.; In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients . Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF . We examined the frequency of correct identification of Alcaligenes spp . by microbiology laboratories affiliated with American CF patient care centers . Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification . The activity of antimicrobial agents against these clinical isolates was determined . A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied . Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans . However, 12 (11%) strains were misidentified; these were found to be P . aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1) . Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited . High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains . Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains . Selective media and biochemical identification proved to be useful strategies for distinguishing A . xylosoxidans from other CF pathogens . Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A . xylosoxidans should be determined.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 141 - 6
Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867; Chua CH et al.; Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product . Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867 . Alignment of known GDO sequences indicated the presence of a conserved central core region . Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78% . Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region . Conversion of these His residues to Asp resulted in the complete loss of catalytic activity . Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding . A mutant enzyme with increased catalytic activities was also characterized.

Ukr Biokhim Zh, 2001 Jan-Feb, 73(1), 142 - 7
{Preparation of bacteria--degraders of alkylnaphthalenesulfonate by a molecular breeding method}; Dybkova SN; Transconjugant strain of bacteria degraders of alkylnaphthalenesulphonate(ANS)--P Pseudomonas alcaligenes TR (NPL-41) was obtained under conditions of ANS selective pressure and flow cultivation of bacteria destructors of alkylbenzene sulphonate (ABS) P . alcaligenes TR (pABS) and naphthalene P . putida BS438 (NPL-41) and their immobilization on an inert carrier . Transconjugant P . alcaligenes TR(NPL-41) was prepared by means of conjugative transfer of plasmid NPL-41 from P . putida BS438 to P . alcaligenes TR . The check out of transconjugant under conditions of flow cultivation showed their ability to degradation of ANS . Investigation of the stability of plasmid NPL-41 in the transconjugant P . alcaligenes TR(NPL-41), obtained by molecular breeding under conditions of flow cultivation and by conjugative transfer, showed the transconjugant prepared by the former method to be more stable.

Sci Total Environ, 2001 Sep 28, 277(1-3), 169 - 80
Runoff rates and ecotoxicity of zinc induced by atmospheric corrosion; Karlen C et al.; Initiated by regulatory restrictions on the use of zinc for various building and construction applications, together with a lack of knowledge related to the release of zinc induced by atmospheric corrosion, a major interdisciplinary research project was implemented to generate data to be used in future risk assessment . Runoff rates from a large number of commercially available zinc-based materials have been determined on panels inclined 45 degrees from the horizon, facing south, during a 1-year atmospheric exposure in an urban environment in Sweden . Possible environmental effects of runoff water immediately after leaving the surface of the various materials have been evaluated during two different sampling periods of varying season and zinc concentration, using the standard growth inhibition test with algae . Raphidocelis subcapitata (formerly Selenastrum capricornutum) . Zinc-specific biosensors with the bacterial strain of Alcaligenes eutrophus, and computer modeling using the water-ligand model MINTEQA2 and the humic aquatic model WHAM, have been used to assess the bioavailability and chemical speciation of zinc in the runoff water . An excellent consistency between the different methods was observed . The results show considerably lower runoff rates of zinc (0.07-3.5 g m(-2) year(-1)) than previously being used for regulatory restrictions, and the concentration of zinc to be predominantly responsible for the observed toxicity of the runoff water towards the green algae . The majority of the released zinc quantity was found to be present as free hydrated zinc ions and, hence, bioavailable . The data do not consider changes in bioavailability and chemical speciation or dilution effects during entry into the environment, and should therefore only be used as an initial assessment of the potential environmental effect of zinc runoff from building applications . This interdisciplinary approach has the potential for studies on the environmental fate of zinc in soil or aquatic systems.

Biotechnol Prog, 2001 Sep-Oct, 17(5), 798 - 808
Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli; Bollinger CJ et al.; Limited oxygen availability is a prevalent problem in microbial biotechnology . Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions . We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E . coli . Several finished and unfinished bacterial genomes were screened using R . eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins . Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni . Previously characterized hmp genes from E . coli and Bacillus subtilis and the novel hmp genes from P . aeruginosa, S . typhi, C . jejuni, K . pneumoniae, and D . radiodurans were PCR amplified and introduced into a plasmid for expression in E . coli . Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide . Growth behavior and byproduct formation of E . coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E . coli cells expressing VHb . The clones expressing hemoglobins from E . coli, D . radiodurans, P.aeruginosa, and S . typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E . coli control (A(600) 5.5) . E . coli cells expressing hemoproteins from P . aeruginosa, S . typhi, and D . radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E . coli strain . Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D . radiodurans hemoprotein-expressing E . coli relative to the E . coli control carrying the parental plasmid without any hemoglobin gene.

J Clin Pathol, 2001 Oct, 54(10), 803 - 5
Improved cultural detection of Burkholderia cepacia from sputum in patients with cystic fibrosis; Wright RM et al.; AIMS: To evaluate the sensitivity and specificity of two selective media for the isolation of Burkholderia cepacia from sputum specimens in patients with cystic fibrosis (CF) . METHODS: In total, 149 expectorated sputum specimens from 113 patients with CF (32 cepacia colonised patients and 81 non-cepacia colonised patients) attending three CF centres were examined for the presence of B cepacia on two selective media: (1) MAST selective agar, a commercially available selective medium widely used in the UK and (2) BCSA (B cepacia selective agar), a new medium recently described, which is used predominantly in North America . RESULTS: Burkholderia cepacia was isolated from 53 of 149 (35.6%) specimens examined, representing 32 of 113 (28.3%) patients, using both the MAST and BCSA media . Growth was most rapid on BCSA with all (53 of 53) isolates detectable after 48 hours, compared with 50 of the 53 isolates on MAST agar, with the remaining three isolates detectable at five days . Twenty eight contaminants were identified on MAST agar and 13 on BCSA agar; mainly Alcaligenes xylosoxidans and yeast on MAST agar and Flavobacterium indologenes on BCSA medium . BCSA was equivalent to MAST agar in its ability to isolate B cepacia from patients with CF with a history of B cepacia infection . CONCLUSIONS: The increased selectivity and reduced time to detection of BCSA makes it an attractive alternative to MAST . However, its present limited commercial availability in the UK may delay its use in routine diagnostic laboratories because of complications with media preparation and quality control.

J Appl Microbiol, 2001 Oct, 91(4), 742 - 9
Diversity of pseudomonads isolated from three different plant rhizospheres; Rangarajan S et al.; AIMS: To study the diversity of the Pseudomonas populations isolated from three different plant rhizospheres, namely pearl millet, cotton and paddy, grown in saline soils along the coastline of Southern India . METHODS AND RESULTS: The Pseudomonas populations were analysed for their biochemical characters and genetic diversity using molecular tools including RAPD and PCR-RFLP . The biochemical characterization, antibiotic resistance assay and RAPD profiles revealed a largely homogeneous population . Even in PCR-RFLP restriction studies, only two groups of isolates were seen . One group was predominant in all three rhizospheres, while the other minor group consisted of salt-sensitive isolates restricted to the paddy rhizosphere alone . CONCLUSIONS: It was observed that increasing salinity caused a predominant selection of salt-tolerant species, in particular Ps . pseudoalcaligenes and Ps . alcaligenes, irrespective of the host rhizosphere . SIGNIFICANCE AND IMPACT OF THE STUDY: This study has reinstated the importance of the soil over the host plant with regard to rhizosphere populations . It has also resulted in the isolation of several salt-tolerant Pseudomonas strains, which are being screened for their biological control activity against common plant pathogens of the coastal agri-ecosystem.

Can J Microbiol, 2001 Jul, 47(7), 642 - 52
Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase; Belimov AA et al.; Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals . The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp . by determination of 16S rRNA gene sequences . The root elongation of Indian mustard and rape (Brassica napus var . oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively . The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution . The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis . A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil . The biomass of pea cv . Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture . The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions . The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions.

Antibiot Khimioter, 2001, 46(2), 26 - 32
{Antibiotic therapy of cystic fibrosis in children}; Kapranov NI et al.; It is postulated that P . aeruginosa in monoculture or in association with Staphylococcus aureus keeps its leading position in chronic bacterial inflammatory broncho-pulmonary processes in children with cystic fibrosis . Antibiotic resistant strains of Burkholderia cepacia, Stenotrophomonas maltophila, Alcaligenes xylosoxidans were revealed (7.1% of the strains) . P . aeruginosa strains were susceptible to aminoglycosides, ciprofloxacin, and polymixin B . Susceptibility of smooth and mucoid forms of P . aeruginosa to ceftazidime stayed at the level of 49.6-57.1% . Such microbial associations as P . aeruginosa sm . + S . aureus, P . aeruginosa sm . + P . aeruginosa muc . + S . aureus were mainly susceptible to ciprofloxacin, aminoglycosides and resistant to ceftasidime . Meropenem, cefepim and ciprofloxacin are highly effective antibiotics for the treatment of broncho-pulmonary processes exacerbations at children with chronic P . aeruginosa cystic fibrosis . Intravenous use of antibiotics out of hospital for the treatment of the children with cystic fibrosis is clinically effective, and is economically and psychologically reasonable . It should be used more widely in medical practice.

J Bacteriol, 2001 Oct, 183(19), 5651 - 8
Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34; Borremans B et al.; The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II) . The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase . Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration . Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins . This is the first report of a mechanism for specific lead resistance in any bacterial genus.

Prikl Biokhim Mikrobiol, 2001 Jul-Aug, 37(4), 436 - 8
{Optimization of conditions for transformation of maleic acid by immobilized cells of Alcaligenes xylosoxidans subspecies xylosoxidans 260}; Safronova IIu et al.; Immobilized cells of Alcaligenes xylosoxidans subsp . xylosoxidans 260 used 98% of maleic acid (initial concentration of 5.0 g/l medium) in periodic conditions for 48 h . Free cells transformed only 26% of substrate in 96 h . Immobilized cells of a selected S-variant of A . xylosoxidans used maleate (30.0 g/l) entirely in 96 h during periodic cultivation and only 15.0 g/l of maleate in continuous cultivation at a flow rate of 0.03 h-1.

Org Lett, 2001 Sep 6, 3(18), 2923 - 6
Synthesis of a broad array of highly functionalized, enantiomerically pure cyclohexanecarboxylic acid derivatives by microbial dihydroxylation of benzoic acid and subsequent oxidative and rearrangement reactions; Myers AG et al.; {reaction: see text} . We have found that the 1,2-dihydroxylation of benzoic acid with Alcaligenes eutrophus strain B9, first reported in 1971 by Reiner and Hegeman, is readily adapted for the preparation of tens to hundreds of grams of (1S,2R)-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylic acid of >95% ee . This unique substrate undergoes many specific oxidative and rearrangement processes . Among these are transformations of unanticipated chemical novelty and many products that have not been previously described.

J AOAC Int, 2001 Jul-Aug, 84(4), 1109 - 15
Characterization of partially transesterified poly(beta-hydroxyalkanoate)s by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Saeed KA et al.; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for the characterization of a partially transesterified poly(beta-hydroxyalkanoate) (PHA), a polymer produced by the bacterial strain Alcaligenes eutrophus with saponified vegetable oils as the sole carbon sources . The transesterification was carried out separately under acidic and basic conditions to obtain PHA oligomers weighing <10 kDa . The intact oligomers were detected in their cationized forms, {M + Na}+ and {M + K}+, by MALDI-TOFMS . A composition analysis, using the MALDI-TOF spectra, indicated that the oligomers obtained via acid catalysis contained a methyl 3-hydroxybutyrate end group, and those obtained by base catalysis had a methyl crotonate (olefinic) end group . In addition to hydroxybutyrate (HB), the oligomers were found to contain a small percentage of hydroxyvalerate, which was independently confirmed by gas chromatography/mass spectrometry . In comparison, analysis of a commercial PHA polymer, transesterified under identical conditions, showed only the presence of HB, i.e., a pure poly(HB) homopolymer.

Res Microbiol, 2001 Jul-Aug, 152(6), 583 - 92
Polychlorinated biphenyl degradation activities and hybridization analyses of fifteen aerobic strains isolated from a PCB-contaminated site; Fedi S et al.; Fifteen bacterial strains using biphenyl as sole carbon and energy source, obtained from different positions and depths of a polychlorinated biphenyl (PCB)-contaminated area, were analyzed for their basic metabolic phenotypes and subjected to genomic DNA hybridization screening for the presence of well characterized bph operons such as those of Pseudomonas pseudoalcaligenes KF707 and Rhodococcus globerulus P6 . Most of the isolates belonged to the gamma-subdivision (Pseudomonas stutzeri, P . plutida, P . fluorescens and Vibrio logei species) and to the beta-subdivision (genera Alcaligenes, Comamonas, Ralstonia) of the Proteobacteria . All the isolates were able to cometabolize different low chlorinated PCB congeners . Among the dichlorinated biphenyls tested, a lower degradation capacity was observed for the di-ortho substituted congeners, whereas high levels of degradation were observed for the di-meta and di-para isomers, whether they were chlorinated on one or on both rings . The PCB congeners nonsubstituted in the 2,3 or 2,3 and 3,4 positions were also degraded by most of the isolated strains, which were, however, unable to significantly metabolize PCBs with more than 3 chlorine atoms . Five of the isolated strains were also able to degrade some of the tri- and tetrachlorobiphenyls tested . Southern hybridization analysis showed a strong homology between four of the fifteen isolated strains and the bph operon obtained from P . pseudoalcaligenes strain KF707 . Conversely, none of the isolates here examined showed homology with the bph operon of R . globerulus strain P6 . In line with this, the KF707 bph probe strongly hybridized with DNA of a significant number of bacterial colonies obtained from selected locations in the contaminated area using biphenyl-supplemented minimal medium agar plates.

Environ Toxicol, 2001, 16(4), 306 - 13
Factors affecting biodegradation of 2-chlorophenol by Alcaligenes sp . in aerobic reactors; Gallego A et al.; The influence of variations in carbon source concentration, cell inocula, pH, presence of other substrates, and other organisms on the biodegradation of 2-chlorophenol (2-CP) was studied for Alcaligenes sp . isolated from natural sources . Assays of biodegradation were performed in batch and continuous-flow fluidized-bed aerobic reactors . Evaluation of biodegradation was performed by determining total phenols, chemical oxygen demand (COD), and 2-CP by ultraviolet (UV) spectrophotometry . Measurement of microbial growth was carried out by the plate count method . Bioassays of acute toxicity were performed to evaluate detoxification by using Daphnia magna . Results obtained show that under batch conditions with initial inocula of 10(6) cells/mL the strain grew exponentially with 100, 200, and 300 mg/L of 2-CP within 48 hr . A lag period was observed with low cell density inocula (10(5) cells/mL) . The strain showed marked delay in the biodegradation of 2-CP at pH 5 . Removal of target substrate from mixtures containing other carbon sources demonstrated the possibility of concurrent growth . Mineralization of 2-CP was assessed by gas chromatography carried out at the end of the batch assays and at the exit of the continuous-flow reactor . The presence of other organisms (bacteria, rotifers, ciliate, and algae) that developed in the fluidized-bed reactor did not affect the efficacy of the biodegradation of 2-CP . The removal of 2-CP in the two assayed systems was over 97% in all cases . Toxicity was not detected at the exit of the continuous reactor.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 270 - 5
Isolation and characterization of a thermotolerant bacterium Ralstonia sp . strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene; Lee SK et al.; A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source . Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 degrees C and pH 7.2 . According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus) . Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol . The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source . It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring . The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.

