Alcaligenes

Biochem J, 1989 Jul 15, 261(2), 495 - 9
Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J; Ambler RP et al.; Methylomonas J is an obligate methylotroph although it is unable to grow on methane . Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase . When grown on methanol, only the other blue copper protein is produced . We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins . The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences . The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity . The proteins show 52% identity with each other . The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins . Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem . J . (1989) 257, 5.

Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4996 - 9
Gene for the ribulose-1,5-bisphosphate carboxylase small subunit protein of the marine chromophyte Olisthodiscus luteus is similar to that of a chemoautotrophic bacterium; Boczar BA et al.; The photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase {3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39} small subunit protein is encoded by the gene rbcS in the chloroplast genome of the unicellular alga Olisthodiscus luteus . This observation contrasts sharply with that seen in terrestrial plants and green algae, where rbcS is nuclear-localized . In this study, the O . luteus rbcS gene has been sequenced . The predicted primary structure of the protein sequence is 139 amino acids in length and lacks an N-terminal signal sequence . Unexpectedly, the O . luteus rbcS amino acid sequence shows the greatest similarity (56% identity) to that of the chemolithotrophic bacterium Alcaligenes eutrophus . A comparison of the N-terminal amino acid rbcS sequence of A . eutrophus to those of O . luteus and brown alga Fucus species shows extensive sequence similarity (68.3% identity) . This observation suggests that the rbcS genes of these organisms are evolutionary homologues and may provide useful information in the study of small-subunit function.

Rev Infect Dis, 1989 Jul-Aug, 11 Suppl 5, S917 - 24
Laboratory survey of fluoroquinolone activity; Bellido F et al.; Fluoroquinolones are active against a wide variety of bacteria . The antibacterial spectra of fluoroquinolones encompass staphylococci, Bacillus species, and Corynebacterium species implicated in infections of the immunocompromised host; Enterobacteriaceae; most intestinal pathogens; and many gram-negative organisms commonly causing nosocomial infections . Haemophilus influenzae, Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis are highly susceptible to this class of drugs . Because of their ability to penetrate into phagocytes, fluoroquinolones have been tested against intracellular pathogens: Legionella species, Rickettsia conorii, Rickettsia rickettsii, and Brucella melitensis are very sensitive; Chlamydia trachomatis and the mycoplasmas are borderline; and some antimycobacterial activities deserve further investigation . Species that are generally resistant include Pseudomonas maltophilia, Pseudomonas cepacia, Pseudomonas pseudomallei, Alcaligenes species, Nocardia species, Bordetella bronchiseptica, and most anaerobes.

Pneumonol Pol, 1989 Jul-Sep, 57(7-9), 409 - 13
{Sensitivity to cefoperazone (CEFOBID) of microorganisms isolated from diagnostic specimens}; Janowiec M et al.; Sensitivity of cephaperasone (CEFOBID) of 966 straits of microorganisms, isolated from 661 patients was assessed . The study was carried out according to the trial described by Bauer et al, using the Mueller-Hinton medium and original cephaperasone tests (Pfizer) . Full sensitivity was found in 87.1% of the organisms . The following conclusions were made: 1 . Most isolated organisms were sensitivite to cephaperasone (87.1%); E . coli (98.5%), Kl . Pneumoniae (94.8%), Atrobacter (93.1%), Enterobacter (92.8%), Klebsiella sp (92.5%) . Lowered sensitivity was seen in Pseudomonas sp . (70.9%), Acinobacter (70.8%) and Alcaligenes (58.3%) . 2 . Cross-resistance with other cephalosporines was found in 27.8% of the cases . In 52.4% full sensitivity to cephaperasone was found, and resistance to other clinically used cephalosporines.

