FEBS Lett, 1998 Nov 6, 438(3), 231 - 5 Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus; Pierik AJ et al.; A third hydrogenase was recently identified in the proteobacterium Alcaligenes eutrophus as a constituent of a novel H2-sensing multicomponent regulatory system . This regulatory hydrogenase (RH) has been overexpressed in cells deficient in both the NAD+-reducing {NiFe}-hydrogenase and the membrane-bound {NiFe}-hydrogenase . EPR, FTIR and activity studies of membrane-free extracts revealed that the RH has an active site much like that of standard {NiFe}-hydrogenases, i.e . a Ni-Fe site with two CN- groups and one CO molecule . Its catalytic power is low, but the RH is always active, insensitive to oxygen, and occurs in only two redox states. J Mol Biol, 1998 Dec 4, 284(3), 751 - 60 Basis for monomer stabilization in Rhodopseudomonas palustris cytochrome c' derived from the crystal structure; Shibata N et al.; The crystal structure of an unusual monomeric cytochrome c' from Rhodopseudomonas palustris (RPCP) has been determined at 2.3 A resolution . RPCP has the four-helix (helices A, B, C and D) bundle structure similar to dimeric cytochromes c' . However the amino acid composition of the surface of helices A and B in RPCP is remarkably different from that of the dimeric cytochromes c' . This surface forms the dimer interface in the latter proteins . RPCP has seven charged residues on this surface contrary to the dimeric cytochromes c', which have only two or three charged groups on the corresponding surface . Moreover, hydrophobic residues on this surface of RPCP are two to three times fewer than in dimeric cytochromes c' . As a result of the difference in amino acid composition, the A-B surface of RPCP is rather hydrophilic compared with dimeric cytochromes c' . We thus suggest that RPCP is monomeric in solution because of the hydrophilic nature of the A-B surface . The amino acid composition of the A-B surface is similar to that of Rhodobacter capsulatus cytochrome c' (RCCP), which is an equilibrium admixture of monomer and dimer . The charge distribution of the A-B surface in RCCP, however, is considerably different from that of RPCP . Due to the difference, RCCP can form dimers by both ionic and hydrophobic interactions . These dimers are quite different from those in proteins which form strong dimers such as in Chromatium vinosum, Rhodospirillum rubrum, Rhodospirillum molischianum and Alcaligenes . Cytochrome c' can be classified into two types . Type 1 cytochromes c' have hydrophobic A-B surfaces and they are globular . The A-B surface of type 2 cytochromes c' is hydrophilic and they take a monomeric or flattened dimeric form . J Biotechnol, 1998 Sep 17, 64(1), 23 - 38 The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production; Gerritse G et al.; Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations . The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids . In order to improve the lipase yield further, the phenotype enhancement method has been developed . This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P . alcaligenes strain with already high lipase productivity . One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes . These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa . Remarkably and dissimilar to P . aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present . For OrfX no equivalent can be found in the known protein data bases . On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK . Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons . A model is proposed for the regulation of the secretion system from P . alcaligenes. J Biotechnol, 1998 Oct 8, 64(2-3), 177 - 86 Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha; Dennis D et al.; The acetoacetyl-CoA reductase and the polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (formerly Alcaligenes eutrophus) were expressed in Escherichia coli, Klebsiella aerogenes, and PHA-negative mutants of R . eutropha and Pseudomonas putida . While expression in E . coli strains resulted in the accumulation of poly(3-hydroxybutyrate) {PHB}, strains of R . eutropha, P . putida and K . aerogenes accumulated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) {poly(3HB-co-3HHx)} when even chain fatty acids were provided as carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) {poly(3HB-co-3HV)} when odd chain fatty acids were provided as carbon source . This suggests that fatty acid degradation can be directly accessed employing only the acetoacetyl-CoA reductase and the PHA synthase . This is also the first proof that the PHA synthase from R . eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described. J Biotechnol, 1998 Oct 8, 64(2-3), 125 - 35 Molecular cloning, sequencing and expression in Escherichia coli of the poly(3-hydroxyalkanoate) synthesis genes from Alcaligenes latus DSM1124; Genser KF et al.; Fragments of chromosomal DNA from Alcaligenes latus DSM1124 were cloned into Escherichia coli and transformants were screened for poly(D(-)-3-hydroxybutyrate) {P(3HB)} production during excess carbon supply . A plasmid harboring a 5.5-kb insert of A . latus DNA was isolated from a P(3HB)-producing bacterial colony . The insert was partially sequenced and three major open reading frames (ORFs) were found, representing the PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) genes . They show striking homology to the Ralstonia eutropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way . The only major difference is the replacement of 560 nucleotides by 160 non-homologous nucleotides in the 5' region of phaC in A . latus . The phaC ORF lacks 29 amino acids at the N-terminus, compared to that of R . eutropha, and starts with a GTG codon . The transcription start points of the operon were determined . P(3HB) production of recombinant E . coli strains harboring the pha operons of A . latus DSM1124 or R . eutropha H16 was investigated . Both operons gave rise to less than 5% P(3HB) formation during exponential growth . At the end of the growth phase, the P(3HB) content reached approximately 20% of cell dry mass . Under nitrogen-depleted conditions, the A . latus pha genes gave rise to 50-52% P(3HB), compared to 33-38% for the R . eutropha pha genes . No NADH oxidase activity was detectable in A . latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth. Plasmid, 1998 Nov, 40(3), 203 - 13 Characterization of the Pac25I restriction-modification genes isolated from the endogenous pRA2 plasmid of Pseudomonas alcaligenes NCIB 9867; Yeo CC et al.; Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-modification (R-M) system, Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2 . The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp . The deduced amino acid sequence of the Pac25I methylase revealed significant similarity with the XcyI, XmaI, Cfr9I, and SmaI methylases . High sequence similarity was displayed between the Pac25I endonuclease and the XcyI, XmaI, and Cfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5'-C CCGGG-3') and are thus perfect isoschizomers . However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5'-CCCGGG-3') . Both the Pac25I methylase and endonuclease were expressed in Escherichia coli . An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of the Pac25I R-M operon . In addition, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes . The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria . CLAO J, 1998 Oct, 24(4), 239 - 41 Alcaligenes xylosoxidans keratitis post penetrating keratoplasty in a rigid gas permeable lens wearer; Lin A et al.; PURPOSE: We report a case of Alcaligenes xylosoxidans keratitis following penetrating keratoplasty in a rigid gas permeable (RGP) lens wearer . METHODS: A 61 year old RGP lens wearer with a history of nonresponsive keratitis of the right eye which involved the graft margin was referred to us for treatment . Corneal cultures revealed growth of a gram-negative rod on the fifth day and the organism was subsequently identified as Alcaligenes xylosoxidans, which was resistant to most antibiotics and sensitive only to Bactrim, Timentin, and imipenem . RESULTS: Clinical improvement was observed within 24 hours after treatment with the use of topical i.v . Bactrim and topical i.v . Timentin 2% alternating every 30 minutes . Complete resolution of the infection with mild scarring was observed 6 weeks after treatment . CONCLUSIONS: Alcaligenes xylosoxidans is a potential cause of bacterial keratitis which should be considered in cases of nonresponsive gram-negative keratitis . The addition of topical Bactrim or Timentin may need to be considered in such cases. Arch Microbiol, 1998 Nov, 170(6), 460 - 3 hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster; Buhrke T et al.; The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined . The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied . The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase . The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected . We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant . The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor . Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX. Arch Microbiol, 1998 Nov, 170(6), 451 - 9 Duplication of hyp genes involved in maturation of {NiFe} hydrogenases in Alcaligenes eutrophus H16; Wolf I et al.; Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1 . Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A . eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies . Mutants with lesions in both copies showed clear alterations in hydrogenase activities . Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H2 . Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium . Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor . Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes . HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms . Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site. J Biochem (Tokyo), 1998 Nov, 124(5), 876 - 9 Type 1 Cu structure of blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 at 2.05 A resolution: comparison of blue and green nitrite reductases; Inoue T et al.; The crystal structure of the blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) has been determined at 2.05 A resolution . AxgNIR contains both type 1 and 2 Cu sites, the geometry of the former being distorted tetrahedral . The superpositioning of the type 1 Cu sites in the blue enzyme and a green nitrite reductase revealed that the orientation of the Met150 side chain differed . The deviation of the Sdelta(Met150) atom from the axial position of the NNS plane formed by two Ndelta(His95 and His145) and one Sgamma(Cys136) atom caused the difference in the colors of the enzymes, i.e . blue and green. FEBS Lett, 1998 Oct 2, 436(2), 239 - 42 The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase; Farver O et al.; The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO) . This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis . The reversible ET reaction proceeds with a rate constant kET = k(1-->2) + k(2-->1) of 450 +/- 30 s(-1) at pH 7.0 and 298 K . The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k(1-->2) = 185 +/- 12 s(-1) and k(2-->1) 265 +/- 18 s(-1) . The temperature dependence of K allowed us to determine the deltaH(o) value of the ET equilibrium to be 12.1 kJ mol(-1) . Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, deltaH* = 22.7 +/- 3.4 kJ mol(-1) and deltaS* = -126 +/- 11 J K(-1) mol(-1); backward reaction, deltaH* = 10.6 +/- 1.7 kJ mol(-1) and deltaS* = -164 +/- 15 J K(-1) mol(-1) . X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion . This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared . The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase. Tsitologiia, 1998, 40(6), 579 - 84 {Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction}; Ponomarev AG et al.; Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean . The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach . Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12474 - 9 A novel multicomponent regulatory system mediates H2 sensing in Alcaligenes eutrophus; Lenz O et al.; Oxidation of molecular hydrogen catalyzed by {NiFe} hydrogenases is a widespread mechanism of energy generation among prokaryotes . Biosynthesis of the H2-oxidizing enzymes is a complex process subject to positive control by H2 and negative control by organic energy sources . In this report we describe a novel signal transduction system regulating hydrogenase gene (hox) expression in the proteobacterium Alcaligenes eutrophus . This multicomponent system consists of the proteins HoxB, HoxC, HoxJ*, and HoxA . HoxB and HoxC share characteristic features of dimeric {NiFe} hydrogenases and form the putative H2 receptor that interacts directly or indirectly with the histidine protein kinase HoxJ* . A single amino acid substitution (HoxJ*G422S) in a conserved C-terminal glycine-rich motif of HoxJ* resulted in a loss of H2-dependent signal transduction and a concomitant block in autophosphorylating activity, suggesting that autokinase activity is essential for the response to H2 . Whereas deletions in hoxB or hoxC abolished hydrogenase synthesis almost completely, the autokinase-deficient strain maintained high-level hox gene expression, indicating that the active sensor kinase exerts a negative effect on hox gene expression in the absence of H2 . Substitutions of the conserved phosphoryl acceptor residue Asp55 in the response regulator HoxA (HoxAD55E and HoxAD55N) disrupted the H2 signal-transduction chain . Unlike other NtrC-like regulators, the altered HoxA proteins still allowed high-level transcriptional activation . The data presented here suggest a model in which the nonphosphorylated form of HoxA stimulates transcription in concert with a yet unknown global energy-responsive factor. Biotechnol Prog, 1998 Sep-Oct, 14(5), 680 - 8 Novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol; Leonard D et al.; The use of a membrane bioreactor with cell retention to achieve high biomass concentrations has been examined for phenol degradation by the bacteria Alcaligenes eutrophus . This process is particularly interesting for toxic substrates as the hydraulic dilution rate and the growth rate are independently controlled . In the case of a transitory excess of phenol, this potentially toxic situation can be overcome by modifying the substrate concentration or the dilution rate without any loss of cells . The injection of a gas phase at the filter inlet increased both the permeate flow rate (by a factor of 1 . 75) and the oxygen transfer capacity (by a factor of 1.5) . This has enabled the cell concentration to reach a maximal value of 60 g L-1 with a hydraulic dilution rate of 0.5 h-1 and a phenol feed concentration of 8 g L-1 . The volumetric productivity of this process corresponds to a phenol degradation rate approaching 100 kg m-3 day-1 . The on-line measurement of the characteristic yellow color of 2-hydroxymuconate semialdehyde, a metabolic intermediate of the phenol degradation pathway, in the permeate provides an interesting basis for process control of phenol supply into the reactor since the color intensity correlates directly to the specific rate of phenol degradation. Lett Appl Microbiol, 1998 Aug, 27(2), 86 - 92 Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration; Andreoni V et al.; A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6,-TCP with stoichiometric release of Cl- . In cultures of Alc . eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation . Experiments conducted with soil microcosms gave evidence that: the degradative process had a biotic nature and was accompanied by microbial growth; the soil used presented an intrinsic degradative capacity versus 2,4,6-TCP; the specialized organism used as inoculum was effective in degrading 2,4,6-TCP in a short time . These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil. FEMS Microbiol Lett, 1998 Aug 15, 165(2), 253 - 60 Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes; Yeo CC et al.; Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563 . Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons . However, no other mer operon genes were found on Tn5563 . Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563. Can J Microbiol, 1998 Jun, 44(6), 554 - 64 The phbC (poly-beta-hydroxybutyrate synthase) gene of Rhizobium (Sinorhizobium) meliloti and characterization of phbC mutants; Willis LB et al.; Defined insertion mutations have been constructed in the Rhizobium (Sinorhizobium) meliloti phbC gene, which encodes poly-beta-hydroxybutyrate (PHB) synthase . The locus was isolated and subcloned from a genomic library of R . meliloti Rm1021 by complementation of phbC mutation of Alcaligenes eutrophus . PHB production was detected in wild-type R . meliloti under nutrient-limited conditions but not in rich medium . No PHB production was detected in the R . meliloti phbC mutants . The DNA sequence of the R . meliloti phbC gene was determined . The deduced polypeptide sequence is homologous to previously identified PhbCs from other bacteria . The R . meliloti phbC locus maps to pRmeSU47a, the smaller of the two megaplasmids in this strain. Am J Nephrol, 1998, 18(5), 452 - 5 Peritoneal dialysis-associated peritonitis caused by Alcaligenes xylosoxidans; El-Shahawy MA et al.; Despite significant progress to decrease its incidence, peritonitis remains the main source of morbidity and treatment failure in patients on continuous ambulatory peritoneal dialysis (CAPD) . The majority of cases of peritonitis result from infection with aerobic gram-positive (Staphylococcus epidermidis and Staphylococcus aureus), or gram-negative organisms . Less common organisms that are also reported include anaerobic bacteria, fungi, and mycobacteria, which collectively account for less than 10% of isolates cultured . We report a case of peritoneal dialysis-associated peritonitis, and review the literature on peritonitis caused by Alcaligenes species . Alcaligenes xylosoxidans is a nonfermenting gram-negative rod and opportunistic pathogen that is motile with peritrichous flagella . The clinical features and microbiological data of our case, as well as the other previously reported cases of peritonitis caused by Alcaligenes species show no particular pattern of peritoneal dialysate cell count . However, the rate of recurrence of peritonitis is characteristically high . The cause of such a high rate of recurrence of peritonitis is probably a reflection of the predilection of Alcaligenes species to cause infection in the 'sicker' patients, and the almost universal resistance of this species to most antimicrobial agents . We, therefore, recommend that catheter removal be undertaken as early as the identification of the organism is made . Whether patients should be allowed to return to CAPD after recovery is a more difficult question . We suggest that a reevaluation of the patient's overall status be undertaken, including personal hygiene, exchange technique, presence of diabetes mellitus, malnutrition, and/or other factors that may render the patient more prone to infection with opportunistic pathogens. Zentralbl Bakteriol, 1998 Jul, 288(1), 145 - 57 Epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans by antibacterial susceptibility testing, fatty acid analysis, PAGE of whole-cell protein and pulsed-field gel electrophoresis; Knippschild M et al.; Antibacterial susceptibility testing, fatty acid analysis, protein analysis and DNA analysis of Alcaligenes xylosoxidans subsp . xylosoxidans were compared to determine the efficiency of the methods available for strain typing . Thirty isolates were investigated: 20 clinical isolates from a