Appl Environ Microbiol, 2001 Aug, 67(8), 3530 - 41
Identification and functional characterization of CbaR, a MarR-like modulator of the cbaABC-encoded chlorobenzoate catabolism pathway; Providenti MA et al.; In Comamonas testosteroni BR60 (formerly Alcaligenes sp . strain BR60), catabolism of the pollutant 3-chlorobenzoate (3CBA) is initiated by enzymes encoded by cbaABC, an operon found on composite transposon Tn5271 of plasmid pBRC60 . The cbaABC gene product CbaABC converts 3CBA to protocatechuate (PCA) and 5-Cl-PCA, which are then metabolized by the chromosomal PCA meta (extradiol) ring fission pathway . In this study, cbaA was found to possess a sigma(70) type promoter . O(2) uptake experiments with whole cells and expression studies with cbaA-lacZ constructs showed that cbaABC was induced by 3CBA . Benzoate, which is not a substrate of the 3CBA pathway, was a gratuitous inducer, and CbaR, a MarR family repressor coded for by a divergently transcribed gene upstream of cbaABC, could modulate induction mediated by benzoate . Purified CbaR bound specifically to two regions of the cbaA promoter (P(cbaA)); site I, a high-affinity site, is between the transcriptional start point (position +1) and the start codon of cbaA, while site II, a lower-affinity site, overlaps position +1 . 3CBA at concentrations as low as 40 microM interfered with binding to P(cbaA) . PCA also interfered with binding, while benzoate only weakly disrupted binding . Unexpectedly, benzoate with a hydroxyl or carboxyl at position 3 improved CbaR binding . Data are also presented that suggest that an unidentified regulator is encoded on the chromosome that induces cbaABC in response to benzoate and 3CBA.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1110 - 8 Epub 2001 Jul 23.
X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 A resolution; Ellis MJ et al.; Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes . Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8 . The structure of this nitrite reductase has previously been determined at pH 4.6 . It has now been crystallized under new conditions at pH 8.5 . Its crystallographic structure provides a structural explanation for the greatly reduced activity of the enzyme at high pH . Characterization of overexpressed protein in solution by EXAFS suggested that the protein lacked Cu in the catalytic type 2 Cu site and that the site was most probably occupied by Zn . Using the anomalous signals from Cu and Zn, the crystal structure revealed that the expressed protein was devoid of Cu in the catalytic site and that only a trace amount (<10%) of Zn was present at this site in the crystal . Despite the close structural similarity of the catalytic site to a number of Zn enzymes, these data suggest that Zn, if it binds at the catalytic copper site, binds weakly in nitrite reductase.

Org Lett, 2001 Jan 25, 3(2), 283 - 5
A thioesterase for chemoselective hydrolysis of S-acyl sulfanylalkanoates; Kumar I et al.; {figure: see text} A thioesterase, isolated from a strain of Alcaligenes sp . ISH108, chemoselectively hydrolyzes thiol esters . The application of the enzyme has been demonstrated in the preparation of the antihypertensive agent captopril.

Br J Ophthalmol, 2001 Jul, 85(7), 842 - 7
Bacterial keratitis: a prospective clinical and microbiological study; Schaefer F et al.; AIM: To define the clinical and microbiological profile of bacterial keratitis at the Jules Gonin Eye Hospital and to test the in vitro bacterial resistance . METHODS: Patients presenting with bacterial keratitis were prospectively followed; clinical features (age, risk factors, visual acuity) and response to therapy were analysed . Bacteriological profile was determined and the sensitivity/resistance of isolated strains were tested towards 12 ocular antibiotics (NCCLS disc diffusion test) . RESULTS: 85 consecutive patients (mean age 44.3 (SD 20.7) years) were prospectively enrolled from 1 March 1997 to 30 November 1998 . The following risk factors were identified: contact lens wear, 36%; blepharitis, 21%; trauma, 20%; xerophthalmia, 15%; keratopathies, 8%; and eyelid abnormalities, 6% . The most commonly isolated bacteria were Staphylococcus epidermidis, 40%; Staphylococcus aureus, 22%; Streptococcus pneumoniae, 8%; others Streptococcus species, 5%; Pseudomonas, 9%; Moraxella and Serratia marcescens, 5% each; Bacillus, Corynebacterium, Alcaligenes xyloxidans, Morganella morganii, and Haemophilus influenza, 1% each . 1-15% of strains were resistant to fluoroquinolones, 13-22% to aminoglycosides, 37% to cefazolin, 18% to chloramphenicol, 54% to polymyxin B, 51% to fusidic acid, and 45% to bacitracin . Five of the 85 patients (5.8%) had a poor clinical outcome with a visual loss of one or more lines of visual acuity . CONCLUSION: Fluoroquinolones appear to be the therapy of choice for bacterial keratitis, but, based upon these in vitro studies, some strains may be resistant.

Plasmid, 2001 May, 45(3), 233 - 9
Isolation and characterization of group II introns from Pseudomonas alcaligenes and Pseudomonas putida; Yeo CC et al.; Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P . putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96% . The genome of P . alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP) . Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators . These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition . One of the group II introns found in P . alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.

Curr Microbiol, 2001 Sep, 43(3), 158 - 62
Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA; Qin X et al.; We have isolated, cloned, and sequenced a 5823-bp cryptic plasmid from a strain of Xylella fastidiosa . This plasmid encodes five open reading frames (ORF) greater than 400 nucleotides each . ORF 2 encodes a protein with 37% amino acid identity to the replication initiator protein of plasmid pECB2 from Pseudomonas alcaligenes . This RepA protein from X . fastidiosa contains both a leucine zipper and helix turn helix motif characteristic of proteins involved in DNA replication . The sequence 5' of ORF 2 has all of the features characteristic of plasmid origins of replication as well as regulatory elements required for transcription of ORF 2 . Open reading frame 2, along with the upstream origin of replication, was cloned as an EcoRI fragment into pUC19 to create a shuttle vector . This construct was introduced into Xylella fastidiosa by electroporation, with selection for carbenicillin resistance . Transformation was verified by both PCR and Southern hybridization experiments . Frequency of transformation was low, but increased ten-fold when the plasmid was grown in X . fastidiosa rather than Escherichia coli prior to transformation . This work represents the first step towards the development of a system for genetic analysis of this important plant pathogen of citrus, grapevines, and other horticultural crops.

Curr Microbiol, 2001 Jun, 42(6), 438 - 41
Frankia KB5 possesses a hydrogenase immunologically related to membrane-bound; Mattsson U et al.; The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-{NiFe}-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum . The antisera raised against the A . latus, R . eutropha, and B . japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5 . None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B . japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5 . An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity.

Environ Microbiol, 2001 Apr, 3(4), 288 - 93
Characterization of (R/S)-mecoprop {2-(2-methyl-4-chlorophenoxy) propionic acid}-degrading Alcaligenes sp.CS1 and Ralstonia sp . CS2 isolated from agricultural soils; Smejkal CW et al.; The herbicide mecoprop {2-(2-methyl-4-chlorophenoxy) propionic acid} is widely applied to corn fields in order to control broad-leaved weeds . However, it is often detected in groundwater where it can be a persistent contaminant . Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade (R/S)-mecoprop rapidly . 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp . (designated CS1) and the other to Ralstonia sp . (designated CS2) . Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4-D (2,4-dichlorophenoxyacetic acid), MCPA {4-chloro-2-methyl phenoxy acetic acid} and (R/S)-2,4-DP {2-(2,4-dichlorophenoxy)propionic acid} as sole carbon sources . tfdABC gene-specific probes derived from the 2,4-D-degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd-like genes are present in mecoprop-degrading bacteria . Hybridization analysis demonstrated that both Alcaligenes sp . CS1 and Ralstonia sp . CS2 harboured tfdA, tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA, tfdB and tfdC genes of V . paradoxus . It is therefore likely that tfd-like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.

Can J Microbiol, 2001 Mar, 47(3), 229 - 36
Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks; Araujo WL et al.; Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks . The principal bacterial species isolated were Alcaligenes sp., Bacillus spp . (including B . cereus, B . lentus, B . megaterium, B . pumilus, and B . subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P . agglomerans and B . pumilus being the most frequently isolated species . The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp . Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B . pumilus isolates were more diverse than P . agglomerans isolates, although genetic diversity was not related to the host plants . In vitro interaction studies between G . citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G . citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P . agglomerans.

Biochemistry, 2001 Apr 3, 40(13), 4115 - 22
Resonance Raman studies of cytochrome c' support the binding of NO and CO to opposite sides of the heme: implications for ligand discrimination in heme-based sensors; Andrew CR et al.; Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2) . Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center . The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors . Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond . RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket . On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme . Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.

J Bacteriol, 2001 Apr, 183(8), 2560 - 9
Mutually exclusive distribution of IS1548 and GBSi1, an active group II intron identified in human isolates of group B streptococci; Granlund M et al.; The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene, scpB, in some group B streptococcus (GBS) isolates that lack insertion sequence IS1548 . IS1548 was previously reported to be often present at the scpB locus in GBS isolated in association with endocarditis . Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS1548 and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III . The DNA region downstream of scpB in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins . IS1548 is inserted 9 bp upstream of the putative promoter for lmb, while the insertion site for GBSi1 is located 88 bp further upstream . Sequences highly similar to GBSi1 exist also in Streptococcus pneumoniae . An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and S . pneumoniae sequences . This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in Bacillus halodurans, Pseudomonas alcaligenes, and Pseudomonas putida . When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated . Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.

Int J Biol Macromol, 2001 Mar 14, 28(3), 235 - 43
Effect of dissolved oxygen concentration in the fermentation medium on transformation of the carbon sources during the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxypropionate) by Alcaligenes latus; Wang Y et al.; Effects of fermentation conditions on the comonomer composition and its distribution of poly(3-hydroxybutyrate-co-3-hydroxypropionate) {P(3HB-co-3HP)} have been investigated for bacterial synthesis of P(3HB-co-3HP)s by Alcaligenes latus from sucrose and 3-hydroxypropionate (3HPA) mixed carbon sources . Comparison of the microstructures of these samples drew a conclusion that when the concentration of oxygen dissolved (DO) in the fermentation medium was controlled between 5 and 20% (based on the concentration at saturation), the 3HP content and the comonomer compositional distribution (CCD) of the copolymer would not be influenced by the DO values . The concentration of the carbon sources was monitored during the fermentation . The results indicated that the comonomer composition and its distribution of P(3HB-co-3HP)s were interrelated to the amounts of carbon sources transported into the bacterial cells . When the bacteria consumed more sucrose, the more 3HPA they would utilize, and the broader the CCD of the copolymer would be . Furthermore, the efficiencies of the transformation of the two carbon sources to the copolymer constituents were found to be similar.

Syst Appl Microbiol, 2000 Dec, 23(4), 556 - 62
How quantitative is quantitative PCR with respect to cell counts?
Ludwig W, Schleifer KH.
Quantitative diagnostic PCR systems based upon rDNA targeted primer and probe combinations were developed for the detection of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, enterococci, Staphylococcus aureus, and Staphylococcus epidermidis . Primers and probes were designed in silico using the ARB software package (TU Munich) in combination with Primer Design software of PE Applied Biosystems . Purified genomic DNA or bacterial cells of target and reference organisms were used for the evaluation of the PCR assays applying the TaqMan technique on an ABI PRISM TM 7700 Sequence Detection System (PE Applied Biosystems) . Sensitive, reliable and reproducible quantification of target rDNA could be achieved applying primer-probe combinations that mediate in vitro amplification of DNA fragments smaller than 100 base pairs . Large amounts of non target DNA (1 mg per sample) remarkably affected the quantification potential of the approach resulting in an underestimation of the amounts of target DNA . One of the principal goals was to use quantitative PCR to study the correlation of gene and cell numbers depending on the growth behavior of target organisms and to explore the potential to estimate cell numbers from target DNA quantification . A clear correlation of rDNA quantification and bacterial growth was observed, however, cell numbers cannot directly be estimated from quantitative PCR data, given that the cellular genome content varies with the growth phase of the organisms . In the case of Escherichia coli the cell numbers which could be assigned to a certain number of rDNA targets varied reasonably depending upon the growth phase of batch cultures.

J Biotechnol, 2001 Mar 9, 86(1), 9 - 17
Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene; Cox M et al.; Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents . The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil . The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated . The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters . Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region . In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified . It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase . A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.

Bioelectrochemistry, 2001 Jan, 53(1), 61 - 71
Enzymatic oxidation of cadmium and lead metals photodeposited on cadmium sulfide; Nedoluzhko AI et al.; Cadmium and lead metals deposited on CdS particles are shown to act as substrates--electron donors for enzymes, hydrogenase from Thiocapsa roseopersicina (HG), NAD-dependent hydrogenase from Alcaligenes eutrophus (NLH), and ferredoxin:NADP oxidoreductase (FNR) from Chlorella in the formation of hydrogen, NADH and NADPH, respectively . Adsorption of the enzyme on the surface of the metallized CdS particle is required for enzymatic oxidation of metal . The maximum rates for the formation of hydrogen and NADH catalyzed by hydrogenase and NAD-dependent hydrogenase with metals as electron donors are comparable with the rates obtained for these enzymes using soluble substrates . Kinetic analysis of the enzymatic oxidation of cadmium metal has revealed that the rate decreases mainly due to the formation of a solid product, which is supposed to be Cd(OH)2 . The deceleration of lead oxidation catalyzed by hydrogenase proceeds at the expense of the inhibitory effect of the formed Pb2+ . The enzymatic oxidation of electrochemically prepared cadmium metal is also shown . Based on these results, a new mechanism of action of the enzymes involved in anaerobic biocorrosion is proposed . By this mechanism, the enzyme accelerates the process of metal dissolution through a mediatorless catalysis of the reduction of the enzyme substrate.

Z Naturforsch {C}, 2000 Nov-Dec, 55(11-12), 890 - 7
Degradation of anthracene by bacteria isolated from oil polluted tropical soils; Ilori MO et al.; Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source . All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources . None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources . Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 10(7) (cfu/ml), 8.6 x 10(6) (cfu/ml), 5.4 x 10(6) and 2.4 x 10(6) (cfu/ml) respectively, for P . aeruginosa, A . eutrophus, B . subtilis and M . luteus after 30 days incubation . Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3%, 44% and 91.7% respectively, for P . aeruginosa, A . eutrophus, B . subtilis, M . luteus and the noninoculated control on the 14 th day . Ring fission enzymes of the meta pathway were detected in induced cells of P . aeruginosa and A . eutrophus while ortho pathway enzymes were detected in B . subtilis and M . luteus . P . aeruginosa and A . eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 +/- 0.183 and 0.64 +/- 0.032 micromol/min x mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 +/- 0.029 and 1.89 +/- 0.026 micromol/min x mg protein were detected in B . subtilis and M . luteus respectively . This work, highlights the capability of these unreported tropical strains of A . eutrophus, B . subtilis and M . luteus as anthracene degraders.

J Bacteriol, 2001 Feb, 183(3), 959 - 67
Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition; de Groot A et al.; Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z) . Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species . Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M . Linderberg, G . P . Salmond, and A . Collmer, Mol . Microbiol . 20:175-190, 1996) . In the present study, we report that XcpP and XcpQ of P . alcaligenes could not substitute for their respective P . aeruginosa counterparts . However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other . Moreover, when P . alcaligenes xcpP and xcpQ were expressed simultaneously in a P . aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures . After growth in liquid culture the heat-stable P . alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible . Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

Appl Environ Microbiol, 2001 Feb, 67(2), 769 - 73
Mobilization of selenite by Ralstonia metallidurans CH34; Roux M et al.; Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals . R . metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis . Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period . Depending on the culture conditions, the medium can be completely depleted of selenite . Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis . Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium . The ability of R . metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.