J Bacteriol, 1989 Jul, 171(7), 4073 - 5
Metal ion uptake by a plasmid-free metal-sensitive Alcaligenes eutrophus strain; Nies DH et al.; The divalent metal cations of zinc, cadmium, cobalt, nickel, and manganese are transported into cells of Alcaligenes eutrophus strain AE104 by the energy-dependent magnesium transport system . Chromate is transported by the sulfate uptake system.

J Mol Biol, 1989 Jun 5, 207(3), 621 - 3
Comparative studies of ribulose-1,5-biphosphate carboxylase/oxygenase from Alcaligenes eutrophus H16 cells, in the active and CABP-inhibited forms; Choe HW et al.; RuBisCO (D-ribulose-1,5-biphosphate carboxylase/oxygenase; EC 4.1.1.39) has been isolated from the autotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 . Combining photon correlation and sedimentation analysis transport parameters of the enzyme were investigated in the active, (E.CO2.Mg2+) as a ternary complex, and inactive state, (E.CO2.Mg2+.CABP) as a quaternary complex, where RuBisCO is complexed with the transition state analogue CABP (2-C-carboxy-D-arabinitol-1,5-biphosphate) . Within experimental error, no difference has been detected between the diffusion and sedimentation coefficients (D020,w = 2.72(+/- 0.07) x 10(-7) cm2 s-1, s020,w = 17.8(+/- 0.5)S) of active and CABP-complexed enzyme thus leading to the conclusion that the molecule, at least in solution, does not assume a different conformation when complexed with CABP.

J Gen Microbiol, 1989 Jun, 135 ( Pt 6), 1479 - 87
Cepabactin from Pseudomonas cepacia, a new type of siderophore; Meyer JM et al.; In iron-deficient conditions of growth Pseudomonas cepacia ATCC 25416 excreted both pyochelin and a low-molecular-mass compound which strongly chelated iron(III), and facilitated iron translocation as demonstrated by growth and uptake experiments . The name cepabactin is proposed for this new siderophore . Comparisons of UV-visible spectra and chromatographic behaviour, together with 1H-NMR spectra, led to the conclusion that cepabactin is 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone, a compound which can be considered as a cyclic hydroxamate, but also as a heterocyclic analogue of catechol . This pyridinone has already been described by other workers as an antibiotic produced by Pseudomonas alcaligenes, and by a soil isolate closely related to Pseudomonas cepacia . Thus, cepabactin appears to act as a siderophore for more than one species of non-fluorescent pseudomonad.

J Bacteriol, 1989 May, 171(5), 2391 - 400
Expressed genes for plant-type ribulose 1,5-bisphosphate carboxylase/oxygenase in the photosynthetic bacterium Chromatium vinosum, which possesses two complete sets of the genes; Viale AM et al.; Two sets of genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were detected in the photosynthetic purple sulfur bacterium Chromatium vinosum by hybridization analysis with RuBisCO gene probes, cloned by using the lambda Fix vector, and designated rbcL-rbcS and rbcA-rbcB . rbcL and rbcA encode the large subunits, and rbcS and rbcB encode the small subunits . rbcL-rbcS was the same as that reported previously (A . M . Viale, H . Kobayashi, T . Takabe, and T . Akazawa, FEBS Lett . 192:283-288, 1985) . A DNA fragment bearing rbcA-rbcB was subcloned in plasmid vectors and sequenced . We found that rbcB was located 177 base pairs downstream of the rbcA coding region, and both genes were preceded by plausible procaryotic ribosome-binding sites . rbcA and rbcD encoded polypeptides of 472 and 118 amino acids, respectively . Edman degradation analysis of the subunits of RuBisCO isolated from C . vinosum showed that rbcA-rbcB encoded the enzyme present in this bacterium . The large- and small-subunit polypeptides were posttranslationally processed to remove 2 and 1 amino acid residues from their N-termini, respectively . Among hetero-oligomeric RuBisCOs, the C . vinosum large subunit exhibited higher homology to that from cyanobacteria, eucaryotic algae, and higher plants (71.6 to 74.2%) than to that from the chemolithotrophic bacterium Alcaligenes eutrophus (56.6%) . A similar situation has been observed for the C . vinosum small subunit, although the homology among small subunits from different organisms was lower than that among the large subunits.