nonsocomial outbreak in Essen (Germany), 9 clinical isolates from sporadic nosocomial cases in Paris (France) and reference strain ATCC 2402 . The highest microbiological discriminative power was exhibited by pulsed-field gel electrophoresis (PFGE) yielding nine types, followed by fatty acid methyl ester (FAME) analysis with six types, and antibacterial susceptibility testing and polyacrylamide gel electrophoresis with five types each . By combining the results of the four typing methods, 14 varieties could be differentiated . Protein analysis and fatty acid analysis failed to discriminate between isolates from Essen and Paris and the reference strain, while antibacterial susceptibility testing and DNA analysis clearly discriminated them . It is concluded that a combination of antibacterial susceptibility testing and PFGE typing is most suitable for epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans strains. J Appl Microbiol, 1998 May, 84(5), 859 - 64 Change in bacterial community during biodegradation of aniline; Tani K et al.; The response of river water microbial communities to chemical compounds was monitored under laboratory conditions using aniline as a model . Bacteria were collected from unpolluted and polluted sites . Bacterial abundance (plate and total direct counting) and its relation to aniline biodegradation was examined . Colony hybridization with 16S rRNA oligonucleotide probes was used to study the changes in microbial community structure during biodegradation of aniline . The changes in bacterial abundance and community structure were related to biodegradation of aniline . Burkholderia-Pseudomonas (rRNA group III), an authentic Alcaligenes group became dominant despite the initial differences in the microbial communities, suggesting that these genera are the main aniline degraders in the aquatic environment. Appl Environ Microbiol, 1998 Sep, 64(9), 3437 - 43 Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer; Sudesh K et al.; The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe . Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa) . Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A . eutrophus . The arrangement of PHA biosynthesis genes in C . acidovorans was also similar to that in A . eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa . The cloned fragment complemented a PHA-negative mutant of A . eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source . The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers . The PHA synthase of C . acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate . This leads to the suggestion that in C . acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content. J Clin Microbiol, 1998 Sep, 36(9), 2618 - 22 Phenotypic and genotypic characterization of clinical strains of CDC group IVc-2; Osterhout GJ et al.; CDC group IVc-2 is a gram-negative, oxidase-positive, nonfermentative bacillus that has been implicated in human infections, including septicemia and peritonitis . Biochemically it most closely resembles Bordetella bronchiseptica and Alcaligenes sp . Results of cellular fatty acid (CFA) and 16S rRNA gene analysis were combined with biochemical data to assist in identification and classification . The predominant CFAs were hexadecanoic acid (16:0), cis-9-hexadecanoic acid (16:1omega7c), cis-11-octadecanoic acid (18:1omega7c), and Delta-cis-9,10-methylenehexadecanoic acid (17:0cyc) . Small amounts (2 to 5%) of 3-hydroxytetradecanoic acid (3-OH-14:0), tetradecanoic acid (14:0), 2-hydroxyhexadecanoic acid (2-OH-16:0), and Delta-cis-11,12-methyleneoctadecanoic acid (19:0cyc) were also consistently present . The highest 16S rRNA gene similarity was with Ralstonia eutropha and Ralstonia solanacearum . The CFA and 16S rRNA gene sequence data support the inclusion of CDC group IVc-2 in the recently created genus Ralstonia, which includes R . eutropha, R . pickettii, and R . solanacearum. Microbiology, 1998 Jul, 144 ( Pt 7), 1765 - 72 Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties; Hino S et al.; Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low Ks (the apparent half-saturation constant in Haldane's equation) and an extremely high KSI (the apparent inhibition constant) . To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl- Cat+) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614 . PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase . The cloned genes consist of eight complete ORFs, designated poxRABCDEFG . The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp . CF600, sharing 30-65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme . The kinetic constants for phenol-oxygenating activity of PAO1c(pROE217) were determined, and these were compared with those of strain E2 . The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined . A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines Ks and KSI values for the cellular phenol-oxygenating activity . It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2. Kansenshogaku Zasshi, 1998 Jun, 72(6), 631 - 4 {Case report: subcutaneous abscess and thoracic empyema caused by Alcaligenes xylosoxidans}; Mizunoe S et al.; Alcaligenes xylosoxidans is a glucose-nonfermentative gram-negative rod which usually exists in the environment . This organism while causing pneumonia, sepsis, meningitis and urinary tract infection in the compromised host, rarely causes thoracic empyema . We report a case of thoracic empyema and subcutaneous abscess due to A . xylosoxidans . A 74-year-old male, who had undergone right total pneumonectomy for chronic necrotizing pulmonary aspergillosis a year ago, was admitted to our hospital because of fever . CT scans of the chest revealed a subcutaneous abscess and empyema . Empyema and subcutaneous pus were aspirated . Culture of materials produced A . xylosoxidans . There was no significant change on symptoms and examinations despite therapy with PIPC 4 g/day and thoracic drainage . Finally, surgical treatment was required and the patient was cured. Appl Environ Microbiol, 1998 Aug, 64(8), 3014 - 22 Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments; Wagner-Dobler I et al.; A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities . Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals . After 6 months, stable population densities between 8 x 10(9) and 2 x 10(11) CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates . A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl . Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region . In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences . The R . opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used . In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives . Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R . opacus, independent of the pollution history of the original sample . R . opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation. Arch Microbiol, 1998 Sep, 170(3), 162 - 70 Synechocystis sp . PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria; Hein S et al.; During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp . PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight . Our analysis of the complete Synechocystis sp . PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene . The open reading frame slr1829 was therefore designated as phaE . The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii . The Synechocystis sp . PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus . Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A . eutrophus PHB-4 . These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp . PCC6803 PHA synthase . PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied . Western blot analysis of Synechocystis sp . PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A . eutrophus (PhaC) and of C . vinosum (PhaE and PhaC) revealed no immunoreaction. Mol Microbiol, 1998 Jun, 28(6), 1059 - 66 The extracytoplasmic function sigma factors: role and regulation; Missiakas D et al.; Alternative sigma factors provide a means of regulating gene expression in response to various extracellular changes . One such class of sigma factors appears to control a variety of functions, including expression of heat-shock genes in Escherichia coli, biosynthesis of alginates and carotenoids in Pseudomonas aeruginosa and Myxococcus xanthus, respectively, iron uptake in E . coli and Pseudomonas spp., nickel and cobalt efflux in Alcaligenes europhus, plant pathogenicity in Pseudomonas syringae and synthesis of outer membrane proteins in Photobacterium sp . strain SS9 . Most of these activities deal with extracytoplasmic functions, and such sigmas have been designated as ECF sigma factors . They have also been characterized in Mycobacteria as well as gram-positive bacteria such as Streptomyces coelicolor and Bacillus subtilus and the archaea Sulpholobus acidocaldarius . ECF factors belong to a subfamily of the sigma 70 class, based on their sequence conservation and function across bacterial species . The promoter consensus sequences recognized by the ECF factors are also highly conserved . In most of the cases, the activity of these factors is modulated by a cognate inner membrane protein that has been shown, both in E . coli and in P . aeruginosa, to act as an anti-sigma activity . This inner membrane protein is presumed to serve as a sensor and signalling molecule, allowing an adaptive response to specific environmental change . Presumably, an on-and-off switch of the anti-sigma activity leads to the release of the sigma factor and thereby to the co-ordinate transcription of the specific regulon it governs. Clin Infect Dis, 1998 Jul, 27(1), 158 - 63 Microbiology of sputum from patients at cystic fibrosis centers in the United States; Burns JL et al.; During a phase III national collaborative study of aerosolized tobramycin (1 July 1995 through 30 