Appl Environ Microbiol, 2001 Feb, 67(2), 680 - 7
Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by 13C nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses; Frey AD et al.; Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and the A . eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional (13)C-labeling of the proteinogenic amino acids and two-dimensional {(13)C, (1)H}-correlation nuclear magnetic resonance (NMR) spectroscopy . The NMR data revealed differences in the intracellular carbon fluxes between E . coli cells expressing either VHb or VHb-Red and cells expressing A . eutrophus FHP or the truncated heme domain (FHPg) . E . coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle . Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis . In contrast, in E . coli cells expressing A . eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation . Consistently, E . coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and D-lactate . The implications of these observations for biotechnological applications are discussed.

Biochem J, 2001 Jan 15, 353(Pt 2), 259 - 66
Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction; Prudencio M et al.; The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres . The latter have been implicated in the binding and reduction of nitrite . The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp(92) {Dodd, Van Beeumen, Eady and Hasnain (1998), J . Mol . Biol . 282, 369-382} . Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed . EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g( parallel)=2.12 (A( parallel)=0 mT) and g( perpendicular)=2.30 (A( perpendicular)=6.4 mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres . The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre . Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite . As isolated, the EPR spectrum of the Asp(92)-->Asn (D92N) mutant showed no characteristic type 2 hyperfine lines . However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form . Like the Asp(92)-->Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite . Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity . The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx . 60% of the activity seen for the wild-type NiR . These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.

Int Microbiol, 1998 Mar, 1(1), 45 - 51
Numerical taxonomy of heavy metal-tolerant nonhalophilic bacteria isolated from hypersaline environments; Rios M et al.; A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cadiz, Huelva and Moron de la Frontera (Spain) . They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc . These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm . At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Gram-positive halotolerant microorganisms . Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria . The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned . Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.

Rev Saude Publica, 2000 Oct, 34(5), 444 - 8
{Evaluation of bacterial contamination in disinfectants for domestic use}; Miyagi F et al.; OBJECTIVE: To evaluate disinfectants for domestic use for the presence of bacteria, identify them, and determine their tolerance level to benzalkonium chloride . METHODS: Fifty-two samples of commercially available disinfectants for domestic use were acquired at random in the metropolitan area of Sao Paulo, Brazil, and analyzed to detect the presence of bacterial contaminants . The isolated organisms were identified and their tolerance level to benzalkonium chloride was determined by broth macrodilution method . RESULTS: Sixteen (30.77%) of fifty-two disinfectants sampled were contaminated by Gram-negative bacteria, with counts varying between 10(4) and 10(6) UFC/ml . Alcaligenes xylosoxidans, Burkholderia cepacia and Serratia marcescens were the predominant organisms found . The minimum inhibitory concentration (MIC: mg/ml) of benzalkonium chloride for these bacteria were 2.48, 1.23 and 0.30 to S . marcescens, A . xylosoxidans and B . cepacia, respectively . CONCLUSIONS: The disinfectant formulation containing quaternary ammonium compounds (QACs) may be exposed to contamination by Gram-negative bacteria . The MICs of benzalkonium chloride against the isolated bacteria were low, indicating that the bacteria grown in culture media without QACs lost their tolerance to this biocide.

Microbiology, 2000 Dec, 146 Pt 12, 3081 - 90
The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri; Lorenz MG et al.; The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated . Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (Rif(R) marker) . The transformation frequencies varied over more than three orders of magnitude . With three highly transformable strains (ATCC 17587, ATCC 17641, JM300) from two gvs and all other strains as DNA donors, sexual isolation from other pseudomonad species (Pseudomonas alcaligenes, Pseudomonas mendocina) and also from other P . stutzeri gvs was observed (i.e . heterogamic transformation was reduced) . For ATCC 17587 (gv 2) and ATCC 17641 (gv 8), heterogamic transformation was up to two and three orders of magnitude lower with other P . stutzeri gv and the other species employed, respectively, than in homogamic transformations . Interestingly, whereas with ATCC 17587 and ATCC 17641 heterogamic transformation with donors of the same gv was as high as homogamic transformation, JM300 (gv 8) was sexually isolated from its nearest relative (ATCC 17641) . Also, sexual isolation of JM300 from other P . stutzeri gvs was most pronounced among the recipients tested, in some cases reaching the highest levels found with the other species as DNA donors (reduction of heterogamic transformation by 4000-fold) . Results obtained here from nucleotide sequence analysis of part (422 nt) of the gene for the RNA polymerase ss subunit (rpoB) from various strains indicated that sexual isolation of ATCC 17641 increased with nucleotide sequence divergence . Implications of the observed great heterogeneity in transformability, competence levels and sexual isolation among strains are discussed with regard to the evolution of P . stutzeri.

Biochimie, 2000 Nov, 82(11), 1023 - 32
Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion; Rosenau F et al.; Lipases from Pseudomonas bacteria are widely used for a variety of biotechnological applications . Overexpression in heterologous hosts like Escherichia coli failed to produce enzymatically active lipase prompting to study the molecular mechanisms underlying the regulation of lipase gene expression and secretion . The prototype lipase from P . aeruginosa is encoded in a bicistronic operon which is transcribed from two different promotors, one of which depends on the alternative sigma factor RpoN (sigma(54)) . Recently, a two-component regulatory system was identified as an element controlling transcription of the lipase operon . P . aeruginosa lipase is secreted via a type II pathway . The cytoplasmic prelipase contains a 26 amino acid N-terminal signal sequence mediating secretion across the inner membrane via the Sec-machinery . In the periplasm, lipase folds into an enzymatically active conformation assisted by its specific intermolecular chaperone Lif and by unspecific accessory folding catalysts including Dsb-proteins which catalyze the formation of a disulfide bond . Enzymatically active and secretion-competent lipase is finally transported through a complex secretion machinery consisting of 12 different Xcp-proteins of which XcpQ forms a pore-like structure in the outer membrane allowing the release of lipase into the extracellular medium . Biotechnologically important lipases from Burkholderia glumae and P . alcaligenes also use such a type II secretion pathway whereas lipases from P . fluorescens and Serratia marcescens, which lack a typical signal sequence are secreted via a type I pathway . Future challenges to produce Pseudomonas lipases may include artificial up-regulation of lipase gene transcription and construction of more efficient expression strains in which both folding and secretion of lipase are optimized.

Curr Opin Pulm Med, 2000 Nov, 6(6), 545 - 50
Unusual respiratory bacterial flora in cystic fibrosis: microbiologic and clinical features; Beringer PM et al.; Pulmonary infections continue to be a significant source of morbidity and mortality among patients with cystic fibrosis . Although our understanding of the pathogenesis and clinical consequences of pulmonary infections with Pseudomonas aeruginosa has increased greatly in recent years, very little is known about potentially emerging pathogens such as Burkholderia cepacia complex, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and methicillin-resistant Staphylococcus aureus . In this review, the authors discuss methods for appropriate identification of these "unusual" organisms and their epidemiologic and clinical features . Multicenter surveillance studies are needed to more clearly establish the pathogenicity of these organisms.

Appl Environ Microbiol, 2000 Dec, 66(12), 5282 - 9
Composition of soil microbial communities enriched on a mixture of aromatic hydrocarbons; Greene EA et al.; Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards) . Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44) . Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP) . The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site . The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp . before convergence on a composition dominated by Alcaligenes spp . The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession . The enrichments effectively degraded all C5+ components except DCPD . Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.

J Agric Food Chem, 2000 Nov, 48(11), 5307 - 11
2,4-Dichlorophenoxyacetic acid metabolism in transgenic tolerant cotton (Gossypium hirsutum); Laurent F et al.; The metabolic fate of 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in leaves of transgenic 2,4-D-tolerant cotton (Gossypium hirsutum), which is obtained by transfer of the tfdA gene from the bacterium Alcaligenes eutrophus . The tdfA gene codes for a dioxygenase catalyzing the degradation of 2,4-D to 2, 4-dichlorophenol (2,4-DCP) . {phenyl-(14)C}-2,4-D was administered by petiolar absorption followed by an 18 h water chase or converted to the isopropyl ester and sprayed onto the leaf surface; the leaves were harvested 48 h later . The herbicide was degraded to 2,4-DCP by the bacterial enzyme expressed in the plants . 2,4-DCP was rapidly converted to more polar metabolites and was never found in detectable amounts . Metabolite structures were deduced from enzymatic hydrolysis studies and mass spectrometric analyses . The first metabolite was the glucoside conjugate of 2,4-DCP (2, 4-DCP-beta-O-glucoside) . The major terminal metabolites were two more complex glucosides: 2,4-DCP-(6-O-malonyl)glucoside and 2, 4-DCP-(6-O-sulfate)glucoside.

Int J Food Microbiol, 2000 Nov 1, 61(2-3), 137 - 45
Microbiological hazard identification and exposure assessment of street food vending in Johannesburg, South Africa; Mosupye FM et al.; One hundred and thirty-two samples of beef, chicken, salad and gravy were collected from two street vendors over eleven replicate surveys to assess microbiological safety and quality . For each food type samples were collected during preparation and holding . Dish water was also collected and food preparation surfaces swabbed during preparation and display . Standard methods were used to determine aerobic plate counts, Enterobacteriaceae counts, coliform counts and spore counts . Six hundred and seventy-five predominant colonies were isolated from aerobic plate counts of all samples and characterised . The incidence of selected foodborne bacterial pathogens and non-pathogenic E . coli 1 was also determined . In most cases mean bacterial counts of the raw materials were significantly higher (P < 0.05) than those of corresponding cooked foods . No significant differences (P > 0.05) in all count types were observed between food samples collected during cooking and those collected during holding . In addition, no significant differences (P > 0.05) in all count types were observed between prepared salads and their raw materials . Mean bacterial counts of water and swab samples collected from vendor 1 were lower than those of water and swab samples collected from vendor 2.The predominant populations isolated from the aerobic plate counts were Bacillus spp., Staphylococcus spp., Enterobacteriaceae and Alcaligenes spp . Bacillus cereus was detected in 17%, Clostridium perfringens in 1%, Staphylococcus aureus in 3% and Vibrio metchnikovii in 2% of the food samples . Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli O157:H7 were not detected . Non-pathogenic E . coli 1 was detected in 13% of food samples, in 86 and 36% of dish water samples collected from vendors 1 and 2, respectively, and in 36% of surface swab samples from vendor 2.

J Bacteriol, 2000 Dec, 182(23), 6651 - 8
Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6; Masai E et al.; Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway . We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC) . CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC . We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase . The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da . The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp . strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M . F . Reid and C . A . Fewson, Crit . Rev . Microbiol . 20:13-56, 1994) . The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity . Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP(+) by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry . LigC is a homodimer . The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25 degrees C, respectively . The K(m) for NADP(+) was estimated to be 24.6 +/- 1.5 microM, which was approximately 10 times lower than that for NAD(+) (252 +/- 3.9 microM) . The K(m)s for CHMS in the presence of NADP(+) and NAD(+) are 26.0 +/- 0.5 and 20.6 +/- 1.0 microM, respectively . Disruption of ligC in S . paucimobilis SYK-6 prevented growth with vanillate . Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC . However, the introduction of ligC into DLC restored its ability to grow on vanillate . PDC was suggested to be an inducer for ligAB gene expression.

Mol Microbiol, 2000 Oct, 38(2), 359 - 67
Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system; Garg RP et al.; In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system . Phc uses a novel self-produced signal molecule {3-hydroxypalmitic acid methyl ester (3-OH PAME)}, an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states . While Phc is present in most R . solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced . Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R . solanacearum phcA mutants . We also demonstrate that some R . eutropha produce an extracellular factor that complements R . solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA . Additionally, Southern blot hybridization analysis suggested that R . eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase) . Analysis of a phcA-null mutant of R . eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R . solanacearum where it represses motility . Similarly, the R . eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R . solanacearum increases siderophore production . Although our data strongly suggest that R . eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R . eutropha.

EMBO J, 2000 Nov 1, 19(21), 5661 - 71
Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase; Lawson DM et al.; Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen . We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans . NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species . In contrast, CO binds 6-coordinate on the distal side . A second CO molecule, not bound to the heme, is located in the proximal pocket . Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.

Biofizika, 2000 Jul-Aug, 45(4), 636 - 40
{Study of the dielectric permeability in the superhigh frequency range of a degraded polyoxybutyrate biopolymer}; Beliaev GA et al.; The dielectric permeability of the degradable biopolymer polyhydroxybutyrate synthesized by hydrogen-oxidizing bacteria Alcaligenes eutrophus was investigated by the resonance method using original highly sensitive microstrip sensors . For the first time, a linear growth of dielectric permeability (delta epsilon/delta T = 7 x 10(-4) degree-1) due to the flexibility of the polymer chain in the temperature range from 10 to 70 degrees C was revealed . The energy of a bend of the nearest fragments was evaluated (E = 392 K), and its correspondence to the energies of bends of the alcyl groups of low-molecular substances like liquid crystals was established . It was shown that at low values of dielectric permeability in the high-frequency range (epsilon' = 2.25 +/- 0.02), which are stable, in a wide range of frequencies of the electromagnetic field (1 MHz - 1 Hz), polyoxybutyrate can be used in the microwave equipment.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12768 - 12773
Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells; John ME et al.; Alcaligenes eutrophus genes encoding the enzymes, beta-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(-)-3-hydroxybutyrate (PHB) from acetyl-CoA . PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton . Endogenous beta-ketothiolase activity is present in cotton fibers . Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, beta-glucuronidase (GUS), expression . Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes . Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm . High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB . Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 x 10(6) to 1.8 x 10(6) Da . The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties . The fibers exhibited better insulating characteristics . The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity . These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources . This is an important step toward producing new generation fibers for the textile industry.

Microbiology, 2000 Oct, 146 ( Pt 10), 2385 - 94
Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes; Yamamoto S et al.; Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains) . Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II) . IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri . IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex' . The 'P: putida complex' included P: putida and P: fulva . The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P . savastanoi and P . syringae . The 'P . fluorescens complex' was further divided into two subpopulations, the 'P . fluorescens lineage' and the 'P . chlororaphis lineage' . The 'P . fluorescens lineage' contained P . fluorescens biotypes A, B and C, P . azotoformans, P . marginalis pathovars, P . mucidolens, P . synxantha and P . tolaasii, while the 'P . chlororaphis lineage' included P . chlororaphis, P . agarici, P . asplenii, P . corrugata, P . fluorescens biotypes B and G and P . putida biovar B . The strains of P . fluorescens biotypes formed a polyphyletic group within the 'P . fluorescens complex'.

Biometals, 2000 Jun, 13(2), 135 - 9
DNA sequence analysis of the tellurite-resistance determinant from clinical strain of Escherichia coli and identification of essential genes; Kormutakova R et al.; The tellurite-resistant Escherichia coli strain KL53 was found during testing of the group of clinical isolates for antibiotics and heavy metal ion resistance (Burian et al . 1990) . Determinant of the tellurite resistance of the strain was located on the large conjugative plasmid pTE53 and cloned into pACYC184 . Three different Ter clones harboring pLK2, pLK18 and pLK20 were isolated (Burian et al . 1998) . The smallest functional Ter clone harboring pLK18 was chosen for further analysis . Plasmid pLK18 have been subcloned to obtain convenient DNA fragments for sequencing of tellurite-resistance determinant . Sequencing of this DNA fragments provided complete DNA sequence of the determinant, 5,250 bp in size . The sequence has been compared with nucleotide and protein databank (BLAST programs) and significant homology with the three known operons coding for tellurite resistance has been found (determinat on plasmid pR478 from Serratia marcescens, on plasmid pMER610 from Alcaligenes sp . and chromosomal tellurite resistance genes from Proteus mirabilis) . We identified 5 ORFs coding for 5 genes named terB to terF . The clone harboring pLK18 was subjected to the transposition with Tn1737Km to disrupt determinant of the tellurite resistance . Plasmid DNA of several clones containing pLK18 with Tn1737Km was isolated to locate the target site of Tn1737Km . Analyses showed, the genes terB, terC, terD and terE are essential for conservation of the resistance whereas the gene terF is not important in this respect.