Eur J Biochem, 1989 Apr 15, 181(1), 175 - 80
Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16; Zaborosch C et al.; The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16 . The hydrogenase of N . opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series . A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated . The A . eutrophus enzyme was purified as previously published . Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta) . The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism . Among themselves, the four subunits do not have any serological relationship . The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions . Strong sequence similarities exist between the analogous subunits isolated from the two bacteria . Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively . No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.

Appl Environ Microbiol, 1989 Apr, 55(4), 1037 - 9
Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9; Carney BF et al.; The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P . putida R5-3 parent . Chromosomal DNA of P . alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2 . A single clone from a genomic library of P . alcaligenes C-0 hybridized to EcoRI-digested pKFL3 . Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3 . The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P . alcaligenes into a Tol-like plasmid of P . putida R5-3.

J Bacteriol, 1989 Apr, 171(4), 2096 - 100
Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase; Lee HS et al.; A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity . The purified protein could form channels in black lipid bilayers . Like N2O reductase, NosA contained copper and was only made anaerobically . In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe) . Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P . stutzeri.

J Bacteriol, 1989 Mar, 171(3), 1428 - 34
Microbial degradation of beta-chlorinated four-carbon aliphatic acids; Kohler-Staub D et al.; Alcaligenes sp . strain CC1 is able to grow on several alpha-chlorinated aliphatic acids (2-chlorobutyrate, 2-chloropropionate, and chloroacetate), as well as on the beta-chlorinated four-carbon aliphatic acids trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate as sole carbon and energy sources . Dehalogenation of alpha-chlorinated acids could be measured by using resting cells grown on all the different carbon sources, whereas dehalogenation of beta-chlorinated four-carbon acids could be detected only by using resting cells grown on four-carbon compounds . A constitutive 2-haloacid dehalogenase, which did not show any activity with beta-chlorinated four-carbon acids, was detected in cell extracts . Cell extracts of crotonate-grown cells additionally contained a beta-haloacid dechlorination activity, which acted on trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate and was strictly dependent on coenzyme A, ATP, and Mg2+ . Dechlorination of beta-chlorinated four-carbon acids takes place after activation of the acids to their coenzyme A derivatives and seems to be independent of the constitutive 2-haloacid dehalogenase.

J Bacteriol, 1989 Mar, 171(3), 1340 - 5
Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus; Eberz G et al.; Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized . The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid . Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease . The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A . eutrophus H16 . hoxN is part of the hydrogenase gene cluster . The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium . This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.

J Bacteriol, 1989 Mar, 171(3), 1733 - 5
Sequence similarities in the genes encoding polychlorinated biphenyl degradation by Pseudomonas strain LB400 and Alcaligenes eutrophus H850; Yates JR et al.; DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains . Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability . These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism . No other sequence similarities were detected in the two genomes . DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

J Biol Chem, 1989 Feb 25, 264(6), 3286 - 91
The poly-beta-hydroxybutyrate granule in vivo . A new insight based on NMR spectroscopy of whole cells; Barnard GN et al.; High resolution 13C NMR spectroscopy of live cells has been used to show that poly-beta-hydroxybutyrate (PHB) is predominantly in a mobile state within the storage granules of Alcaligenes eutrophus, Methylobacterium extorquens, and Methylobacterium AM1 . Comparison of chemical and NMR analysis of PHB indicates that about 70% of the polymer in A . eutrophus gives sharp observable resonances . Temperature-dependent line widths and relaxation rates together with nuclear Overhauser effect measurements demonstrate that the observed material is effectively a mobile amorphous elastomer that is well above its glass transition temperature . The hydroxyvalerate-hydroxybutyrate copolymer produced by propionate-fed A . eutrophus has virtually the same mobility as the homopolymer . Evidence is presented indicating that water is an integral component of the PHB granule and that this component acts as a plasticizer for the polymer . These observations strongly suggest that the enzyme(s) responsible for PHB biosynthesis and consumption operate only on mobile hydrated material and that the solid granules characteristic of dried cells are partially artifactual . This model is supported by a reinterpretation of previously inexplicable biochemical results.