September 1996), the microbiology of specimens from 595 patients at 69 cystic fibrosis (CF) centers was examined . Samples from three screening visits were processed in a single laboratory by means of standardized techniques for identification and susceptibility testing . From 1,753 pretreatment specimens, 5,128 pathogens were isolated (average, 2.9/specimen) . Of the 3,936 Pseudomonas aeruginosa isolates, 56.7% were mucoid . The specimens of 125 patients (21.0%) yielded tobramycin-resistant P . aeruginosa (213 isolates); 61 (10.3%), Stenotrophomonas maltophilia; and 52 (8.7%), Alcaligenes xylosoxidans . Isolation of Burkholderia cepacia was an exclusion criterion . Only visit 3 sputum samples were cultured for gram-positive organisms and fungi (n = 465 patients); samples from 201 patients (43.2%) yielded Staphylococcus aureus (18.8% of isolates were oxacillin-resistant), and those from 114 (24.5%) yielded an Aspergillus species . Compared with the Cystic Fibrosis Foundation Patient Registry, the current study identified many more patients colonized with S . maltophilia, A . xylosoxidans, Aspergillus species, and oxacillin-resistant S . aureus, suggesting the utility of standardized processing of CF specimens. Biochim Biophys Acta, 1998 May 19, 1384(2), 197 - 203 Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus; Willis LB et al.; A screen for Rhizobium meliloti genes which improve the growth of Alcaligenes eutrophus on sucrose identified the first alcohol dehydrogenase gene (adhA) isolated from the Rhizobiaceae . R . meliloti adhA is constitutively expressed in A . eutrophus and has alcohol dehydrogenase activity . R . meliloti adhA mutants retain some alcohol dehydrogenase activity. Appl Environ Microbiol, 1998 Jul, 64(7), 2644 - 51 Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain; Gerritse G et al.; Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions . A fed-batch fermentation process based on the secretion of the alkaline lipase from P . alcaligenes was developed . Due to the inability of P . alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively . The gene encoding the high-alkaline lipase from P . alcaligenes was isolated and characterized . Amplification of lipase gene copies in P . alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number . It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA . In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only . During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed . Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses . A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss. Appl Environ Microbiol, 1998 Jul, 64(7), 2566 - 71 Aerobic mineralization of 2,6-dichlorophenol by Ralstonia sp . strain RK1; Steinle P et al.; A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France) . It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP)as the sole carbon and energy source at pH 7.5 and room temperature . The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations . At concentrations above 300 microM (50 mg.liter-1), 2,6-DCP increasingly inhibited its own degradation . The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes) . The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers . Substituents other than chlorine prevented degradation . The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors . The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 microM in continuous reactor culture supplied with H2O2 as an oxygen source . Lack of peroxide completely stopped further degradation of 2,6-DCP . Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity . A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 microM) with hydraulic residence times of 8 to 30 min. Appl Environ Microbiol, 1998 Jul, 64(7), 2520 - 7 Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme; Peng X et al.; Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15) . The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway . A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA . Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA . An open reading frame encoding 334 amino acids was identified and designated ligZ . The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y . Noda, S . Nishikawa, K.-I . Shiozuka, H . Kadokura, H . Nakajima, K . Yano, Y . Katayama, N . Morohoshi, T . Haraguchi, and M . Yamasaki, J . Bacteriol . 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M . Kabisch and P . Fortnagel, Nucleic Acids Res . 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp . strain CA10 (S . I . Sato, N . Ouchiyama, T . Kimura, H . Nojiri, H . Yamane, and T . Omori, J . Bacteriol . 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E . L . Spence, M . Kawamukai, J . Sanvoisin, H . Braven, and T . D . H . Bugg, J . Bacteriol . 178:5249-5256, 1996) . The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage . Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase . LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion. Appl Biochem Biotechnol, 1998 Spring, 70-72, 929 - 35 Accumulation of biodegradable copolyesters of 3-hydroxy-butyrate and 3-hydroxyvalerate in Alcaligenes eutrophus; Chua H et al.; Biodegradable copolyesters of 3-hydroxybutyrate-co-3-hydroxyvalerate (3HB-3HV) were produced by Alcaligenes eutrophus in a two-staged process, namely growth stage and nitrogen-deficient polyester-accumulation stage . When C5 was used as the sole carbon source, the copolyester contained 43 mol % of 3HV . A range of copolyesters with 0-43 mol % of 3HV could be produced by using a medium containing different concentration ratios of butyric acid C4 and C5 . Tm of PHB homopolymer was 177.6 degrees C and that of copolyester with highest 3HV mol fraction of 43% was 99.0 degrees C . C5 concentration in the medium could be an effective means to control the polymeric composition and mechanical properties of the copolyesters accumulated in A . eutrophus. Appl Biochem Biotechnol, 1998 Spring, 70-72, 341 - 52 Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila; Umeda F et al.; Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus . H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P . acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA) . In this study, the pha genes of P . acidophila were cloned and a sequence analysis was carried out . A gene library was constructed using the cosmid vector pVK102 . A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16 . The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert . Subcloning was done . and the recombinant plasmid pEH74 was selected by hybridization with the A . eutrophus H16 pha genes . Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P . acidophila . The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined . The homologies of phaA, phaB, and phaC between P . acidophila and A . eutrophus H16 were 64.7, 76.1 and 56.6%, respectively. J Bacteriol, 1998 Jun, 180(12), 3197 - 204 Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes; Schwartz E et al.; Alcaligenes eutrophus H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H2, supplying the organism with energy for autotrophic growth . The promoters of the structural genes for the SH and the MBH, PSH and PMBH, respectively, were identified by means of the primer extension technique . Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under condition which repressed hydrogenase synthesis . The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters . Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system . Both promoters were dependent on the minor sigma factor sigma 54 and on the hydrogenase regulator HoxA in vivo . PSH was stronger than PMBH under both heterotrophic and autotrophic growth conditions . The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures . The response regulator HoxA mediated low-level activation of PSH and PMBH in a heterologous system. Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 791 - 6 Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335; Kang BS et al.; Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde . As our ongoing study to characterize biochemical and genetic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned . The C23O gene was localized in an 1.4-kb PstI fragment from A . eutrophus 335, and was expressed in E . coli HB101 . The C23O exhibited the highest aromatic ring-fission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol >> 4-methylcatechol > 3-methylcatechol, protocatechuate, 4-chlorocatechol > 3,4-dihydroxy-phenylacetate > 2,3-dihydroxybiphenyl . Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading frame (ORF) corresponding to the C23O gene was composed of 930 base pairs . A putative ribosome-binding sequence of AGGAG was found at about 10 nucleotides upstream the ORF which can encode a polypeptide of molecular weight 34 kDa consisting of 309 amino acid residues . The deduced amino acid sequence of C23O from A . eutrophus 335 exhibited the highest 59% identity with those of corresponding enzymes from Pseudomonas sp . CF600 (p VI150), P . putida HS1 (pDK1), and P . putida PpG7 (NAH7) . An alignment of amino acid sequences of extradiol-type dioxygenases including C23O from A . eutrophus 335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution. Am J Infect Control, 1998 Apr, 26(2), 146 - 8 A pseudoepidemic of Alcaligenes xylosoxidans attributable to contaminated saline; Granowitz EV et al.; Alcaligenes xylosoxidans is an uncommon but serious cause of nosocomial epidemics . This report describes a cluster of two patients who underwent revision of hip arthroplasties and one patient who had a lumbar puncture . Cultures obtained during all three procedures showed A . xylosoxidans with similar antibiotic sensitivity patterns . An investigation found that saline used to process these specimens was contaminated with this organism. Plasmid, 1998, 39(3), 187 - 95 IS1491 from Pseudomonas alcaligenes NCIB 9867: characterization and distribution among Pseudomonas species; Yeo CC et al.; A new insertion sequence, IS1491, has been cloned and sequenced . The 2489-bp IS1491 was isolated from a Pseudomonas alcaligenes NCIB 9867 (strain P25X) 4.8-kb PstI chromosomal fragment . IS1491 is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2 . Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21 family of insertion sequences, which include two IS elements previously isolated from P . alcaligenes P25X, IS1474, and IS1475 (Yeo, C . C., and Poh, C . L . (1997) . FEMS Microbiol . Lett . 149, 257-263) . Transposition assays showed that IS1491 transposed at a frequency of approximately 1.4 x 10(-6) . Transposition of IS1491 into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected . IS1491-like sequences were detected in total DNA of Pseudomonas putida NCIB 9869 (strain P35X), Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans, and Comomonas testosteroni by hybridization with IS1491 DNA . Appl Microbiol Biotechnol, 1998 Mar, 49(3), 333 - 6 Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain; Fukui T et al.; The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated . When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A . eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase . In addition, a recombinant strain of A . eutrophus PHB-4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w) . The mole fraction of 3-hydroxyhexanoate units was 4-5 mol% whatever the structure of the triglycerides fed . The polyesters produced by the A . eutrophus strains from olive oil were 200-400 kDa (the number-average molecular mass) . The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A . eutrophus strains. Appl Microbiol Biotechnol, 1998 Mar, 49(3), 258 - 66 In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum; Jossek R et al.; Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaECCv) was used to examine in vitro the specific synthase activity, turnover of R-(-)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions . The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase . Salts (MgCl2, CaCl2, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold . Specific PHA synthase activity was only partially affected by the added components . In general, a higher concentration of salt often inhibited the activity of PhaECCv without affecting the yield according to 3HB-CoA turnover . NAD+ and NADP+ (2 mM) inhibited PhaECCv completely, whereas NADH and NADPH did not . Macroscopic poly(3HB) granules were formed in vitro if PhaECCv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl2 was present . The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus . Scanning electron micrographs from the synthesized granules were obtained . The granules consisted of poly(3HB) that had a molar mass in the range (1-2) x 10(6) g/mol. Mol Cells, 1998 Feb 28, 8(1), 62 - 7 Cloning of genes specifically expressed in rice embryogenic cells; Jung BK et al.; We have examined differences in gene expression pattern between embryogenic callus (EC) and nonembryogenic callus (NEC) derived from mature seed embryo of rice (Oryza sativa L . cv Donggin) . Three EC-specific transcripts were identified by differential display of amplified cDNAs . Specific expression of two partial cDNAs, designated as REC1 and REC2, respectively, was confirmed by a northern blot analysis . Partial nucleotide sequence of the clone REC1 showed no homology with any known genes, but partial amino acid sequence deduced from the clone REC2 exhibited 55-82% homology with nickel-cobalt-resistant proteins identified from a bacterium, Alcaligenes eutrophus CH34. Biochemistry, 1998 Mar 3, 37(9), 3035 - 42 Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid; Saari RE et al.; The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 . This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate . TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine . In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation . The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA . The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases . Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D . Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate . The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions . On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D . The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events. Biochem Mol Biol Int, 1998 Feb, 44(2), 235 - 43 Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes; Etchegaray A et al.; The superfamily of adenylate forming enzymes including peptide synthetases, acyl-CoA synthetases and insect luciferases is readily identified by the signature sequence SGTTGXPKG . This sequence including an invariant lysyl residue is located in a disordered loop region and was predicted to be of significant antigenicity . Antibodies were generated employing YTSGTTGRPKGC attached to bovine serum albumin and have been successfully used to identify respective enzymes and adenylate forming domains in multienzyme systems . These include the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetases of Aspergillus nidulans and Acremonium chrysogenum, gramicidin S synthetase 1 and tyrocidine synthetase 1 from Bacillus brevis, acetyl-CoA synthetase from Alcaligenes eutrophus and a putative peptide synthetase from Metarhizium anisopliae . Weaker or no reactions are observed when the amino acid in position X in the protein is non-basic or hydrophobic, which is respectively the case for gramicidin S synthetase 1 and luciferase. Gene, 1998 Feb 27, 208(2), 243 - 51 Characterisation of the urease gene cluster in Bordetella bronchiseptica; McMillan DJ et al.; Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen . The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes . Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively . The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon . UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv . viciae, and may potentially be involved in nickel transport . A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon . BbuR shares homology with members of the LysR regulatory protein family . LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage . A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B . bronchiseptica . We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B . bronchiseptica from phagolysosomal damage . Comparison of the urease promoter regions of B . bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B . pertussis Tohama I revealed no differences in the ureD open reading frame between each species . A cluster of mutations in both B . pertussis and B . parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter . The inability of B . pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself. J Ind Microbiol Biotechnol, 1998 Jan, 20(1), 61 - 8 Effect of the siderophore alcaligin E on the bioavailability of Cd to Alcaligenes eutrophus CH34; Gilis A et al.; Alcaligin E, the siderophore of the heavy metal-resistant A . eutrophus strain CH34, was shown to interact with Cd and consequently affect its bioavailability and toxicity . The addition of alcaligin E markedly stimulated the growth in the presence of Cd of an alcaligin E-deficient CH34 derivative . Using bioluminescence assays, this effect could be assigned to a decrease in bioavailability of Cd in the presence of alcaligin E . However, Cd-uptake studies showed no influence of alcaligin E on the cellular concentration of Cd . Furthermore, by scanning electron microscopy, the morphology of precipitated Cd crystals was shown to be altered by alcaligin E . These data suggest that alcaligin E, besides its function in iron supply to the cell, provides a protection against heavy metal toxicity . A link between the A . eutrophus CH34 siderophore system and the czc-mediated Cd-efflux system is hypothesized. Gene, 1998 Jan 19, 207(1), 9 - 18 Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10; Bergeron H et al.; The degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde (CAA), a toxic intermediate in the cells if it is not metabolized further by the NAD(+)-dependent CAA dehydrogenases . Here, we describe the cloning, sequence and expression in Escherichia coli of aldA, a plasmid-located CAA dehydrogenase-encoding gene of GJ10 as well as a chromosomal homolog, designated aldB . The DNA-predicted amino acid (aa) sequences of the two proteins (505 aa in AldA and 506 aa in AldB) are 84% identical . The cloned aldA and aldB genes were verified by their expression in the E . coli T7 polymerase/promoter and the pUC lac promoter systems . The expression level of AldA and its enzymatic activity towards CAA were both higher than those of AldB . In a hybrid construct, the 3'end of aldB was able to complement, although not completely, the corresponding portion of aldA to produce a functional gene . Both AldA and AldB proteins of GJ10 share the highest degree of sequence identity with an acetaldehyde dehydrogenase (ALDH) encoded by acoD of Alcaligenes eutrophus (77.3-78% identity) . Together with at least three other ALDHs of prokaryotic origin, these proteins apparently form a special class of ALDHs whose expressions are dependent on RpoN factors . By pulsed-field gel electrophoresis the 225-kb pXAU1 plasmid encoding aldA was shown to be linear. Appl Environ Microbiol, 1998 Mar, 64(3), 930 - 9 Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds; Stoffels M et al.; This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100) . The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source . The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides . Two significant shifts in the bacterial community structure could be demonstrated . The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria . A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor . The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined . Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor . In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis . Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor. J Bacteriol, 1998 Mar, 180(5), 1023 - 9 Subforms and in vitro reconstitution of the NAD-reducing hydrogenase of Alcaligenes eutrophus; Massanz C et al.; The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis . HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer . HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation . A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms . A deletion removing most of hoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide . While the hydrogenase dimer, produced by a strain deleted of hoxF and hoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH . Deletion of hoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity . Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme. Eur J Clin Microbiol Infect Dis, 1997 Dec, 16(12), 933 - 8 Bacteremia and respiratory involvement by Alcaligenes xylosoxidans in patients infected with the human immunodeficiency virus; Manfredi R et al.; Seven cases of Alcaligenes xylosoxidans bacteremia and/or respiratory disease in patients infected with the human immunodeficiency virus (HIV) are described . Reported only thrice previously in this setting, these bacterial complications occurred during different phases of HIV infection and were associated with leukopenia-neutropenia in four patients and a central vascular catheter in two . Although the majority of cases were diagnosed after day 3 of hospitalization, a distinct source of infection was never identified . In four patients with advanced underlying disease, a polymicrobial infection was present . In vitro resistance to aminoglycosides, first-generation cephalosporins, and aztreonam was identified, but treatment with fluoroquinolones, piperacillin, or an aminoglycoside in combination with either ceftazidime or pefloxacin was successful in all cases . The relevance of Alcaligenes xylosoxidans and related species of gram-negative non-glucose fermenting bacilli as opportunistic pathogens in the immunocompromised host and in the setting of HIV infection is briefly reviewed. Arch Microbiol, 1998 Jan, 169(1), 52 - 60 The Alcaligenes eutrophus hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase, is required for heme biosynthesis during anaerobic growth; Lieb C et al.; The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite . When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis . With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment . Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa . In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified . The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN . HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX . Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A . eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector . Likewise, the A . eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth . Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A . eutrophus is nitrate-independent and repressed by oxygen. Arch Microbiol, 1997 Dec, 168(6), 486 - 92 Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase; Henderson RA et al.; Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, DL-lactate or L-lactate . Unlike growing cultures, washed cells excreted significant amounts of pyruvate . The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production . The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA) . Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP . Citrate synthase was purified and shown to be a "large" form of the enzyme (Mr 227,000), comprising a single type of subunit (Mr 47,000) as found in several other gram-negative aerobes . The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed. J Colloid Interface Sci, 1998 Jan 15, 197(2), 185 - 90 Biosorption of Heavy Metal Ions on Rhodobacter sphaeroides and Alcaligenes eutrophus H16 Seki H, Suzuki A, Mitsueda SI. A fundamental study of the application of bacteria to the recovery of toxic heavy metals from aqueous environments was carried out . The biosorption characteristics of cadmium and lead ions were determined with purple nonsulfur bacteria, Rhodobacter sphaeroides and hydrogen bacteria, Alcaligenes eutrophus H16 that were inactivated by steam sterilization . A simplified version of the metal binding model proposed by Plette et al . was used for the description of metal binding data . The results showed that the biosorption of bivalent metal ions to whole cell bodies of the bacteria was due to monodentate binding to two different types of acidic sites: carboxilic and phosphatic-type sites . The number of metal binding sites of A . eutrophus was 2.4-fold larger than that of R . sphaeroides . FEMS Microbiol Lett, 1998 Jan 15, 158(2), 159 - 65 Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region; Kwong SM et al.; The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined . An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length . The pRA2 replication region has two open reading frames (ORFs) . ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end . ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively . The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1. Appl Environ Microbiol, 1998 Feb, 64(2), 453 - 8 Alcaligenes eutrophus as a bacterial chromate sensor; Peitzsch N et al.; In Alcaligenes eutrophus CH34, determinants encoding inducible resistance to chromate (chr) and to cobalt and nickel (cnr) are located adjacent to each other on plasmid pMOL28 . To develop metal-sensing bacterial strains, a cloned part of plasmid pMOL28, which contains both determinants, was mutated with Tn5-lacZ . The chr::lacZ fusions were specifically induced by chromium; cnr was induced best by Ni2+ but was also induced by Co2+, Mn2+, chromate, Cu2+, Cd2+, and Zn2+ . The broad-host-range IncP1 plasmid pEBZ141, which contains a chr::lux fusion, was constructed . A . eutrophus AE104(pEBZ141), carrying a chr::lux transcriptional fusion, could be used as a biosensor for chromate when cultivated in glycerol as an optimal carbon source . Chromate and bichromate were the best inducers; induction by Cr3+ was 10 times lower, and other ions induced only a little or not at all . Interactions among induction of the chr resistance determinant, chromate reduction, chromate accumulation, and the sulfate concentration of the growth medium were demonstrated. Appl Environ Microbiol, 1998 Jan, 64(1), 1 - 6 Repression of phenol catabolism by organic acids in Ralstonia eutropha; Ampe F et al.; During batch growth of Ralstonia eutropha (previously named Alcaligenes eutrophus) on phenol in the presence of acetate, acetate was found to be the preferred substrate; this organic acid was rapidly metabolized, and the specific rate of phenol consumption was considerably decreased, although phenol consumption was not abolished . This decrease corresponded to a drop in phenol hydroxylase and catechol-2,3-dioxygenase specific activities, and the synthesis of the latter was repressed at the transcriptional level . Studies with a mutant not able to consume acetate indicated that the organic acid itself triggers the repression . Other organic acids were also found to repress phenol degradation . One of these, benzoate, was found to completely block the catabolism of phenol (diauxic growth) . A mutant unable to metabolize benzoate was also unable to develop on benzoate-phenol mixtures, indicating that the organic acid rather than a metabolite involved in benzoate degradation was responsible for the repression observed. Gene, 1997 Nov 20, 202(1-2), 103 - 14 Genetic characterization of insertion sequence ISJP4 on plasmid pJP4 from Ralstonia eutropha JMP134; Leveau JH et al.; Directly adjacent to the (tfdT-) tfdCDEF gene cluster for chlorocatechol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134, we identified a 0.9-kb DNA element, designated ISJP4, with the typical features of a bacterial insertion sequence . ISJP4 occurs as a single complete copy on plasmid pJP4 . About 9 kb away from this copy, in the tfdA-tfdS intergenic region, we found a 71-bp duplication of the ISJP4 right-hand extremity . In addition, we discovered a complete copy of ISJP4 on the chromosome of the R . eutropha JMP134 strain that we use routinely in our laboratory . We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R . eutropha JMP222 and JMP289, two previously pJP4-cured derivatives of JMP134 . By comparing both complete copies and their flanking regions, we could establish that element ISJP4 has a size of 915 bp and is bordered by 18-bp inverted repeats with one mismatch . Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of the IS4 family of bacterial insertion sequences, where it is mostly related to IS402 of Burkholderia cepacia . A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromosomal copy of ISJP4 . We could demonstrate that a piece of DNA that is flanked by two complete copies of ISJP4 can be transposed . Even more so, one complete ISJP4 plus its tfdA-tfdS intergenic remnant were sufficient to mediate transposition of intervening DNA . A possible role of ISJP4 in the formation of the tfd pathway genes will be discussed. Appl Environ Microbiol, 1997 Dec, 63(12), 4765 - 9 Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli; Wang F et al.; Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium . One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium . By overexpressing an essential cell division protein, FtsZ, in recombinant E . coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium . A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E . coli in a defined medium . It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production . By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained . For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter . However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E . coli. Mol Cells, 1997 Oct 31, 7(5), 620 - 9 Cloning and characterization of the regulatory genes phlR1 and phlR2 involved in phenol metabolism from Alcaligenes eutrophus JMP134; Kim Y et al.; One mutant (AEK201) of Alcaligenes eutrophus JMP134 deficient in phenol metabolism was isolated by transposon mutagenesis using pSUP2021, a suicide plasmid . The 14.5 kb EcoRI fragment containing Tn5 and flanking DNA was cloned from AEK201 and used to probe a gene bank of wild type by colony hybridization . All five positive cosmids isolated rendered AEK201 to grow on phenol . The data from subcloning revealed that a trans-acting factor encoded on the 2.3 kb SalI-HindIII fragment, which is common to all cosmids, allowed the mutant to restore three enzyme activities tested (phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase) . This fragment seems to act as a positive regulator on the entire phenol pathway . Another regulatory segment was subcloned from the 16.8 kb HindIII fragment on which phenol hydroxylase and catechol 2,3-dioxygenase activities were carried {Kim, Y., Ayoubi, P., and Harker, A . R . (1996) Appl . Environ . Microbiol . 62, 3227-3233} . The expression of phenol hydroxylase activity was entirely repressed in the presence of this segment in Pseudomonas aeruginosa PAO1c, but the enzyme activity was increased in A . eutrophus AEK301, suggesting that this trans-acting factor is both an activator and a repressor for phenol hydroxylase . Possible regulatory mechanisms for the phenol pathway in A . eutrophus JMP134 are discussed. J Bacteriol, 1997 Nov, 179(22), 6871 - 9 New functions for the three subunits of the CzcCBA cation-proton antiporter; Rensing C et al.; The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport . The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit . The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation . Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter . Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins . Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein . Based on these data, a new working model for the function of the Czc system is discussed. Microbiol Res, 1997 Sep, 152(3), 233 - 7 Rapid physiological characterization of microorganisms by biosensor technique; Riedel K et al.; Eleven microorganisms, Arxula adeninivorans LS3, Candida boidinii DSM 70034, Candida lactis-condensi DSM 70635, Pichia jadinii DSM 2361, Pichia minuta DSM 7018, Kluyveromyces lactis DSM 4394, Pseudomonas putida DSM 50026, Alcaligenes sp . DSM 30002, Arthrobacter nicotianae DSM 20123 as well as Issatchenkia orientalis DSM 70077 and Rhodococcus erythropolis DSM 311 were characterized by the sensor technique by injection of 30 different substrates and substrate mixtures . The obtained data which are based on the determination of respiratory rate of microorganisms are similar to physiological characteristics obtained with conventional methods . In comparison to these conventional methods the sensor technique works much more rapid and permits quantification of the data . Therefore, the described technique provides an alternative method for the characterization of microorganisms. FEMS Microbiol Lett, 1997 Oct 15, 155(2), 179 - 84 Bacterial flavohaemoglobins: a consensus sequence and identification of a discrete enterobacterial group and of further bacterial globins; Membrillo-Hernandez J et al.; The amino acid sequences of haemoglobin-like proteins from the bacteria Alcaligenes eutrophus, Bacillus subtilis, Erwinia chrysanthemi, Escherichia coli, Vibrio parahaemolyticus, Vitreoscilla sp . and the yeast Saccharomyces cerevisiae were studied . Phylogenies based on distance and parsimony analysis showed that the eubacterial group can be easily distinguished from the other haemoglobin-like proteins . The construction of a consensus bacterial flavohaemoglobin based on the alignment of six bacterial and one yeast globins allowed the design of consensus primers to search for haemoglobin-like genes in other bacteria . PCR products of the expected size were found in Campylobacter jejuni, Salmonella typhimurium, Listeria monocytogenes, Rhizobium leguminosarum, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Eur J Biochem, 1997 Sep 1, 248(2), 385 - 93 Isolation and characterization of D-threonine aldolase, a pyridoxal-5'-phosphate-dependent enzyme from Arthrobacter sp . DK-38; Kataoka M et al.; D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde . Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonas, and Pseudomonas, but not in yeasts or fungi . The enzyme was purified to homogeneity from one strain, Arthrobacter sp . DK-38 . The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa . This enzyme, as well as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme . Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity . The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation . A divalent cation was also essential for the thermal stability of the enzyme . The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity . The enzyme is strictly D-specific for the alpha-position, whereas it cannot distinguish between threo and erythro forms at the beta-position . Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different; the Km and Vmax values are 3.81 mM and 38.8 micromol x min(-1) x mg(-1) toward D-threonine, and 14.0 mM and 102 micromol x min(-1) x mg(-1) toward D-allothreonine . respectively . The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde . The enzyme also acts, in the same manner, on several other D-beta-hydroxy-alpha-amino acids, including D-beta-phenylserine, D-beta-hydroxy-alpha-aminovaleric acid, D-beta-3,4-dihydroxyphenylserine, and D-beta-3,4-methylenedioxyphenylserine. Diagn Microbiol Infect Dis, 1997 Aug, 28(4), 173 - 8 Comparison of polymerase chain reaction and pulsed-field gel electrophoresis for the epidemiological typing of Alcaligenes xylosoxidans subsp . xylosoxidans in a burn unit; Lin YH et al.; Eighteen isolates of Alcaligenes xylosoxidans subsp . xylosoxidans were collected from clinical specimens of 15 patients in a burn unit and a plastic surgery ward over a 16-month period . Pulsed-field gel electrophoresis and polymerase chain reaction (PCR) were compared for the epidemiologic typing of these 18 isolates and fifteen epidemiologically unrelated strains . These 18 isolates demonstrated an identical fingerprint pattern and were easily distinguished from the 15 epidemiologically unrelated strains by pulsed-field gel electrophoresis typing and both enterobacterial repetitive intergenic concensus and repetitive extragenic palindrome-primed PCR fingerprinting . We conclude that pulsed-field gel electrophoresis analysis of XbaI-digested genomic DNA is a highly discriminatory and reproducible method for epidemiological typing of A . xylosoxidans subsp . xylosoxidans isolates . However, poor resolution due to frequent cutting in the smaller fragments (< 145.5 Kb) may lead to difficulty in interpretation . PCR is a rapid and highly discriminatory, but less reproducible, technique with occasional loss of major bands . The fingerprints produced by repetitive extragenic palindrome primed PCR had more intense bands and were easier to read than those produced by enterobacterial repetitive intergenic concensus-primed PCR in this study. Biodegradation, 1997, 8(2), 113 - 24 Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site; Trajanovska S et al.; Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead . Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate . Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates . Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems . Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes . Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants . These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil . In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates. Plasmid, 1997, 38(2), 129 - 34 Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans; Chakraborty R et al.; Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans . The 1.4-kb fragment hybridized with IST2, an insertion sequence of T . ferrooxidans . The 1.2-kb fragment was cloned and sequenced . The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively . It displays 54.4% identity in 967 nucleotides of overlap with ISAE1 of Alcaligenes eutrophus . The IST445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T . ferrooxidans . In Southern blots of restricted genomic DNAs probed with IST445, each of the several strains of T . ferrooxidans gives a distinctive fingerprint . IST445 is present in the range of 10-20 copies per genome in the four strains studied. J Biotechnol, 1997 Oct 2, 58(1), 33 - 8 Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose; Valentin HE et al.; A recombinant Escherichia coli strain has been developed that produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) when grown in complex medium containing glucose . This has been accomplished by introducing into E . coli DH5 alpha separate plasmids harboring the polyhydroxyalkanoate (PHA) biosynthesis genes from Ralstonia eutropha (formerly named Alcaligenes eutrophus) and the succinate degradation genes from Clostridium kluyveri, respectively . Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels reached 50% of the cell dry weight and contained up to 2.8 mol.% 4-hydroxybutyrate . The molecular weight of the polymer was 1.8 x 10(6). Gene, 1997 Sep 1, 196(1-2), 209 - 18 The cis-diol dehydrogenase cbaC gene of Tn5271 is required for growth on 3-chlorobenzoate but not 3,4-dichlorobenzoate; Nakatsu CH et al.; The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined . The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III . Expression studies were carried out with Alcaligenes sp . strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60 . BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite . These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently . These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite . Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp . BR60 . The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains . o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins . These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III). Gene, 1997 Sep 1, 196(1-2), 69 - 74 Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications; Suarez A et al.; The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria . This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions . To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP . This modified gfp gene was also equipped with the strong translation signals of the atpE gene . Transfer of the mini-transposon into two different Pseudomonas spp . and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye . Individual GFP+ cells were readily observed by fluorescence microscopy. Eur J Biochem, 1997 Aug 15, 248(1), 179 - 86 Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16; Bernhard M et al.; This study shows that the product of the hoxZ gene of Alcaligenes eutrophus H16 is a b-type cytochrome (cytochrome b(z)), which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H2-coupled respiration . The hoxZ product is not required for MBH translocation and H2-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride . The lack of cytochrome b(z) does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZdelta membranes . We show that the hoxZ product is a dihaem cytochrome b (haems with E(m7.0) of +10 mV and +166 mV) involved in H2-dependent electron transfer . We conclude that cytochrome b(z) of the A . eutrophus MBH complex is the link necessary for transfer of electrons from H2 to the ubiquinone pool and that it is required for attachment of MBH to the membrane. FEMS Microbiol Lett, 1997 Sep 1, 154(1), 131 - 7 Monoclonal antibody against species-specific epitope of Pseudomonas aeruginosa Hsp60 protein cross-reacts with Pseudomonas stutzeri and other Pseudomonas species; Luneberg E et al.; In a previous study, we determined the epitope of the Pseudomonas aeruginosa Hsp60 heat shock protein which is recognized by the specific monoclonal antibody (MAb) 2528 . Subsequent investigations revealed a weak cross-reactivity of MAb 2528 with P . stutzeri, P . alcaligenes, P . mendocina and P . pseudoalcaligenes . To elucidate the molecular structure for these cross-reactions, we cloned the P . stutzeri hsp60 gene in Escherichia coli and determined the nucleotide sequence of the gene . In addition, the hsp60 gene of further Pseudomonas species was amplified and sequenced and amino acid substitutions within the epitope recognized by MAb 2528 were determined . The decapeptide QADIEARVLQ is unique to the P . aeruginosa Hsp60 protein, and cross-reaction of MAb 2528 reflects the phylogenetic relationship of Pseudomonas species as P . aeruginosa and all four cross-reacting species constitute a DNA homology group within the rRNA group I of the family Pseudomonadaceae, which belong to the gamma-subclass of the Proteobacteria. J Biol Chem, 1997 Sep 12, 272(37), 23031 - 6 Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli; Eichhorn E et al.; The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation . The sequence relatedness of the TauD protein to the alpha-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an alpha-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J . R., Weiss, M . A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M . A., and Leisinger, T . (1996) J . Bacteriol . 178, 5438-5446) . TauD was overexpressed in E . coli to approximately 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure . The apparent Mr of 81,000 of the native protein and the subunit Mr of 37,400 were consistent with a homodimeric structure . The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine . The reaction also consumed equimolar amounts of oxygen and alpha-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction . The properties and amino acid sequence of this enzyme thus define it as a new member of the alpha-ketoglutarate-dependent dioxygenase family . The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at -20 degrees C for several weeks . Taurine (Km = 55 microM) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates . Among the cosubstrates tested, only alpha-ketoglutarate (Km = 11 microM) supported significant dioxygenase activity. FEMS Microbiol Lett, 1997 Aug 15, 153(2), 411 - 8 Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli; Hein S et al.; The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli . Wild-type strains and other widely used non-recombinant strains of E . coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source . Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase . A 3.5-kb genomic SmaI/ApaI fragment from A . eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively . The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E . coli XL1-Blue . Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources . If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated . The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources . Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E . coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen . 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source . If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents. Microbiology, 1997 Aug, 143 ( Pt 8), 2833 - 40 Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis; Yeo CC et al.; Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol . Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated Xin) which ranged in size from 2 kb to approximately 26 kb instability of DNA inserts in RP4::Xin hybrid plasmids was observed . The smallest insert present in a stable RP4::Xin6 hybrid plasmid, termed Xin6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA . Sequence analysis of Xin6 led to the discovery of an open reading frame with homology to the maturases of group II introns . The Xin6 insert also exhibited several features characteristic of a group II intron . These included the presence of the consensus sequence GUGYG at the 5' and and RAY at the 3' end of the intron . RNA secondary structure modelling of Xin6 also revealed the presence of perfectly conserved domains V and VI . Differences were detected in the Xin6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol . In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the Xin6 group II intron . This is the first reported group II intron isolated from Pseudomonas spp . and the first time that the mobility of a bacterial group II intron has been demonstrated. J Bacteriol, 1997 Aug, 179(15), 4821 - 30 Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae; Fukui T et al.; A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) {P(3HB-co-3HHx)} from alkanoic acids or oils . The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) {i.e., the phaC gene from A . caviae}) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region . The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts . Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A . eutrophus mutant resulted in a decrease in the polyester content of the cells . Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A . caviae . The transconjugant of the A . eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation. J Biol Chem, 1997 Jul 4, 272(27), 17139 - 44 A Ni2+ binding motif is the basis of high affinity transport of the Alcaligenes eutrophus nickel permease; Eitinger T et al.; Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated . Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T . (1995) J . Bacteriol . 177, 1840-1843) . Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake . This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges . Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E . coli system . The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease . These mutations were reintroduced into the A . eutrophus wild-type strain . Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion. Biotechnol Prog, 1997 Jul-Aug, 13(4), 347 - 54 Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures; Hahn JJ et al.; Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells . Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks . Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days) . Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots . However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins . Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture . These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable . A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation . Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA . The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium . This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells. Appl Environ Microbiol, 1997 Jul, 63(7), 2765 - 70 Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate; Ampe F et al.; During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (mu = 0.56 h-1) than when a single substrate was used (mu = 0.51 h-1 for benzoate alone and 0.44 h-1 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol) . Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol.g-1.h-1) . It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the beta-ketoadipate pathway . Indeed, the in vitro beta-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction. Arch Microbiol, 1997 Jul, 168(1), 33 - 8 Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134; Prucha M et al.; An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134