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 262 - 7
Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand; Andres Y et al.; The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd3+) . The uptake by sand of this element was also measured . Saturation curves and Scatchard models were established for all biosorbants used in this work . The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd3+, which ranged from 350 micromol g(-1) for B . subtilis to 5.1 micromol g(-1) for S . cerevisiae . This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations . Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells . Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 44 - 51
Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug; Liu JQ et al.; The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669 . It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues . The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Arthrobacter sp . DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes . This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract . The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps . The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 81 - 6
Conversion of solvent evaporation residues from the AB- (acetone-butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters; Parrer G et al.; In a bioconversion study based on utilisation of by-products from the AB- (acetone-butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents . The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp . G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate- (PHA-)accumulation up to 40% per dry weight . A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs . Concentrations of 1-butanol, which is known for its membrane damaging properties in micro-organisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth . No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate . Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process . The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 81 - 6
Conversion of solvent evaporation residues from the AB- (acetone - butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters; Parrer G et al.; In a bioconversion study based on utilisation of by-products from the AB- (acetone - butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents . The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp . G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate-(PHA-)accumulation up to 40% per dry weight . A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs . Concentrations of 1-butanol, which is known for its membrane damaging properties in microorganisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth . No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate . Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process . The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.

Am J Ophthalmol, 2000 Jun, 129(6), 813 - 5
Alcaligenes xylosoxidans and Propionibacterium acnes postoperative endophthalmitis in a pseudophakic eye; Rahman MK et al.; PURPOSE: To report a case of persistent polymicrobial postoperative endophthalmitis caused by Alcaligenes xylosoxidans and Propionibacterium acnes in a pseudophakic eye . A . xylosoxidans is a gram-negative bacteria resistant to most antibiotics . METHODS: Case report . RESULTS: A 72-year-old man presented with clinical signs of endophthalmitis on the first postoperative day after a phacoemulsification procedure with posterior chamber intraocular lens, left eye . Initial treatment included topical, subconjunctival, and oral antibiotics . After initial clearing, there was recrudescence of infection on postoperative day 37 that prompted referral of the patient to the Cullen Eye Institute . Treatment at that time included anterior chamber and vitreous taps with intravitreal antibiotic injections . Complete pars plana vitrectomy and intraocular lens explantation were eventually required because of persistent infection with a resistant organism . Cultures from the first procedure grew A . xylosoxidans and P . acnes . Cultures from the vitrectomy grew only A . xylosoxidans . At the final follow-up visit 6 months after the initial procedure . The eye was without inflammation with best-corrected visual acuity of 20/40 . CONCLUSION: Both A . xylosoxidans and P . acnes can cause chronic progressive endophthalmitis after cataract extraction often resistant to corrective antibiotic therapy . Successful intervention may require complete vitrectomy with intraocular lens and capsule removal.

Appl Environ Microbiol, 2000 Aug, 66(8), 3624 - 7
Production of Poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution; Ahn WS et al.; Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes were developed . The pH-stat fed-batch cultures of E . coli CGSC 4401 harboring pJC4, a stable plasmid containing the A . latus PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter . Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.

J Biol Chem, 2000 Oct 13, 275(41), 31581 - 7
Nitric-oxide dioxygenase activity and function of flavohemoglobins . sensitivity to nitric oxide and carbon monoxide inhibition; Gardner PR et al.; Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity . FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics . Turnover numbers (V(max)) for S . cerevisiae, A . eutrophus, and E . coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2) . The K(M) values for NO are low and range from 0.1 to 0.25 microm . V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism . Approximate K(M) values for O(2) range from 60 to 90 microm . NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine . High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes . Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm) . These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.

Anal Chem, 2000 Jul 1, 72(13), 2937 - 42
Analysis of ternary mixtures with a single dynamic microbial sensor and chemometrics using a nonlinear multivariate calibration; Plegge V et al.; An amperometric biosensor based on immobilized bacterial cells of Alcaligenes eutrophus KT02 and an oxygen electrode was integrated in a flow-through system . Because microorganisms metabolize various organic analytes in a specific manner, the sensor shows for different pure analytes distinct time-dependent oxygen consumption rates that can be treated as characteristic patterns . This behavior is conserved also when the biosensor is exposed to a mixture of these organic analytes; the sensor with a particular type of microorganisms responds with a total signal . The respiration curves as time-dependent amplitudes were subdivided into several time channels . This procedure creates an additional data dimension and makes the single sensor "dynamic" . Using multivariate calibration models with only one single biosensor, simultaneous quantitative analysis of ternary mixtures of acetate, L-lactate, and succinate was realized . A nonlinear algorithm that compensated for conceivable interactions of the analytes was superior to a partial least-squares algorithm . Each analyte was predicted more precisely by the nonlinear approach resulting in root-mean-square errors of prediction of 0.20 mg/L for acetate, 0.43 mg/L for L-lactate, and 0.73 mg/L for succinate.

Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 103 - 7
{Studies on fermentation conditions for the accumulation of poly-beta-hydroxybutyrate in Alcaligenes eutrophus}; Du GC et al.; The results of the cultivation of Alcaligenes eutrophus showed that nitrogen limitation or exhaustion could stimulate the substantial accumulation of PHB . But the exhaustion of nitrogen source in PHB formation period would result in the rapid drop of PHB synthetic rate . Oxygen limitation could also stimulate the formation of PHB, but the content of PHB in the cell was much less than that in case of nitrogen controlled conditions . Obvious influences were observed on PHB fermentation when ammonia water feeding was stopped at different cell growth phases, and better results could be obtained when it was performed at 20 g/L to 30 g/L of residual biomass . Cell dry weight, PHB content and PHB concentration reached 61.9 g/L, 80.5% and 49.0 g/L, respectively under desired conditions.

Appl Environ Microbiol, 2000 Jul, 66(7), 2959 - 64
Molecular analysis of surfactant-driven microbial population shifts in hydrocarbon-contaminated soil; Colores GM et al.; We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil . A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels . Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC') (determined to be 13 mg g(-1)) . Addition of the surfactant at a concentration below the CMC' (2 mg g(-1)) did not affect the mineralization rates of either hydrocarbon . However, when surfactant was added at a concentration approaching the CMC' (10 mg g(-1)), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited . Addition of surfactant at concentrations above the CMC' (40 mg g(-1)) completely inhibited mineralization of both phenanthrene and hexadecane . Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels . Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth . The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.

Biofizika, 2000 May-Jun, 45(3), 445 - 51
{Study of molecular structure of polyhydroxybutyrate-a termoplastic anddegradable biopolymer}; Volova TG et al.; The molecular structure of polyhydroxybutyrate from hydrogen-oxidizing bacteria Alcaligenes eutrophus was studied by X-ray diffraction analysis . It was shown that the degree of crystallinity of various samples depends little on the conditions of their preparation and is equal to 0.62-0.76 . The molecular structure of solid samples and solution of polyhydroxybutyrate in chloroform were studied by the NMR and EPR methods . The conclusion is made that the molecular structure of polyhydroxybutyrate does not depend on the features of the strain and conditions of carbon nutrition of microorganisms producing polyhydroxybutyrate . Defects induced by gamma-radiation in polyhydroxybutyrate were studied . Free radicals were isolated, and their structure was decoded.

Nat Biotechnol, 2000 Jun, 18(6), 661 - 5
Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil; Valls M et al.; Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions . DNA sequences encoding MT were fused to the autotransporter beta-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane . The translocation, surface display, and functionality of the chimeric MTbeta protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R . eutropha . The resulting bacterial strain, named R . eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media . Furthermore, the inoculation of Cd2+-polluted soil with R . eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana).

Biotechnol Prog, 2000 May-Jun, 16(3), 363 - 7
Biocatalytic transformation of {(2-Hydroxyethyl)thio}acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxydans ssp . xylosoxydans (SH91); Lee T et al.; A Gram-negative bacterium, Alcaligenes xylosoxydans ssp . xylosoxydans (SH91), consumed thiodiglycol (TDG), the nontoxic hydrolysis product of sulfur mustard, as a primary carbon source and transformed TDG to commercially relevant chemical precursors, {(2-hydroxyethyl)thio}acetic acid (HETA) and thiodiglycolic acid (TDGA) . Aerobic fed batch and repeated batch experiments were run to compare the molar yields of HETA and TDGA that result under different operating policies . In repeated batch experiments, 35% of the TDG was converted to HETA . Under the conventional batch process and a repeated fed batch process, the HETA yields were reduced (21% and 18%, respectively), while the yield of TDGA was increased (47% and 31%,respectively) . This work demonstrated that cell growth associated biocatalytic transformations were manipulated to achieve a desired byproducts profile through an understanding of the specific reaction and cell growth kinetics and by altering the reaction operating policy accordingly.

Appl Environ Microbiol, 2000 Jun, 66(6), 2641 - 6
Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates; Duetz WA et al.; Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable . We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen . Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite . Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml . A shaking diameter of 2.5 cm resulted in threefold-lower values . These high oxygen transfer rates allowed P . putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml . The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media . Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

Acta Crystallogr D Biol Crystallogr, 2000 Jun, 56 ( Pt 6), 690 - 6
Structures of oxidized and reduced azurin II from Alcaligenes xylosoxidans at 1.75 A resolution; Dodd FE et al.; Crystallographic structures of oxidized and reduced forms of azurin II are reported at 1.75 A resolution . Data were collected using one crystal in each case and by translating the crystal after each oscillation range to minimize photoreduction . Very small differences are observed at the Cu site upon reduction and these cannot be determined with confidence at current resolution . A comparison with the three-dimensional EXAFS reveals a good correspondence for all the ligand distances except for Cu-His46, where a larger deviation of approximately 0.12-0.18 A is observed, indicating that this ligand is more tightly restrained in the crystallographic refinement at the current resolution.

Appl Microbiol Biotechnol, 2000 Apr, 53(4), 420 - 9
Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems; Clemente T et al.; A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes . This ORF has lower homology to the R . rubrum strain Ha PHA synthase than would be expected within the same species . We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104 . The N . corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source . When the T . violacea and R . sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R . eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate . Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N . corrallina, T . violacea, and R . eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate . These studies demonstrate that the PHA synthase from N . corrallina, T . violacea, and R . eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R . sphaeroides lacks this ability and only produces a short-chain-length polymer . These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.

Theriogenology, 2000 Mar 15, 53(5), 1167 - 76
Field investigations of bacterial contaminants and their effects on extended porcine semen; Althouse GC et al.; Field investigations (n=23) were made over a 3-yr period at North American boar studs and farms in which the primary complaint was sperm agglutination in association with decreased sperm longevity of extended semen, and increased regular returns to estrus and/or vaginal discharges across parity . Microscopic examination of extended semen from these units revealed depressed gross motility (usually <30%), sperm agglutination, and sperm cell death occurring within 2 d of semen collection and processing regardless of the semen extender used . The extended semen exhibited a high number of induced acrosome abnormalities (>20%) . Sample pH was acidic (5.7 to 6.4) in 93% of the submitted samples . Aerobic culture yielded a variety of bacteria from different genera . A single bacterial contaminant was obtained from 66% of the submitted samples (n=37 doses); 34% contained 2 or more different bacterial genera . The most frequently isolated contaminant bacteria from porcine extended semen were Alcaligenes xylosoxydans (n=3), Burkholderia cepacia (n=6), Enterobacter cloacae (n=6), Escherichia coli (n=6), Serratia marcescens (n=5), and Stenotrophomonas {Xanthomonas} maltophilia (n=6); these 6 bacteria accounted for 71% of all contaminated samples, and were spermicidal when re-inoculated and incubated in fresh, high quality extended semen . All contaminant bacteria were found to be resistant to the aminoglycoside gentamicin, a common preservative antibiotic used in commercial porcine semen extenders . Eleven genera were spermicidal in conjunction with an acidic environment, while 2 strains (E . coli, S . maltophilia) were spermicidal without this characteristic acidic environment . Bacteria originated from multiple sources at the stud/farm, and were of animal and nonanimal origin . A minimum contamination technique (MCT) protocol was developed to standardize hygiene and sanitation . This protocol focused on MCT's during boar preparation, semen collection, semen processing and laboratory sanitation . Implementation of the MCT, in addition to specific recommendations in stud management, resulted in the control of bacterial contamination in the extended semen.

J Appl Microbiol, 2000 May, 88(5), 914 - 8
Multiple chromosomes in Burkholderia cepacia and B . gladioli and their distribution in clinical and environmental strains of B . cepacia; Wigley P et al.; Burkholderia cepacia is found in soils and waters, it can be used in biocontrol and bioremediation but is also a human pathogen . It is not yet clear what differentiates pathogenic from non-pathogenic strains of the organism . In this study the multiple replicon structure was investigated in 28 strains of B . cepacia by pulsed field gel electrophoresis . All strains examined, whether of clinical, environmental or plant pathogenic origin, were found to have two, three or four large (> 500 kbp) replicons . Many strains also contained small replicons . Clinical strains were more likely to have three or four large replicons than non-clinical strains . Multiple replicon structure was also demonstrated in B . gladioli and Alcaligenes eutrophus.

J Bacteriol, 2000 May, 182(10), 2716 - 24
The H(2) sensor of Ralstonia eutropha is a member of the subclass of regulatory {NiFe} hydrogenases; Kleihues L et al.; Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source . The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H(2), which is available in low amounts in aerobic environments . H(2) sensing in R . eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system . In this study we show that the H(2) sensor of R . eutropha is a cytoplasmic protein . Although capable of H(2) oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases . A specifically designed overexpression system for R . eutropha provided the basis for identifying the H(2) sensor as a nickel-containing regulatory protein . The data support previous results which showed that the sensor has an active site similar to that of prototypic {NiFe} hydrogenases (A . J . Pierik, M . Schmelz, O . Lenz, B . Friedrich, and S . P . J . Albracht, FEBS Lett . 438:231-235, 1998) . It is demonstrated that in addition to the enzymatic activity the regulatory function of the H(2) sensor is nickel dependent . The results suggest that H(2) sensing requires an active {NiFe} hydrogenase, leaving the question open whether only H(2) binding or subsequent H(2) oxidation and electron transfer processes are necessary for signaling . The regulatory role of the H(2)-sensing hydrogenase of R . eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features . Thus, the family of H(2) sensors represents a novel subclass of {NiFe} hydrogenases denoted as the "regulatory hydrogenases."

Biotechnol Prog, 2000 Mar-Apr, 16(2), 238 - 43
Separation of Alcaligenes eutrophus cells containing Poly(3-hydroxybutyrate) from fermentation broth with pretreatment using Al- and Fe-based coagulants; Cho KS et al.; Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants . Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6-5.6 for the Al-based coagulants and about 5-8 for the Fe-based coagulants . Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration . In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also . The coagulant requirement increased with the cell concentration regardless of coagulant type . The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate . The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5-210 g/L . It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation.