J Bacteriol, 1989 Feb, 171(2), 896 - 900
Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus; Nies DH et al.; In Alcaligenes eutrophus CH34, resistance to chromate is plasmid determined, inducible, and based on decreased net accumulation of the metal anion . Plasmid-encoded resistances to zinc, cadmium, cobalt, and nickel are resulting from inducible, energy-dependent cation efflux systems.

J Bacteriol, 1989 Jan, 171(1), 314 - 20
Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR; Harker AR et al.; Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD) . Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4 . Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A . eutrophus AEO106, a cured isolate of JMP134 . AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD . Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate . A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer . This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment . Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD . Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101 . Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101 . This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA . Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

Arch Microbiol, 1989, 152(4), 335 - 41
Homology and distribution of CO dehydrogenase structural genes in carboxydotrophic bacteria; Kraut M et al.; The 17 (S), 30 (M) and 87 kDa (L) subunits of CO dehydrogenases from the CO-oxidizing bacteria Pseudomonas carboxydoflava, Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans OM5 were isolated and purified . The N-terminal sequences of same subunits from different bacteria showed distinct homologies . Dot blot hybridization employing oligonucleotide probes derived from the sequences of the S-subunit of P . carboxydovorans OM5 and the M-subunit of P . carboxydohydrogena and DNA of the plasmid-containing CO-oxidizing bacteria Alcaligenes carboxydus, Azomonas B1, P . carboxydoflava, P . carboxydovorans OM2, OM4 and OM5 indicated that all genes encoding these subunits reside on plasmids . That in P . carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing DNA from the cured mutant strain OM5-12 . CO dehydrogenase structural genes could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, P . carboxydohydrogena and P . carboxydovorans OM3 . There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on the plasmid . The N-terminal sequences of CO dehydrogenase structural genes were found to be conserved among carboxydotrophic bacteria of distinct taxonomic position, independent of the presence of plasmids . It is discussed whether this might be the consequence of horizontal gene transfer.

Arch Biochem Biophys, 1989 Jan, 268(1), 298 - 305
Redox-dependent inactivation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1; Petrov RR et al.; A novel inactivation mechanism of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 comprising redox-dependent steps is described . The model of the hydrogenase inactivation process is proposed which implies that the enzyme may exist in several forms which differ in their stability and spectral properties . One of these forms, existing within a limited (approximately -200 +/- 30 mV) potential range, undergoes a rapid and irreversible inactivation . The dissociation of the FMN prosthetic group from the apohydrogenase appears to be the main reason for the enzyme inactivation . The rationale for the enzyme stabilization under real operational conditions based on the chemical modification of the hydrogenase molecule is suggested.

Arch Biochem Biophys, 1989 Jan, 268(1), 287 - 97
Effect of redox potential on the activation of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1; Petrov RR et al.; A formal kinetic treatment of the autocatalytic activation cycle of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1 is presented . The value for the enzyme first-order activation rate constant is estimated to be (2.0 +/- 0.6) s-1 (pH 7.8, 25 degrees C) . The effect of the redox potential on the activation properties of the NAD-dependent hydrogenase is studied . Hydrogenase activation is controlled by a midpoint redox potential of approximately -100 mV (pH 7.8) . Once activated the enzyme is not immediately transformed back into an inactive state on rapid reoxidation and is able to preserve its catalytic properties for at least 3-4 h of intense oxigenation . Several lines of evidence show that the reductive activation of the NAD-dependent hydrogenase is accompanied by a structural reorganization of the protein . A possible origin of the -100 mV transition is discussed . A model for the activation process of the NAD-dependent hydrogenase is suggested.

Arch Biochem Biophys, 1989 Jan, 268(1), 306 - 13
Effect of redox potential on the catalytic properties of the NAD-dependent hydrogenase from Alcaligenes eutrophus Z1; Petrov RR et al.; The effect of redox potential on the catalytic activities of the soluble hydrogenase from the hydrogen bacterium Alcaligenes eutrophus Z1 was studied . Several transitions were observed on the enzyme catalytic activity vs potential profiles . The coenzyme-dependent activities of the hydrogenase, its diaphorase activity and activity toward NAD, are controlled by the Em -300 mV, while the process of hydrogen evolution from reduced methyl viologen is governed by the midpoint redox potential of -435 mV . This value of Em was independent of pH in the range 5 to 8 . The redox potential of the medium appears to be one of the major factors determining the hydrogenase activation, inactivation, and catalytic properties . It is suggested that a change in the redox state of the enzyme electron transport chain is followed by structural rearrangements within the protein affecting both the hydrogenase catalytic activity and stability . The probable mechanism of enzyme activity regulation is discussed.

Electron Microsc Rev, 1989, 2(1), 139 - 69
D-ribulose-1,5-bisphosphate carboxylase/oxygenase: function-dependent structural changes; Holzenburg A et al.; The key carboxylating enzyme of the reductive pentose phosphate cycle, D-ribulose-1,5-bisphosphate carboxylase/oxygenase {RuBisCO} isolated from the chemolithoautotrophic, H2-oxidizing bacterium Alcaligenes eutrophus H16 has been analyzed by several different techniques that allow conclusions about structure and function-dependent structural changes . The techniques include a novel approach in which the enzyme was induced to form 2D-crystals suitable for electron microscopy in each of its three stable functional states: as active enzyme {Ea} (in the presence of Mg2+ and HCO3-); as inactivated enzyme {Eia} (in the absence of Mg2+ and HCO3-) and as enzyme locked in an in vitro transition state {CABP-E} (Ea fully saturated with the transition state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate {CABP-E}) . In conjunction with X-ray crystallography, X-ray small angle scattering and other biophysical and biochemical data, the results obtained by electron microscopy support the idea that drastic configurational changes occur . Upon transition from Ea to the CABP-E the upper and lower L4S4 halves of the molecule consisting of eight large and eight small subunits (L8S8; MW = 536,000 Da) are assumed to be laterally shifted by as much as 3.6 nm relative to one another while the location of the small subunits on top of the large subunits, and relative to them, remains the same . For the Eia a similar sliding-layer configurational change in the range of 2-2.5 nm is proposed and in addition it is suggested that other configurational/conformational changes take place . The proposed structural changes are discussed with respect to the current model for the tobacco enzyme and correlated with data obtained for various other plant and (cyano) bacterial L8S8 RuBisCOs leading to speculations about structure-function relationships.

J Bacteriol, 1988 Dec, 170(12), 5669 - 72
Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4); Perkins EJ et al.; The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid . tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway . The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R . Streber, K . N . Timmis, and M . H . Zenk, J . Bacteriol . 169:2950-2955, 1987) . A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity . Gas chromatographic analysis of the 2,4-D metabolites of A . eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF . This fragment was further characterized in Escherichia coli by deletion and subcloning analysis . A region of 2.5 kilobases, adjacent to tfdC, enabled E . coli extracts to degrade 2,4-D to 2,4-dichlorophenol . Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.

J Bacteriol, 1988 Dec, 170(12), 5837 - 47
Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli; Schubert P et al.; Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011 . EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks . One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A . eutrophus genomic DNA . In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred . In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1 . All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay . In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected . In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1 . The PHB-synthetic pathway of A . eutrophus was heterologously expressed in Escherichia coli . Recombinant strains of E . coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight . Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase . Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A . eutrophus . In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does . These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1 . The rationale for this mutant type remains unknown.