J Biochem (Tokyo), 2000 Feb, 127(2), 345 - 50
Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase; Kataoka K et al.; A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized . All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme . Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR . The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg . The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants . All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 1 - 8
Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6; Wakayama M et al.; D-Aminoacylase from Alcaligenes xylosoxydans subsp . xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC) . An H67N mutant was barely active, with a kcat/Km 6.3 x 10(4) times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity . The H67N mutant had almost constant Km, but greatly decreased kcat . These results suggested that His67 is essential to the catalytic event . Both H69N and H69I mutants were overproduced in the insoluble fraction . The kcat/Km of H250N mutant was reduced by a factor of 2.5 x 10(4)-fold as compared with the wild-type enzyme . No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found . The Zn content of H250N mutant was nearly half of that of wild-type enzyme . These results suggest that the His250 residue might be essential to catalysis via Zn binding.

Enzyme Microb Technol, 2000 Feb 1, 26(2-4), 201 - 208
Metabolic engineering of Alcaligenes eutrophus through the transformation of cloned phbCAB genes for the investigation of the regulatory mechanism of polyhydroxyalkanoate biosynthesis; Jung Y et al.; The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate {PHB} and poly(3-hydroxybutyrate-3-hydroxyvalerate) {P(3HB-3HV)} of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes . The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA . The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV) . The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase . This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor . Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.

Plasmid, 2000 Mar, 43(2), 103 - 10
The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542; Fong KP et al.; Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542 . Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site . The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat . The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P . alcaligenes, P . aeruginosa, P . stutzeri, and Serratia marcescens . The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases . Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1 . Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542 .

FEBS Lett, 2000 Jan 28, 466(2-3), 259 - 63
Unusual FTIR and EPR properties of the H2-activating site of the cytoplasmic NAD-reducing hydrogenase from Ralstonia eutropha; Happe RP et al.; Soluble NAD-reducing {NiFe}-hydrogenase (SH) from Ralstonia eutropha (formerly Alcaligenes eutrophus) has an infrared spectrum with one strong band at 1956 cm(-1) and four weak bands at 2098, 2088, 2081 and 2071 cm(-1) in the 2150-1850 cm(-1) spectral region . Other {NiFe}-hydrogenases only show one strong and two weak bands in this region, attributable to the NiFe(CN)2(CO) active site . The position of these three bands is highly sensitive to redox changes of the active site . In contrast, reduction of the SH resulted in a shift to lower frequencies of the 2098 cm(-1) band only . These and other properties prompted us to propose the presence of a Ni(CN)Fe(CN)3(CO) active site.

J Bacteriol, 2000 Mar, 182(5), 1399 - 409
Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34; Tibazarwa C et al.; The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28 . The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli . CnrY fractionated as a 10.7-kDa protein in in vitro translation assays . CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E . coli . His-CnrX was found to possess a secondary structure content rich in alpha-helical and beta-sheet structures . CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion . In gel shift mobility assays, His-CnrH, in the presence of E . coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus . These promoters and their transcription start sites were confirmed by primer extension . Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry) . To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes . Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni(2+) and 2.0 mM Co(2+) in a noncomplexing medium for metals . The two promoter regions P(Y) (upstream cnrY) and P(H) (upstream cnrH) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the cnr operon . The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter . The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2091 - 6
Cloning and expression of the N,N-dimethylformamidase gene from Alcaligenes sp . strain KUFA-1; Hasegawa Y et al.; N,N-Dimethylformamidase (DMFase) from Alcaligenes sp . strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation . The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced . The deduced amino acid sequence of the enzyme consisted of two alpha- and two beta-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases . The protein may be a new kind of amidase . DMFase activity was detected in E . coli cells transformed with an expression plasmid of the cloned DMFase gene . The properties of recombinant DMFase purified from E . coli were identical to those of Alcaligenes DMFase.

Chem Phys Lipids, 2000 Jan, 104(1), 13 - 21
Enzymatic fatty acid exchange in digalactosyldiacylglycerol; Persson M et al.; Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene . Lipases from Geotrichum candidum, Alcaligenes sp . and Penicillium camembertii did not catalyse the acidolysis reaction . Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG . The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety . Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters . R . arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated . Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R . arrhizus lipase was used as catalyst . At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained . In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.

FEMS Microbiol Ecol, 2000 Feb 1, 31(2), 143 - 152
Humus bacteria of Norway spruce stands: plant growth promoting properties and birch, red fescue and alder colonizing capacity; Elo S et al.; We studied the potential of the humus layer of the Norway spruce stands to supply beneficial rhizobacteria to birch (Betula pendula), alder (Alnus incana) and fescue grass (Festuca rubra), representatives of pioneer vegetation after clear-cutting of the coniferous forest . Axenically grown seedlings of these species were inoculated with the acid spruce humus, pH 3.7-5.3 . Actinorhizal propagules, capable of nodulating alder, were present in high density (10(3) g(-1)) in humus of long-term limed plots, whereas plots with nitrogen fertilization contained almost none (</=10 g(-1)) . The genera most frequently found in the humus were Bacillus, Paenibacillus, Arthrobacter, Nocardia, Rhodococcus and Pseudomonas, independently of prior liming or fertilization of the plots . The taxa found in the seedling roots differed from that in humus by the prevalence of the Gram-negative genera Pseudomonas, Alcaligenes and Comamonas . Enrichment cultures of the roots on nitrogen-free media yielded Paenibacillus and Rhodococcus species . Nitrogen-fixing R . erythropolis and a novel Paenibacillus, closest by full sequence of 16S rDNA to P . durus, represented new classes of nitrogen-fixing rhizosphere bacteria . In addition, nitrogen-fixing R . fascians was found in the humus . The rhizoflora and humus contained high proportions of bacteria antagonistic towards plant pathogenic Rhizoctonia sp., Botrytis cinerea and Fusarium culmorum . The antagonistic isolates also commonly produced siderophores and/or cell wall degrading enzymes.

J Bacteriol, 2000 Feb, 182(3), 581 - 8
Ralstonia eutropha TF93 is blocked in tat-mediated protein export; Bernhard M et al.; Ralstonia eutropha (formerly Alcaligenes eutrophus) TF93 is pleiotropically affected in the translocation of redox enzymes synthesized with an N-terminal signal peptide bearing a twin arginine (S/T-R-R-X-F-L-K) motif . Immunoblot analyses showed that the catalytic subunits of the membrane-bound {NiFe} hydrogenase (MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase (Nap) are mislocalized to the cytoplasm and to the inner membrane, respectively . Moreover, physiological studies showed that the copper-containing nitrous oxide reductase (NosZ) was also not translocated to the periplasm in strain TF93 . The cellular localization of enzymes exported by the general secretion system was unaffected . The translocation-arrested MBH and Nap proteins were enzymatically active, suggesting that twin-arginine signal peptide-dependent redox enzymes may have their cofactors inserted prior to transmembrane export . The periplasmic destination of MBH, Nap, and NosZ was restored by heterologous expression of Azotobacter chroococcum tatA mobilized into TF93 . tatA encodes a bacterial Hcf106-like protein, a component of a novel protein transport system that has been characterized in thylakoids and shown to translocate folded proteins across the membrane.

FEMS Immunol Med Microbiol, 2000 Jan, 27(1), 73 - 7
Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens; Hutchison ML et al.; Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life . Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche . At present, it is unclear from clinical data whether these microbes are commensal or pathogenic . In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients . Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed . Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF) . A bioassay was also used to assess TNF activity . With the exception of S . maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed . This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.

Appl Environ Microbiol, 2000 Jan, 66(1), 98 - 104
Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli; Frey AD et al.; Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp . (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation . The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb . However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities . To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A . eutrophus in frame after the vhb gene . Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed . Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide . The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E . coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb . Approximately the same final optical densities were achieved with the E . coli strains expressing FHPg and VHb . The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions . The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control . The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant beta-lactamase relative to the VHb-expressing control.

J Bacteriol, 2000 Jan, 182(1), 81 - 90
Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer; Kwong SM et al.; The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8% . Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability . The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized . An additional eight open reading frames with unknown functions were also detected . pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination . Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P . alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor) . This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml) . Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed . pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans . Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P . alcaligenes NCIB 9867, as well as in P . putida RA713 after 100 generations of nonselective growth . Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability . This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

J Mol Biol, 1999 Dec 17, 294(5), 1169 - 79
The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity; Brok R et al.; The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway . Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells . However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P . alcaligenes mutant strain 537 . Protease protection experiments with P . alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K . This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation . Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures . Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar . The central cavity of the intact XcpQ complex contains structured mass . Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel . However, the single-channel conductances observed were not uniform . Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported .

Mol Biotechnol, 1999 Sep, 12(2), 149 - 58
Heavy metals bioremediation of soil; Diels L et al.; Historical emissions of old nonferrous factories lead to large geographical areas of metals-contaminated sites . At least 50 sites in Europe are contaminated with metals like Zn, Cd, Cu, and Pb . Several methods, based on granular differentiation, were developed to reduce the metals content . However, the obtained cleaned soil is just sand . Methods based on chemical leaching or extraction or on electrochemistry do release a soil without any salts and with an increased bioavailability of the remaining metals content . In this review a method is presented for the treatment of sandy soil contaminated with heavy metals . The system is based on the metal solubilization on biocyrstallization capacity of Alcaligenes eutrophus CH34 . The bacterium can solubilize the metals (or increase their bioavailability) via the production of siderophores and adsorb the metals in their biomass on metal-induced outer membrane proteins and by bioprecipitation . After the addition of CH34 to a soil slurry, the metals move toward the biomass . As the bacterium tends to float quite easily, the biomass is separated from the water via a flocculation process . The Cd concentration in sandy soils could be reduced from 21 mg Cd/kg to 3.3 mg Cd/kg . At the same time, Zn was reduced from 1070 mg Zn/kg to 172 mg Zn/kg . The lead concentration went down from 459 mg Pb/kg to 74 mg Pb/kg . With the aid of biosensors, a complete decrease in bioavailability of the metals was measured.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Sep-Nov, 27(5-6), 411 - 6
Feasibility study on the utilization of rubber latex effluent for producing bacterial biopolymers; Tang SN et al.; Rubber latex effluent is a polluting source that has a high biochemical oxygen demand (BOD) . It is estimated that about 100 million liters of effluent are discharged daily from rubber processing factories . Utilization of this effluent such as the use of a coupled system not only can reduce the cost of treatment but also yield a fermentation feedstock for the production of bioplastic . This study initially was carried out to increase the production of organic acids by anaerobic treatment of rubber latex effluent . It was found that through anaerobic treatment the concentration of organic acids did not increase . Consequently, separation of organic acids from rubber latex effluent by anion exchange resin was examined as a preliminary study of recovering acetic and propionic acids . However, the suspended solids (SS) content in the raw effluent was rather high which partially blocked the ion-exchange columns . Lime was used to remove the SS in the rubber latex effluent . After the lime precipitation process, organic acids were found to adsorb strongly onto the anion exchange resin . Less adsorption of organic acids onto the resin was observed before the lime precipitation . This was probably due to more sites being occupied by colloidal particles on the resin thus inhibiting the adsorption of organic acids . The initial concentration of organic acids in the raw effluent was 3.9 g/L . After ion exchange, the concentration of the organic acids increased to 27 g/L, which could be utilized for production of polyhydroxyalkanoates (PHA) . For PHA accumulation stage, concentrated rubber latex effluent obtained from ion exchange resins and synthetic acetic acid were used as the carbon source . Quantitative analyses from fed batch culture via HPLC showed that the accumulation of PHA in Alcaligenes eutrophus was maximum with a concentration of 1.182 g/L when cultivated on synthetic acetic acid, corresponding to a yield of 87% based on its cell dry weight . The dry cell weight increased from 0.71 to 1.67 g/L . On the other hand, using concentrated rubber latex effluent containing acetic and propionic acids resulted in reduced PHA content by dry weight (14%) but the dry cell weight increased from 0.49 to 1.30 g/L . The results clearly indicated that the cells grow well in rubber latex effluent but no PHA was accumulated . This could be due to the high concentration of propionic acid in culture broth or other factors such as heavy metals . Thus further work is required before rubber latex effluent can be utilized as a substrate for PHA production industrially.

Microbiology, 1999 Nov, 145 ( Pt 11), 3255 - 64
The oxygenase component of the 2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp . strain O-1; Mampel J et al.; Growth of Alcaligenes sp . strain O-1 with 2-aminobenzenesulfonate (ABS; orthanilate) as sole source of carbon and energy requires expression of the soluble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deaminating) (ABSDOS) which is plasmid-encoded . ABSDOS was separated by anion-exchange chromatography to yield a flavin-dependent reductase component and an iron-dependent oxygenase component . The oxygenase component was purified to about 98% homogeneity and an alpha2beta2 subunit structure was deduced from the molecular masses of 134,45 and 16 kDa for the native complex, and the alpha and beta subunits, respectively . Analysis of the amount of acid labile sulfur and total iron, and the UV spectrum of the purified oxygenase component indicated one {2Fe-2S} Rieske centre per alpha subunit . The inhibition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site . Recovery of active protein required strictly anoxic conditions during all purification steps . The FAD-containing reductase could not be purified . ABSDOS oxygenated nine sulfonated compounds; no oxygen uptake was detected with carboxylated aromatic compounds or with aliphatic sulfonated compounds . Km values of 29, 18 and 108 microM and Vmax values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed . The N-terminal amino acid sequences of the alpha- and beta-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the alpha-subunit was thereafter determined . Both redox centres were detected in the deduced amino acid sequence . Sequence data and biochemical properties of the enzyme system indicate a novel member of the class IB ring-hydroxylating dioxygenases.

Carbohydr Res, 1999 Aug 15, 320(3-4), 192 - 9
Enzymatic synthesis of alpha-L-fucosyl-N-acetyllactosamines and 3'-O-alpha-L-fucosyllactose utilizing alpha-L-fucosidases; Murata T et al.; An alpha-L-fucosidase from porcine liver produced alpha-L-Fuc-(1-->2)-beta-D-Gal-(1-->4)-D-GlcNAc (2'-O-alpha-L-fucosyl-N-acetyllactosamine, 1) together with its isomers alpha-L-Fuc-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc (2) and alpha-L-Fuc-(1-->6)-beta-D-Gal-(1-->4)-D-GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl alpha-L-fucopyranoside and beta-D-Gal-(1-->4)-D-GlcNAc . The enzyme formed the trisaccharides 1-3 in 13% overall yield based on the donor, and in the ratio of 40:37:23 . In contrast, transglycosylation by Alcaligenes sp . alpha-L-fucosidase led to the regioselective synthesis of trisaccharides containing a (1-->3)-linked alpha-L-fucosyl residue . When beta-D-Gal-(1-->4)-D-GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and alpha-L-Fuc-(1-->3)-beta-D-Gal-(1-->4)-D-Glc (3'-O-alpha-L-fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.

Biochemistry, 1999 Oct 26, 38(43), 14330 - 7
Amino acid replacements at the H2-activating site of the NAD-reducing hydrogenase from Alcaligenes eutrophus; Massanz C et al.; The role of amino acid residues in the H(2)-activating subunit (HoxH) of the NAD-reducing hydrogenase (SH) from Alcaligenes eutrophus has been investigated by site-directed mutagenesis . Conserved residues in the N-terminal L1 (RGxE) and L2 (RxCGxCx(3)H) and the C-terminal L5 (DPCx(2)Cx(2)H/R) motifs of the active site-harboring subunit were chosen as targets . Crystal structure analysis of the {NiFe} hydrogenase from Desulfovibrio gigas uncovered two pairs of cysteines (motifs L2 and L5) as coordinating ligands of Ni and Fe . Glutamate (L1) and histidine residues (L2 and L5) were proposed as being involved in proton transfer {Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E . C., Frey, M., and Fontecilla Camps, J . C . (1995) Nature 373, 580-587} . The A . eutrophus mutant proteins fell into three classes . (i) Replacement of the putative four metal-binding cysteines with serine led to the loss of H(2) reactivity and blocked the assembly of the holoenzyme . Exchange of Cys62, Cys65, or Cys458 was accompanied by the failure of the HoxH subunit to incorporate nickel, supporting the essential function of these residues in the formation of the active site . Although the fourth mutant of this class (HoxH{C461S}) exhibited nickel binding, the modified protein was catalytically inactive and unable to oligomerize . (ii) Mutations in residues possibly involved in proton transfer (HoxH{E43V}, HoxH{H69L}, and HoxH{H464L}) yielded Ni-containing proteins with residual low levels of hydrogenase activity . (iii) The most promising mutant protein (HoxH{R40L}), which was identified as a metal-containing tetrametric enzyme, was completely devoid of H(2)-dependent oxidoreductase activity but exhibited a remarkably high level of D(2)-H(+) exchange activity . These characteristics are compatible with the interpretation of a functional proton transfer uncoupled from the flow of electrons.