J Bacteriol, 1988 Nov, 170(11), 5248 - 56
Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions; Jendrossek D et al.; The nucleotide sequence of the gene that encodes the fermentative, multifunctional alcohol dehydrogenase (ADH) in Alcaligenes eutrophus, and of adjacent regions on a 1.8-kilobase-pair PstI fragment was determined . From the deduced amino acid sequence, a molecular weight of 38,549 was calculated for the ADH subunit . The amino acid sequence reveals homologies from 22.3 to 26.3% with zinc-containing alcohol dehydrogenases from eucaryotic organisms (Schizosaccharomyces pombe, Zea mays, mouse, horse liver, and human liver) . Most of the 22 amino acid residues, which are strictly conserved in this group of ADHs (H . Jornvall, B . Persson, and J . Jeffery, Eur . J . Biochem . 167:195-201, 1987), either were present in the A . eutrophus enzyme or had been substituted by related amino acids . The A . eutrophus adh gene was transcribed in Escherichia coli only under the control of the lac promoter, but was not expressed by its own promoter . A sequence resembling the E . coli consensus promoter DNA sequence did not contain the invariant T, but a G, in the potential -10 region . In the transposon-induced mutants HC1409 and HC1421, which form ADH constitutively, the insertions of Tn5::mob were localized 56 and 66 base pairs, respectively, upstream of the presumptive translation initiation codon . In contrast to the promoter, the A . eutrophus ribosome-binding site with a GGAG Shine-Dalgarno sequence 6 base pairs upstream of the translation initiation codon was accepted by the E . coli translation apparatus . A stable hairpin structure, which may provide a transcription termination signal, is predicted to occur in the mRNA, with its starting point 21 base pairs downstream from the translation termination codon.

Gene, 1988 Oct 15, 70(1), 199 - 204
A novel vector allowing the expression of genes in a wide range of gram-negative bacteria; Kozlowski M et al.; The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described . The vector utilizes the regulatory region from IS50 . The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn5 by creating EcoRI and Sa/I or PstI restriction sites by in vitro mutagenesis . This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens . Different strains containing the cat gene behind the regulatory elements of IS50 were able to tolerate high concentrations (300 micrograms/ml) of chloramphenicol in the medium . The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI . Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.

J Bacteriol, 1988 Oct, 170(10), 4431 - 6
Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway; Slater SC et al.; The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli . Initially, an A . eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase . Six enzyme-positive clones were identified . Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB . PHB was produced in the cosmid clones at approximately 50% of the level found in A . eutrophus . One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment . This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E . coli to levels approaching 80% of the bacterial cell dry weight.

J Bacteriol, 1988 Sep, 170(9), 4188 - 93
Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A; Siddiqui RA et al.; The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A . eutrophus N9A . Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail . At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h . When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property . Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM . Two mutants of M220 were isolated which expressed nickel resistance constitutively . When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance . This indicates that the mutation is plasmid located . Both mutants expressed constitutive resistance to nickel and cobalt . Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives . (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM).

Biochem J, 1988 Sep 1, 254(2), 469 - 75
Reversible and irreversible effects of nitric oxide on the soluble hydrogenase from Alcaligenes eutrophus H16; Hyman MR et al.; The effects of NO on the H2-oxidizing and diaphorase activities of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated . With fully activated enzyme, NO (8-150 nM in solution) inhibited H2 oxidation in a time- and NO-concentration-dependent process . Neither H2 nor NAD+ appeared to protect the enzyme against the inhibition . Loss of activity in the absence of an electron acceptor was about 10 times slower than under turnover conditions . The inhibition was partially reversible; approx . 50% of full activity was recoverable after removal of the NO . Recovery was slower in the absence of an electron acceptor than in the presence of H2 plus an electron acceptor . The diaphorase activity of the unactivated hydrogenase was not affected by NO concentrations of up to 200 microM in solution . Exposure of the unactivated hydrogenase to NO irreversibly inhibited the ability of the enzyme to be fully activated for H2-oxidizing activity . The enzyme also lost its ability to respond to H2 during activation in the presence of NADH . The results are interpreted in terms of a complex inhibition that displays elements of (1) a reversible slow-binding inhibition of H2-oxidizing activity, (2) an irreversible effect on H2-oxidizing activity and (30 an irreversible inhibition of a regulatory component of the enzyme . Possible sites of action for NO are discussed.