FEBS Lett, 1999 Oct 22, 460(1), 6 - 10
Purification and characterization of the single-component nitric oxide reductase from Ralstonia eutropha H16; Cramm R et al.; Nitric oxide (NO) reductase was purified from Ralstonia eutropha (formerly Alcaligenes eutrophus) using a two step chromatographic procedure . Unlike the common NO reductases, the enzyme consists of a single subunit of 75 kDa which contains both high-spin and low-spin heme b, but lacks heme c . One additional iron atom, probably a ferric non-heme iron, was identified per enzyme molecule . Whereas reduced cytochrome c was ineffective as electron donor, NO was reduced at a specific activity of 2.3 micromol/min per mg of protein in the presence of 2-methyl-1,4-naphthoquinol.

Biochem J, 1999 Dec 1, 344 Pt 2, 397 - 402
2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp . 4HAP: a novel enzyme with an atypical dioxygenase sequence; Hopper DJ et al.; 2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp . 4HAP grown on 4-hydroxyacetophenone . Measurements of the M(r) of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively . The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme . From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced . The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide . It gave a calculated M(r) of 20364 . The gene was expressed in Escherichia coli and yielded active enzyme . The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.

DNA Seq, 1999, 10(1), 7 - 17
Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869; Cronin CN et al.; The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively . The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway . In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P . alcaligenes . A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.

Arch Microbiol, 1999 Nov, 172(5), 330 - 9
Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway; Davis JK et al.; Nitrobenzene is degraded to pyruvate and acetaldehyde by Pseudomonas pseudoalcaligenes JS45 via a reductive pathway, and by Comamonas sp . JS765 via an oxidative pathway . Although the initial reactions in the degradation of nitrobenzene by the two bacteria are totally different, the lower pathways are similar and converge at the level of 4-oxalocrotonate . In order to further investigate the biochemical properties and reveal the evolutionary relationships between the two lower pathways, the genes encoding the 2-aminophenol 1,6-dioxygenase were cloned and sequenced . 2-Aminophenol 1,6-dioxygenase from P . pseudoalcaligenes JS45 and catechol 2,3-dioxygenase from Comamonas sp . JS765 were able to act on both catechol and 2-aminophenol, but catechol was a suicide substrate of 2-aminophenol 1,6-dioxygenase . The activity of 2-aminophenol 1,6-dioxygenase was restored after removal of catechol and incubation with ascorbate and FeCl(2) . Both the alpha-subunit (AmnA) and the beta-subunit (AmnB) of the dioxygenase from P . pseudoalcaligenes JS45 show a high degree of identity to the corresponding subunits of the ring-fission dioxygenase from Pseudomonas sp . AP-3: 67% for the alpha-subunit, and 84% for the beta-subunit . Sequence similarity studies suggest that the beta-subunits of both 2-aminophenol 1,6-dioxygenases are distantly related to homoprotocatechuate 2,3-dioxygenase from Escherichia coli strains W and C and then to catechol 2, 3-dioxygenase from Alcaligenes eutrophus . Four active-site-relevant histidines are conserved in AmnB, but not in AmnA . The lack of conserved histidines indicates the absence of an Fe(2+) binding site in AmnA, which explains the previous observations of only approximately one Fe(2+) per two subunits in the 2-aminophenol 1, 6-dioxygenases from P . pseudoalcaligenes JS45 . The 2-aminophenol 1, 6-dioxygenase genes are located upstream of the 2-aminomuconic semialdehyde dehydrogenase gene, and a putative member of the YjgF protein family is upstream of the dioxygenase genes . Transcriptional analysis indicates that the YjgF-like protein, 2-aminophenol 1, 6-dioxygenase, and 2-aminomuconic semialdehyde dehydrogenase are coordinately transcribed . A putative ORF similar to part of the RNA helicase genes is downstream of the dehydrogenase gene . Both the novel organization of the genes and the phylogeny of the dioxygenases and dehydrogenase indicate that the 2-aminophenol pathway in P . pseudoalcaligenes JS45 represents an example of a distant divergent evolution of meta-cleavage pathways.

J Bacteriol, 1999 Nov, 181(21), 6697 - 705
Transcriptional activation of the chlorocatechol degradative genes of Ralstonia eutropha NH9; Ogawa N et al.; Ralstonia eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobenzoate via the modified ortho-cleavage pathway . A ca . 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCD operon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbn operon . The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2, 4-trichlorobenzene-degrading bacterium Pseudomonas sp . strain P51 . Transcriptional fusion studies demonstrated that cbnR regulates the expression of cbnABCD positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively . In vitro transcription assays confirmed that 2-chloro-cis,cis-muconate (2-CM) and cis, cis-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon . This inducer-recognizing specificity is different from those of the homologous catechol (catBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which only the intermediates of the regulated pathway, CCM for catBCA and 2-CM for clcABD, act as significant inducers . Specific binding of CbnR protein to the cbnA promoter region was demonstrated by gel shift and DNase I footprinting analysis . In the absence of inducer, a region of ca . 60 bp from position -20 to position -80 upstream of the cbnA transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position -50 . Circular permutation gel shift assays demonstrated that CbnR bent the cbnA promoter region to an angle of 78 degrees and that this angle was relaxed to 54 degrees upon the addition of inducer . While a similar relaxation of bending angles upon the addition of inducer molecules observed with the catBCA and clcABD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.

J Bacteriol, 1999 Nov, 181(21), 6656 - 63
Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival; Chang DE et al.; In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E . coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated . The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate . In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain . The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate . The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux . By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis . These results suggest that E . coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

Microbiology, 1999 Oct, 145 ( Pt 10), 2797 - 802
The effect of motility and cell-surface polymers on bacterial attachment; Morisaki H et al.; Recently it was shown that motility of Vibrio alginolyticus facilitated cell attachment to glass surfaces . In the present study the same relationship between motility and cell attachment was confirmed for Alcaligenes and Alteromonas spp . These findings clearly answer a long-standing question: does motility facilitate attachment? However, they are contradictory to a general view on cell attachment that the energy barrier due to electrostatic repulsion between negatively charged bacterial cells and a glass surface is much greater than both the thermal kinetic energy of the bacterial cell and the bacterial swimming energy . It is shown that the energy barrier becomes far less than that usually estimated when bacterial cells are rich in polymers at their surfaces . This finding reasonably explains the dependence of bacterial attachment rate on cell motility and demands reconsideration of the mechanism of bacterial attachment.

Epidemiol Mikrobiol Imunol, 1999 Aug, 48(3), 117 - 21
{Elastase activity of gram-negative non-fermenting bacteria and two variations of a simple method for their detection}; Ondrovcik P et al.; The ability to produce elastase was examined in 1,168 strains of 11 representative genera of Gram-negative non-fermenting bacteria isolated from weakened patients in the course of eight years . Detection of the elastase activity was performed parallelly using the conventional method and its pulverisation variant . The difference between the results accomplished by the classical method and its pulverisation variant (53.9% versus 60.5% detected elastase-positive strains) was highly significant (p < 0.05) . Among the examined strains 500 (42.8%) displayed elastase activity . The highest proportion of the elastase positive strains was found in Pseudomonas aeruginosa (84.5%), P . fluorescens (60.5%), P . stutzeri (56.4%), Burkholderia cepacia (43.8%) and Shewanella putrefaciens (39.7%) . Elastase activity detected in Burkholderia cepacia, Pseudomonas alcaligenes, P . mendocina, P . putida, P . stutzeri and in Shewanella putrefaciens is apparently described for the first time.

Mol Microbiol, 1999 Sep, 33(6), 1254 - 66
Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria; Holden MT et al.; In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor . Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively . These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans {cyclo(DeltaAla-L-Val) only} . Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro) . Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) . Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site . Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens . Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.

Appl Environ Microbiol, 1999 Oct, 65(10), 4363 - 8
High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli; Choi JI et al.; Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-co-3HV)} with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed . Fed-batch cultures of recombinant E . coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium . When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained . Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content . An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid . When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively . When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.

Curr Microbiol, 1999 Nov, 39(5), 244 - 8
Beta-lactamase-free penicillin amidase from Alcaligenes sp.: isolation strategy, strain characteristics, and enzyme immobilization; Pal A et al.; Isolation and characterization of a beta-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1 . 11)-producing organism is reported . The test strain was isolated by an enrichment technique with a substrate other than penicillins . The isolated strain belongs to the genus Alcaligenes . Phenylacetic acid was found to be the inducer of penicillin amidase . The amidase has a broad substrate spectrum . It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate . Immobilized cells of Alcaligenes sp . were shown to act as a reversible enzyme.

Rapid Commun Mass Spectrom, 1999 Oct 15, 13(19), 1951 - 1957
Characterization of partially transesterified poly(beta-hydroxyalkanoate)s using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Saeed KA et al.; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for the characterization of a partially transesterified poly(beta-hydroxyalkanoate), PHA, polymer produced by the bacterial strain Alcaligenes eutrophus using saponified vegetable oils as the sole carbon sources . The transesterification was carried out separately under acidic and basic conditions to obtain PHA oligomers weighing less than 10 kDa . The intact oligomers were detected in their cationized {M + Na}(+) and {M + K}(+) forms by MALDI-TOFMS . A composition analysis, using the MALDI-TOF spectra, indicate that the oligomers obtained via acid catalysis were terminated with a methyl 3-hydroxybutyrate end group, and those obtained by base catalysis had a methyl crotonate (olefinic) termination . In addition to HB (hydroxy butyrate), the oligomers were found to contain a small percentage of HV (hydroxy valerate) . This was independently confirmed using gas chromatography/mass spectrometry (GC/MS) . In comparison, the analysis of a commercial PHA polymer, transesterified under identical conditions, only showed the presence of HB, i.e . a pure PHB homopolymer .

Biotechnol Bioeng, 1999 Nov 5, 65(3), 363 - 8
Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals; Lee SY et al.; A novel and efficient method for the production of enantiomerically pure (R)-(-)-hydroxycarboxylic acids by in vivo depolymerization of microbial polyester polyhydroxyalkanoates (PHAs) was developed . Using this method, several model compounds, (R)-(-)-3-hydroxyalkanoic acids, consisting of 4 to 12 carbon atoms, and (R)-(-)-3-hydroxy-5-phenylvaleric acid, could be prepared . In particular, (R)-(-)-3-hydroxybutyric acid could be efficiently prepared by this method . By providing the environmental condition in which cells possess high activity of intracellular PHA depolymerase and low activity of (R)-(-)-3-hydroxybutyric acid dehydrogenase, (R)-(-)-3-hydroxybutyric acid could be produced with a yield of 96% in only 30 min by in vivo depolymerization of polyhydroxybutyrate (PHB) accumulated in Alcaligenes latus .

J Bacteriol, 1999 Sep, 181(18), 5684 - 92
Positive transcriptional feedback controls hydrogenase expression in Alcaligenes eutrophus H16; Schwartz E et al.; The protein HoxA is the central regulator of the Alcaligenes eutrophus H16 hox regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis . Expression of the regulatory gene hoxA was analyzed . Screening of an 8-kb region upstream of hoxA with a promoter probe vector localized four promoter activities . One of these was found in the region immediately 5' of hoxA; the others were correlated with the nickel metabolism genes hypA1, hypB1, and hypX . All four activities were independent of HoxA and of the minor transcription factor sigma(54) . Translational fusions revealed that hoxA is expressed constitutively at low levels . In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the hox regulon . Quantitative transcript assays indicated elevated levels of hyp mRNA under hydrogenase-derepressing conditions . Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth . Site-directed mutagenesis revealed that P(MBH), which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of hoxA under hydrogenase-derepressing conditions . Thus, expression of the hox regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA . These results imply the existence of an unusually large (ca . 17,000-nucleotide) transcript.

J Bacteriol, 1999 Aug, 181(16), 4919 - 28
A megaplasmid-borne anaerobic ribonucleotide reductase in Alcaligenes eutrophus H16; Siedow A et al.; The conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth of Alcaligenes eutrophus H16 in the presence of nitrate as the terminal electron acceptor . We identified two megaplasmid-borne genes (nrdD and nrdG) which are indispensable under these conditions . Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases . Deletion of nrdD and nrdG in A . eutrophus abolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked . Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif . A mutant of A . eutrophus with a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical . Analysis of transcriptional and translational fusions indicate that nrdD and nrdG are cotranscribed and that the translation efficiency of nrdD is 40-fold higher than that of nrdG . Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.

Appl Environ Microbiol, 1999 Aug, 65(8), 3561 - 5
Cloning, molecular analysis, and expression of the polyhydroxyalkanoic acid synthase (phaC) gene from Chromobacterium violaceum; Kolibachuk D et al.; The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaC(Cv)) was cloned and characterized . A 6.3-kb BamHI fragment was found to contain both phaC(Cv) and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaA(Cv)) . Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities . While C . violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaC(Cv), accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source . This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C . violaceum PHA synthase . Neither recombinant E . coli nor recombinant Pseudomonas putida harboring phaC(Cv) accumulated significant levels of PHA . Sequence analysis of the phaC(Cv) product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no . AAD10274).

Can J Microbiol, 1999 Apr, 45(4), 326 - 38
Characterization of the bacterial community of a zinc-polluted soil; Brim H et al.; The bacterial community of a zinc-contaminated soil (Maatheide soil in Lommel, Belgium) was studied using cultivation as well as cultivation-independent techniques . Colony-forming units (CFU) were determined by plating on media with or without metals . Dominant isolates were characterized by fatty acid methyl ester analysis (FAME analysis) and PCR fingerprinting using repetitive extragenic palindromic sequences as primers . DNA was directly extracted from soil samples and used as a template for the PCR amplification of the 16S rDNA (8-1511) or a 16S rDNA fragment (968-1401) . Clones resulting from cloning the 16S rDNA from soil DNA were sequenced . Temperature gradient gel electrophoresis (TGGE analysis) was performed for 16S rDNA fragments (968-1401) amplified from the dominant isolates, the clones, and the total soil DNA extracted according to two protocols differing in strength of lysis . Total CFU ranged from 10(4) to 10(5)/g soil . The majority of the isolates were identified by FAME analysis as Arthrobacter spp . (18 out of 23) . None of the isolates were identified as a Ralstonia eutropha like strain (formerly Alcaligenes eutrophus) . Metalloresistant Rastomia eutropha like strains were previously shown to be dominant in the analyzed biotope . Most of the isolates were zinc tolerant but only seven could be considered zinc resistant . Sequences of the 16S rDNA clones obtained from total soil DNA were affiliated with genes of different bacteria such as alpha-proteobacteria, beta-proteobacteria, and the Cytophaga-Flexibacter-Bacteroides group . None of the sequenced clones aligned with the Ralstonia eutropha 16S rRNA gene . TGGE analysis of the 16S rDNA fragments (968-1401) amplified from the dominant strains, the clones, and the total soil DNA showed that isolates and clones represented only a part of the bands present in the TGGE pattern from total DNA . The 968-1401 fragment amplified from all Arthrobacter strains had a similar electrophoretic mobility . This band was seen as a major band in the pattern of DNA extracted from soil using a harsh cell lysis, whereas it did not appear, or appeared only as a weak band, in patterns obtained from soil DNA extracted using gentle lysis . The previously reported predominance of a Ralstonia eutropha like strain in this soil was no longer observed . This may suggest a population replacement by less resistant bacteria, concomitant with a progressive decrease of the zinc toxicity in the Maatheide soil.