Biochem J, 1988 Sep 1, 254(2), 463 - 8
Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16; Hyman MR et al.; The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated . Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation . Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2 . The enzyme could also be partly activated either with NADH alone or with H2 alone . The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone . Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration . The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction . Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity . The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase.

J Bacteriol, 1988 Sep, 170(9), 3891 - 6
Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus; Doyle CM et al.; The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied . In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel . Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+ . The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration . Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+ . Thus, all of the added Ni2+ could be accounted for in hydrogenase . Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium . No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase . We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.

J Bacteriol, 1988 Sep, 170(9), 3897 - 902
Isolation and characterization of a new plasmid from a Flavobacterium sp . which carries the genes for degradation of 2,4-dichlorophenoxyacetate; Chaudhry GR et al.; A Flavobacterium sp . (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10 . Cured strains of the Flavobacterium sp . lost the plasmid as well as the ability to degrade these chlorinated compounds . Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids . Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida . From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB) . In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases) . This may be due to the difference in the microbial background during evolution of the two plasmids.

J Clin Microbiol, 1988 Aug, 26(8), 1580 - 1
Alcaligenes piechaudii from chronic ear discharge; Peel MM et al.; A recently described bacterium, Alcaligenes piechaudii, was isolated repeatedly from the ear discharge of a diabetic man . This appears to be the first report of human infection in which there is clinical evidence of a pathogenic role for this species.

Can J Microbiol, 1988 Jun, 34(6), 709 - 15
Gene duplication in haloaromatic degradative plasmids pJP4 and pJP2; Ghosal D et al.; pJP2 and pJP4 are 2,4-dichlorophenoxyacetic acid catabolic plasmids, and they show DNA sequence homology . Most of the pJP2 molecules (80% or more) isolated from 2,4-dichlorophenoxyacetic acid grown cells of Alcaligenes eutrophus harbor a tandem duplication of a 25-kilobase (kb) segment encoding the catabolic functions . Unlike plasmid pJP4, pJP2 in A . eutrophus gives rise to a 3-chlorobenzoate phenotype without further genetic rearrangement . pJP4 under 3-chlorobenzoate selection contains an inverted duplication of 24.5 kb . Absence of selective pressure results in the prompt loss of one copy of the duplication in pJP4, but not of the tandem duplication in pJP2 . In both pJP4 and pJP2, mutation of the duplicated copy, rather than gene dosage, is likely to be the basis of phenotypic change of catabolic functions . Experiments using the cloned DNA suggest that a tandem duplication is more stable than an inverted duplication.

Infection, 1988 May-Jun, 16(3), 194 - 8
Cefetamet pivoxil: bacteriostatic and bactericidal activity of the free acid against 355 gram-negative rods; Hohl P et al.; The in vitro activity of the free acid of cefetamet pivoxil (Ro 15-8075) was tested against 355 clinical isolates, namely enteropathogenic bacteria, glucose non-fermentative gram-negative rods (excluding Pseudomonas aeruginosa) and Legionella pneumophila . Ceftriaxone was included in the study as reference compound . Although the free acid of the orally active cephalosporin was generally weaker than ceftriaxone, it inhibited 88.2% and 94.5% of Enterobacteriaceae and Vibrionaceae at a concentration of 4 mg/l and 8 mg/l or less, respectively . Campylobacter jejuni proved resistant to both compounds . The activity of the new compound against glucose non-fermentative gram-negative rods was generally insufficient to be of promise for broad clinical use . Although the compound was at least twofold more active than ceftriaxone against Pseudomonas acidovorans, Pseudomonas alcaligenes and Pseudomonas cepacia, the former was at least two dilution steps less active than the latter against 14 species of the other less common glucose non-fermentative organisms.