Int J Biol Macromol, 1999 Jun-Jul, 25(1-3), 55 - 60
Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus; Lenz RW et al.; Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed . In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described . K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated . With a racemic mixture of HBCoA, the enzyme polymerized only the {R} monomer.

Int J Biol Macromol, 1999 Jun-Jul, 25(1-3), 43 - 53
Chain termination in polyhydroxyalkanoate synthesis: involvement of exogenous hydroxy-compounds as chain transfer agents; Madden LA et al.; We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) {P(3HB)} accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization . End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus . All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation . The number-average molecular mass (Mn) of P(3HB) produced by R . eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures . These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups . When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both {R} and {S} configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer . 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively . Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria . It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis.

Biotechnol Bioeng, 1999 Jul 20, 64(2), 240 - 9
Use of exogenous specialised bacteria in the biological detoxification of a dump site-polychlorobiphenyl-contaminated soil in slurry phase conditions
Fava F, Bertin L.
The possibility of biologically detoxifying a contaminated soil from an Italian dump site containing about 1500 mg/kg (in dry soil) of polychlorinated biphenyls was studied in the laboratory in this work . The soil, which contained indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids at concentration of about 300 CFU per g of air-dried soil, was amended with inorganic nutrients, saturated with water and treated in aerobic 3-L batch slurry reactors (soil suspension at 20% w/v) . Either Pseudomonas sp . CPE1 strain, capable of cometabolising low-chlorinated biphenyls into chlorobenzoic acids, or a bacterial co-culture capable of aerobically dechlorinating polychlorobiphenyls constituted by this bacterium and the two chlorobenzoic acid degrading bacteria Pseudomonas sp . CPE2 strain and Alcaligenes sp . CPE3 strain, were used as inocula (final concentration of about 10(8) CFU/mL for each bacterium), in the absence and in the presence of biphenyl (4 g/kg of air dried soil) . Significant soil polychlorobiphenyl depletions were observed in all the reactors after 119 days of treatment . The soil inoculation with the sole CPE1 was found to slightly enhance the polychlorobiphenyl depletions (about 20%) and the soil detoxification; the effect was higher in the presence of biphenyl . The use of the polychlorobiphenyl mineralising bacterial co-culture as inoculum resulted in a strong enhancement of the depletions of both the soil polychlorobiphenyls (from 50 to 65%) and of the original soil ecotoxicity . The bacterial biomass inoculated was found to implant into the soil; the higher specialised biomass availability thus reached in the inoculated soil was probably responsible of a more extensive biodegradation of polychlorobiphenyls and therefore of the higher detoxification yields observed in the inoculated reactors . The soil ecotoxicity, measured through two different soil contact assays, i.e., the Lepidium sativum germination test and the Collembola mortality test, was often found to decrease proportionally with the soil polychlorobiphenyl concentration .

Syst Appl Microbiol, 1999 May, 22(2), 258 - 68
Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs; Brim H et al.; The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis) . The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals . These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes . The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34 . The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans . The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii . The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains . This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia . Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species . Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.

Ecotoxicol Environ Saf, 1999 Jun, 43(2), 222 - 8
Validation of genetically engineered bioluminescent surfactant resistant bacteria as toxicity assessment tools; Layton AC et al.; Bacteria are useful organisms for measuring acute and chronic toxicity . The most popular toxicity tests utilize the inhibition of bioluminescence as an indication of toxicity . An extensive toxicity database on pure chemical compounds has been created using the bioluminescent microorganism, Vibrio fischeri . However, the use of the Microtox assay in applications for environmental samples is not always successful, due to the test organism . Because the genes for bioluminescence have been cloned from V . fischeri, environmentally relevant test strains can be readily constructed . In this study, surfactant-resistant bioluminescent bacterial strains were constructed by transferring a broad host range plasmid containing the bioluminescent genes under the regulation of a constitutive promoter into strains from several bacterial genera . Two test strains, Stenotrophomonas 3664 and Alcaligenes eutrophus 2050, were approximately 400 times more resistant to the nonionic surfactant polyoxyethylene 10 lauryl ether than V . fischeri and are useful for toxicity reduction evaluations of remediation processes which use surfactants for solubilization of hydrophobic pollutants . The use of these strains as alternative test organisms in the Microtox assay was evaluated using nonpolar narcosis as the baseline toxicity mechanisms . The two test strains and V . fischeri indicated linear fits of EC50 values with the octanol/water partition (Kow) for five nonpolar narcotic compounds in acute assays (r2>0.9) with a slope of approximately 1 . For all three strains, the y-intercept values were approximately the same, indicating that sensitivity did not vary . These results indicate that the nonpolar narcosis baseline toxicity mechanism may be useful as a general tool to validate the functioning of genetically engineered bioluminescent microorganisms .

Appl Environ Microbiol, 1999 Jun, 65(6), 2762 - 4
Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria; Lee SY et al.; Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli . PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined . When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell . The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E . coli, was also examined for endotoxin removal . The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB . Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB . It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction . Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E . coli by simple NaOH digestion.

Eur J Biochem, 1999 Jun, 262(2), 396 - 405
Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus; Ollesch G et al.; The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds . The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains . The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity . The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions . The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein . Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers . Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds . Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.

J Mol Biol, 1999 May 14, 288(4), 609 - 21
The crystal structure of rubisco from Alcaligenes eutrophus reveals a novel central eight-stranded beta-barrel formed by beta-strands from four subunits; Hansen S et al.; Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is involved in photosynthesis where it catalyzes the initial step in the fixation of carbon dioxide . The enzyme also catalyzes a competing oxygenation reaction leading to loss of fixed carbon dioxide, thus reducing the net efficiency of photosynthesis significantly . Rubisco has therefore been studied extensively, and a challenging goal is the engineering of a more photosynthetically efficient enzyme . Hexadecameric rubiscos fall in two distinct groups, "green-like" and "red-like" . The ability to discriminate between CO2 and O2 as substrates varies significantly, and some algae have red-like rubisco with even higher specificity for CO2 than the plant enzyme . The structure of unactivated rubisco from Alcaligenes eutrophus has been determined to 2.7 A resolution by molecular replacement and refined to R and Rfree values of 26.6 and 32.2 %, respectively . The overall fold of the protein is very similar to the rubisco structures solved previously for green-like hexadecameric enzymes, except for the extended C-terminal domains of the small subunits which together form an eight-stranded beta-barrel which sits as a plug in the entrance to the central solvent channel in the molecule . The present structure is the first which has been solved for a red-like rubisco and is likely to represent a fold which is common for this group . The small subunits in general are believed to have a stabilizing effect, and the new quaternary structure in the oligomer of the present structure is likely to contribute even more to this stabilization of the assembled rubisco protein .

Microbiol Immunol, 1999, 43(2), 115 - 25
Chromosome-determined zinc-responsible operon czr in Staphylococcus aureus strain 912; Kuroda M et al.; A novel operon, czrAB (zinc-responsible genes), was identified in the chromosome of Staphylococcus aureus . The operon consists of two genes, czrA and czrB . The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S . aureus plasmid pI258 and smtB of Synechococcus PCC7942 . The czrB, coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium-resistant factor of Bacillus subtilis and Alcaligenes eutrophus . In the presence of zinc (0.1-10 mM), the transcription of czrAB was enhanced in a concentration-dependent manner . Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression . The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM) . The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight . The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.

J Mol Biol, 1999 Apr 16, 287(5), 1001 - 9
Structural and kinetic evidence for an ordered mechanism of copper nitrite reductase; Strange RW et al.; The crystallographic structures of several copper-containing nitrite reductases are now available . Despite this wealth of structural data, no definitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, followed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates . We present here the first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy . The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu2Zn2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu2Zn2 SOD . The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre . These observations provide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs . We propose that the two links between the Cu sites of AxNiR, namely His129-Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively . Nitrite binding at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer . The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR .

Arch Microbiol, 1999 Feb, 171(3), 139 - 45
Selective transport of divalent cations by transition metal permeases: the Alcaligenes eutrophus HoxN and the Rhodococcus rhodochrous NhlF; Degen O et al.; nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli . 57CO2+ and 63Ni2+ transport of the recombinants was examined by means of a previously described physiological assay . Although the transporters are highly similar, different preferences for divalent transition metal cations were observed . HoxN was unable to transport 57CO2+, but mediated 63Ni2+ uptake . The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni2+ . In contrast, NhlF transported both 57CO2+ and 63Ni2+ ion . NhlF-mediated 63Ni2+ uptake was markedly reduced in the presence of CO2+, while 57CO2+ uptake was only slightly lower in the presence of Ni2+ . These results indicate different affinities of NhlF for CO2+ and Ni2+ and identified CO2+ ion as the preferred substrate.

J Bacteriol, 1999 Apr, 181(8), 2385 - 93
Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus; Grosse C et al.; The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter . DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc . Additional open reading frames upstream of czc had no homologs in the current databases . Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6, 200-nucleotide czcCBA message (D . Van der Lelie et al., Mol . Microbiol . 23:493-503, 1997) was confirmed . Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 microM zinc, less by 300 microM cobalt, and only slightly by 300 microM cadmium . Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS . The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN . A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were DeltaczcR and DeltaczcS mutants of this strain and of AE128(pMOL30) wild type . Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

J Bacteriol, 1999 Apr, 181(8), 2323 - 9
The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme; Prudencio M et al.; The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced . To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase . The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources . The full-length protein included a 24-amino-acid leader peptide . The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm . Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A . xylosoxidans . The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers . This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity . Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity . The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A . xylosoxidans . This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 325 - 8
High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium; Wang F et al.; A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium . Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E . coli that underwent filamentation . Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E . coli .

J Infect Dis, 1999 May, 179(5), 1190 - 6
Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis; Burns JL et al.; Pseudomonas aeruginosa endobronchial infection causes significant morbidity and mortality among cystic fibrosis patients . Microbiology results from two multicenter, double-blind, placebo-controlled trials of inhaled tobramycin in cystic fibrosis were monitored for longitudinal changes in sputum microbial flora, antibiotic susceptibility, and selection of P . aeruginosa isolates with decreased tobramycin susceptibility . Clinical response was examined to determine whether current susceptibility standards are applicable to aerosolized administration . Treatment with inhaled tobramycin did not increase isolation of Burkholderia cepacia, Stenotrophomonas maltophilia, or Alcaligenes xylosoxidans; however, isolation of Candida albicans and Aspergillus species did increase . Although P . aeruginosa tobramycin susceptibility decreased in the tobramycin group compared with that in the placebo group, there was no evidence of selection for the most resistant isolates to become most prevalent . The definition of resistance for parenteral administration does not apply to inhaled tobramycin: too few patients had P . aeruginosa with a tobramycin MIC >/=16 microgram/mL to define a new break point on the basis of clinical response.

Biotechnol Bioeng, 1999 Mar 5, 62(5), 518 - 25
Maximum production strategy for biodegradable copolymer P(HB-co-HV) in fed-batch culture of Alcaligenes eutrophus; Shimizu H et al.; A novel strategy for the maximum production of a biodegradable copolymer, poly(3-hydroxybutyric-co-hydroxyvaleric) acid, P(HB-co-HV), was developed, based on the kinetic parameters obtained from fed-batch culture experiments of Alcaligenes eutrophus . The effects of various culture conditions such as mole ratio of carbon:nitrogen in feed medium (C/N); total fatty acids concentrations; and addition ratio of fatty acids on cultivation properties such as the specific rates of cell formation, mu (h-1), P(HB-co-HV) production, rho{g.P(HB-co-HV)/g.cell/h}, production yield from fatty acids {g.P(HB-co-HV)/g.fatty acid}, and mole fraction of monomeric units in the copolymer {mol.(HV)/ inverted question markmol.(HB) + mol.(HV) inverted question mark}, were investigated . When nitrogen supply was sufficient for cell growth; that is, C/N (mol.nitrogen atom/mol.carbon atom) was low, mu was high, but rho and the production yield were low, because fatty acids were used mainly for energy formation and anabolic reactions in the cells . On the other hand, when nitrogen supply was limited for cell growth-that is, C/N was high-rho was high . The highest value of rho was obtained when C/N was 75 . As the mole ratio of valeric acid (VA) to butyric acid (BA) in the feed medium was increased, the mole fraction of HV units in P(HB-co-HV) increased linearly . When the ratio of BA to VA in the feed medium was kept at a constant value, but C/N was increased, the mole fraction of HV units decreased . In particular, when C/N was >12, the mole fraction of HV units decreased linearly as C/N increased . When VA was utilized as the sole carbon source and C/N was fixed at 4, P(HB-co-HV) with the highest mole fraction of HV units (67 mol%) was achieved . From these results, it was shown that both C/N and the mole ratio of BA to VA in the feed medium should be well controlled for an optimal production of P(HB-co-HV) with the desired value of the mole fraction of HV units . When the addition ratio of butyric acid was 50 wt% of total fatty acids, a maximum production strategy for P(HB-co-HV) was developed and realized experimentally, which was based on a model of the relationship between mu and rho .

Biotechnol Bioeng, 1999 Jan 5, 62(1), 106 - 13
A tunable switch to regulate the synthesis of low and high molecular weight microbial polyesters; Ashby RD et al.; The addition of poly(ethylene glycol) (Mn = 200 g/mol) (PEG-200) to the fermentation media of Alcaligenes eutrophus and Alcaligenes latus at various stages of growth resulted in the synthesis of poly(3-hydroxybutyrate) (PHB) with bimodal molecular weight distributions . The presence of 2% w/v-PEG-200 did not have deleterious effects on PHB volumetric yields and cell productivity . In general, the Mn values of the high (H) and low (L) fractions showed little variability as a function of the time at which PEG-200 was added to the cultures . By this approach, the H:L ratios (w/w) of the PHB synthesized by A . eutrophus and A . latus were varied from 9:91 to 76:24 and from 16:84 to 88:12, respectively . It is believed that the H fractions were formed prior to the addition of PEG-200 to the cultures . Also, once PEG-200 was made available to the cells, PEG-200 acted as a switch so that the reduced molecular weight fraction was formed . In addition, a necessary requirement for the above is that the frequency of transesterification reactions during polymer synthesis was small . The efficiency that PEG-200 reduced the molecular weight of the PHBs formed by both bacteria appears similar . Indirect evidence suggests that the PHB L fractions formed by A . latus subsequent to PEG-200 addition consist primarily of chains that have PEG terminal groups . This terminal chain structure was not observed for PHB formed by A . eutrophus .

Biotechnol Bioeng, 1998 Mar 5, 57(5), 557 - 70
Metabolic modeling of polyhydroxybutyrate biosynthesis; Leaf TA et al.; A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed . The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior . Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting . Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively . To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms . Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+ . These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium .

Biotechnol Bioeng, 1998 Jan 5, 57(1), 79 - 86
Evaluation of Alcaligenes eutrophus cells as an NADH regenerating catalyst in organic-aqueous two-phase system; Andersson M et al.; A soluble NAD-dependent hydrogenase contained in Alcaligenes eutrophus was evaluated as a coenzyme regenerating catalyst in an organic-aqueous two-phase (predominantly organic) system . The horse-liver alcohol-dehydrogenase (HLADH) catalyzed reduction of cyclohexanone to cyclohexanol was used as a model reaction . The impact of different solvents (selected to span a large variety of principal properties) on the stability and activity of the HLADH, using substrate-driven regeneration, was studied . Solvents suitable for the HLADH were then selected for an evaluation of the hydrogenase-driven coenzyme regeneration . Hydrophobic solvents such as heptane, toluene, and 1,1,1-trichloroethane were found to be suitable for the coupled reactions catalyzed by HLADH and hydrogenase . Nonimmobilized cells, permeabilized with cetyl-trimethyl-ammonium bromide, were the most efficient preparation for the regeneration of NADH . The use of this preparation in heptane (10% water) was optimized with respect to the yield obtained in the HLADH-catalyzed reduction of cyclohexanone . Using the optimized conditions, yields of 99% cyclohexanol were obtained .

Biotechnol Bioeng, 1999 Mar, 62(6), 625 - 631
Control of acetic acid concentration by pH-stat continuous substrate feeding in heterotrophic culture phase of two-stage cultivation of Alcaligenes eutrophus for production of P(3HB) from CO2, H2, and O2 under non-explosive conditions; Sugimoto T et al.; A-two stage culture method of hydrogen-oxidizing bacterium, Alcaligenes eutrophus, is used to produce poly-D-3-hydroxybutyrate, P(3HB) from CO2, O2, and H2 without using a very high oxygen transfer rate while maintaining the O2 concentration in gas phase below 6.9 (v/v)% to prevent detonation of the gas mixture . The two-stage method consists of a heterotrophic culture using fructose as carbon source for exponential cell growth and an autotrophic culture for P(3HB) accumulation . We investigated the use of acetic acid as a cheaper carbon source than fructose for the heterotrophic culture in the two-stage method . However, the acetate concentration in the culture system must be maintained at 1.0 g . dm-3 since its inhibitory effect on the cell growth is very strong . Then, high cell density cultivation of A . eutrophus was investigated by pH-stat continuous feeding of acetic acid to control acetate concentration . As a result, acetate concentration was automatically maintained around 1.0 g . dm-3 by using a feed with a composition in CH3COOH/CH3COONH4/KH2PO4 molar ratio of 5:1:0.084 . Cell concentration increased to 48.6 g . dm-3 after 21 h of cultivation . The cell mass grown in the fed-batch culture on acetic acid was useful for P(3HB) production from CO2 in the subsequent autotrophic culture stage .

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 310 - 3 Epub 1999 Jan 01.
Purification, crystallization and preliminary X-ray studies of two isoforms of Rubisco from Alcaligenes eutrophus; Hansen S et al.; Two different isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Alcaligenes eutrophus have been purified and crystallized . Both isoforms crystallize in space group P43212 . Crystals of isoform I (unit-cell dimensions a = 112.0 and c = 402.7 A) diffract to 2.7 A, whereas isoform II (unit-cell dimensions a = 111.8 and c = 400.0 A) presently diffract to 3.2 A, using synchrotron radiation in both cases.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 307 - 9 Epub 1999 Jan 01.
Crystallization and preliminary X-ray study of iso-2 azurin from the methylotrophic bacterium, Methylomonas J; Inoue T et al.; The obligate methylotroph Methylomonas J possesses two distinct azurins . The iso-2 azurin, which functions as an electron acceptor for methylamine dehydrogenase, has been crystallized using two kinds of precipitants: PEG 4000 and ammonium sulfate . The crystals precipitated with PEG belong to the monoclinic system, space group P21, with unit-cell parameters a = 32.96, b = 33.67, c = 47.34 A and beta = 101.35 degrees . The crystals precipitated with ammonium sulfate belong to the orthorhombic system, space group C2221, with unit-cell parameters a = 31.52, b = 62.49 and c = 135.41 A . The crystals diffract to 1.6 and 1.9 A resolution, respectively, and were suitable for X-ray crystallographic studies . A Patterson search is being conducted using the recently reported structure of Alcaligenes xylosoxidans NCIMB 11015 as a starting model.

Isotopes Environ Health Stud, 1998, 34(4), 325 - 34
Investigation into PCB biodegradation using uniformly 14C-labelled dichlorobiphenyl; Kubatova A et al.; Biodegradation of polychlorinated biphenyls (PCBs) in soil is considered to be very complex due to various physico-chemical factors involved . Isotope labelling technique is the best to trace fate of the xenobiotic in the environment . In this work, the uniformly 14C-labelled PCB congener 11 (3,3'-chlorobiphenyl) was chosen as a low chlorinated coplanar biphenyl which was assumed to be readily degraded by microorganisms . Pleurotus ostreatus and two Pseudomonas species, representing white rot fungi and soil bacteria were used separately or in a consortium . The amount of liberated 14CO2 and radio-HPLC, HPLC, GC-MS, and radio-TLC analyses of extracts at the end of a two-month experiment showed that the mineralization of PCB 11 was < 0.4%, volatilization < 3.1%, and 30% of radioactivity was irreversibly bound to the soil matrix . The respective contents of all intermediate metabolites were 4.7 to 10.5 and 2.5 to 2.7% where Pseudomonas alcaligenes alone or in combination with P . putida was applied . 3-Chlorobenzoic acid was the major biodegradation product.

Am J Ophthalmol, 1999 Mar, 127(3), 345 - 6
Alcaligenes xylosoxidans endophthalmitis 8 months after cataract extraction; Swart J et al.; PURPOSE: To report a case of Alcaligenes xylosoxidans endophthalmitis and to increase awareness of its potential as an intraocular pathogen . METHODS: An 80-year-old woman in good general health developed A . xylosoxidans endophthalmitis 8 months after an uncomplicated cataract extraction performed at another institution . Eventually, vitrectomy with removal of the intraocular lens and capsule was performed because of recurrent disease after intravitreal antibiotic injections . RESULTS: Microbiologic examination of the vitreous biopsies and capsule disclosed A . xylosoxidans, a motile, gram-negative rod resistant to many antibiotics . CONCLUSION: A . xylosoxidans should be considered as a cause of low-grade endophthalmitis after cataract surgery.

Microbiol Mol Biol Rev, 1999 Mar, 63(1), 21 - 53
Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic; Madison LL et al.; Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers . A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways . This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms . Commercial processes for PHA production were initially developed by W . R . Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s . Since the early 1990s, Metabolix Inc . and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States . The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported . The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades . Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism . Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs . This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.

Appl Environ Microbiol, 1999 Mar, 65(3), 946 - 50
Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869; Feng Y et al.; Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively . The estimated molecular mass of the purified P25X gentisate 1, 2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa . Its structure is deduced to be a tetramer . The pI of this enzyme was established to be 4.8 to 5.0 . The subunit mass of P35X gentisate 1, 2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa . The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8 . Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 microM for gentisate, respectively . Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs . Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 x 10(4) s-1 M-1 for the P25X enzyme and 39.34 x 10(4) s-1 M-1 for the P35X enzyme . Higher kcat/Km values were expressed by both enzymes against the substituted gentisates . Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases . The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0 . The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0 . The optimal temperature for both P25X and P35X gentisate 1, 2-dioxygenases was around 50 degrees C, but the P35X enzyme was more heat stable than that from P25X . Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+ . Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

J Biotechnol, 1999 Jan 22, 67(2-3), 113 - 34
Dynamics and modeling on fermentative production of poly (beta-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus; Katoh T et al.; The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor . For the modeling of the effect of NH3 concentration on the cell growth of A . eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods . It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from alpha-ketoglutalate to glutamate when NH3 was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH3 concentration decreased . This phenomenon was reflected in the development of mathematical model . In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L . delbrueckii and the lactate consumption phase due to A . eutrophus . The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level . Several mixed culture experiments were conducted to see the dynamics of the system . Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data . The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.

Adv Biochem Eng Biotechnol, 1999, 63, 1 - 30
Biocatalytic asymmetric decarboxylation; Ohta H; Biocatalytic decarboxylation is a unique reaction, in the sense that it can be considered to be a protonation reaction to a "carbanion equivalent" intermediate in aqueous medium . Thus, if optically active compounds can be prepared via this type of reaction, it would be a very characteristic biotransformation, as compared to ordinary organic reactions . An enzyme isolated from a specific strain of Alcaligenes bronchisepticus catalyzes the asymmetric decarboxylation of alpha-aryl-alpha-methylmalonic acid to give optically active alpha-arylpropionic acids . The effect of additives revealed that this enzyme requires no biotin, no co-enzyme A, and no ATP, as ordinary decarboxylases and transcarboxylases do . Studies on inhibitors of this enzyme and spectroscopic analysis made it clear that the Cys residue plays an essential role in the present reaction . The unique reaction mechanism based on these results and kinetic data in its support are presented.

Appl Environ Microbiol, 1999 Feb, 65(2), 724 - 31
The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp . strain P51, confers the ability to grow on 3-chlorobenzoate; Ogawa N et al.; Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy . Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway . The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp . strain P51, which was isolated in The Netherlands . The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes . Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene . The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols . The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units . Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707 . Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51 . The sequence of the fragment suggested that it might be a remnant of an IS . The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family . The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.

Rev Environ Contam Toxicol, 1999, 159, 1 - 24
Polyhydroxybutyrate: plastic made and degraded by microorganisms; Hankermeyer CR et al.; Polyhydroxybutyrate (PHB) offers many advantages over traditional petrochemically derived plastics . In addition to its complete biodegradability, PHB is formed from renewable resources . It possesses better physical properties than polypropylene for food packaging applications and is completely nontoxic . The poor low-impact strength of PHB is solved by incorporation of hydroxyvalerate monomers into the polymer to produce polyhydroxybutyrate-co-valerate (PHBV), which is commercially marketed under the trade name Biopol . Like PHB, PHBV completely degrades into carbon dioxide and water under aerobic conditions . Microbial synthesis of PHB is the best method for industrial production because it ensures the proper stereochemistry for biodegradation . Microorganisms synthesize and store PHB under nutrient-limited conditions and degrade and metabolize it when the limitation is removed . Current production employs Alcaligenes eutrophus because it grows efficiently on glucose as a carbon source, accumulates PHB up to 80% of its dry weight, and is able to synthesize PHBV when propionic acid is added to the feedstock . PHBV is currently 16 times the price of polypropylene . However, the development of transgenic PHA-producing organisms is expected to greatly reduce its cost . Benefits of using transgenic systems include lack of a depolymerase system, ability to use faster-growing organisms, production of highly purified polymers, and ability to utilize inexpensive carbon sources . Because transgenic plants may someday result in the evolution of plastic crops that could lower the price of PHA to a competitive level, future research will surely focus on such recombinant DNA techniques.

Appl Environ Microbiol, 1999 Jan, 65(1), 131 - 7
Degradation of 3-chlorobenzoate under low-oxygen conditions in pure and mixed cultures of the anoxygenic photoheterotroph Rhodopseudomonas palustris DCP3 and an aerobic Alcaligenes species; Krooneman J et al.; The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds . In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 microM O2 . Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation . Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp . strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur . Under both oxygen limitation (O2 < 0.1 microM) and low oxygen concentrations (3 microM O2), all the 3CBA was metabolized by the aerobic heterotroph . These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R . palustris DCP3 may be restricted to anoxic photic environments.

Arch Microbiol, 1998 Dec, 171(1), 44 - 9
Oxidation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5; Nadeau LJ et al.; Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product . The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes . In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4'- chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization . The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols . 1H-NMR indicated a cis-relative stereochemistry . The results indicate that the biphenyl dioxygenase from A . eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol.

Eur J Clin Microbiol Infect Dis, 1998 Oct, 17(10), 724 - 6
Investigation of an outbreak of wound infections due to Alcaligenes xylosoxidans transmitted by chlorhexidine in a burns unit; Vu-Thien H et al.; Alcaligenes xylosoxidans, an environmental gram-negative bacillus, was isolated within a 1-month period from six patients in a pediatric burns unit . Twelve isolates were studied, one from each of the six patients (five from wound cultures and one from a blood culture) and one from each of six contaminated atomizers containing chlorhexidine diluted to 600 mg/l . The biochemical and susceptibility patterns of all the isolates were similar, and their DNA enzyme restriction patterns were identical . The epidemic strain of Alcaligenes xylosoxidans was probably introduced into the atomizers during handling of the diluted solution, which failed to eliminate it.

Wei Sheng Wu Xue Bao, 1997 Aug, 37(4), 319 - 22
{The experimental study of Pseudomonas contamination in soft drinks}; Zou G et al.; Bymeans of this study, we have found out about the situation of Pseudomons contamination in the soft drinks which are manufactured and sold at the area of Nanchang City . In the course of this study, we examined total 416 specimens, in which we found that 69 specimens were positive reaction . The positive rate made up 16.59% of the total specimens . From these 69 positive specimens we separate isolated andidentified the following Pseudomonas 111 strains including 16 different species: P . aeruginosa, 9; P . fluorescens biovars; 11; P . putida biovars, 14; P . syringae pathovars, 5; P . mendocina, 5; P . alcaligenes, 23; P . pseudoalcaligenes, 10; P . cepacia, 17; P . solanacearum, 1; P . testosteroni, 1; P . delafieldii, 3; P . facilis, 6; P . flava 1; P . psenudoflava, 2; P . palleronii, 1; and a new species of Pseudomonas--P . halosensibilis . The achievement of this study will provide scientific basis for working out the standards of food safety control and inspection, raising the level of food hygieneinspection, and directing the drinks menufacturers and selling units to strengthen the food safety control and inspection.

Appl Environ Microbiol, 1998 Dec, 64(12), 4944 - 9
Detection and isolation of novel rhizopine-catabolizing bacteria from the environment
Gardener BBM, de Bruijn FJ.
Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes . The activity observed generally ranged between 10(6) and 10(7) catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher . A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media . However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30 . Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth . Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set . Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions . Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti . However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.

Appl Environ Microbiol, 1998 Dec, 64(12), 4897 - 903
Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli; Choi JI et al.; Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications . Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria . However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E . coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus . To endow the potentially superior PHA biosynthetic machinery to E . coli, we cloned the PHA biosynthesis genes from A . latus . The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E . coli . Recombinant E . coli strains harboring the A . latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R . eutropha genes . With a pH-stat fed-batch culture of recombinant E . coli harboring a stable plasmid containing the A . latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h . This improvement should allow recombinant E . coli to be used for the production of PHB with a high level of economic competitiveness.

Chemosphere, 1998 Oct-Nov, 37(9-12), 1915 - 22
Biodegradation pathway of 2-chlorodibenzo-p-dioxin and 2-chlorodibenzofuran in the biphenyl-utilising strain JB1; Parsons JR et al.; The biphenyl-utilising Burkholderia (previously Alcaligenes) strain JB1 is also able to degrade a number of chlorinated dibenzo-p-dioxins and dibenzofurans . In this study, 4-chlorocatechol and a chlorotrihydroxydiphenyl ether were identified as metabolites of 2-chlorodibenzo-p-dioxin . 5-Chlorosalicylic acid and a chlorotrihydroxybiphenyl were metabolites of 2-chlorodibenzofuran . These results show that degradation of these compounds follows pathways in which the initial reaction is angular dioxygenation, followed by cleavage of an ether bridge . This pathway is similar to that used by dibenzofuran-degrading strains such as Sphingomonas sp . strain RW1.

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