J Appl Bacteriol, 1988 May, 64(5), 451 - 8
Regulation of isofunctional enzymes in Pseudomonas alcaligenes mutants defective in the gentisate pathway; Poh CL et al.; The regulation of the inducible set of gentisate pathway enzymes used by Pseudomonas alcaligenes (P25X1) has been studied in strains derived from mutant strains of P25X1 that had lost the constitutive enzymes that degrade m-cresol, 2,5-xylenol and 3,5-xylenol . The enzyme, 3-hydroxybenzoate 6-hydroxylase II, that catalyzes the oxidation of 3-hydroxybenzoate to gentisate is substrate- and product-induced while gentisate dioxygenase II is substrate induced . Neither 3-hydroxybenzoate nor gentisate could induce the synthesis of maleylpyruvate hydrolase II and fumarylpyruvate hydrolase II . The results suggest that the structural genes encoding these four inducible enzymes and malepylpyruvate hydrolase I (a constitutive enzyme) exist in at least four operons . There is strict induction specificity of expression of this inducible set of gentisate pathway enzymes . 3-Hydroxy-4-methylbenzoate failed to induce whilst 3-hydroxybenzoate and 3-hydroxy-5-methylbenzoate served as inducers of 6-hydroxylase II . Degradation of 2,5-xylenol is mediated by constitutive enzymes whereas the inducible set of enzymes are responsible for the metabolism of m-cresol and 3,5-xylenol.

Pathol Biol (Paris), 1988 Feb, 36(2), 187 - 92
Identification of Pseudomonas, Flavobacterium, and Alcaligenes with the API 20 NE system; Peladan F et al.; The API 20 NE system is designed for the rapid identification of Gram negative rods other than Enterobacteriaceae . It has been compared to conventional methods in the characterization of 404 strains representative of 23 species belonging to 3 genera: Pseudomonas (236 strains), Flavobacterium (133 strains) and Alcaligenes (35 strains) . The system consists of 9 enzymatic tests and 12 carbohydrate assimilation tests . A 7-digit numerical profile is obtained by an octal coding of the test results . The strains are identified by comparing the numerical profile to those of species listed in a profile index . The API 20 NE tests showed 99-100% correlation with corresponding conventional methods except in indole production (96.5%) for which we observed 14 false negative reactions in testing F . meningosepticum and CDC groups IIb and IIf . The API 20 NE system presented 94.1% identification to the species level and 96.3% to the genus level in agreement with conventional biochemical methods . Among the correctly identified strains, 7.4% (30) gave an erroneous or unlisted numerical profile in the first version of the Profile Index and required the use of the computer . Among the misidentified strains 2%, (8) were assigned to the right genus but wrong species, 0.7% (3) were placed in the wrong genus and 1% (4) gave a doubtful profile with one or more tests compared to the profiles listed.

J Bacteriol, 1988 Feb, 170(2), 685 - 92
Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene; Kuhn M et al.; Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis . The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A . eutrophus . Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin . Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1) . Forty mutants were negative for 2,3-butanediol and for acetoin (class 2) . Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16 . Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol . Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3) . The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library . They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank . The gene which encodes the fermentative alcohol dehydrogenase in A . eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment . The gene was heterologously expressed in A . eutrophus JMP222 and in Pseudomonas oxalaticus . The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.

Int J Clin Pharmacol Res, 1988, 8 Suppl 1, 31 - 7
Bacterial species detected in some bottled mineral waters sold in Italy; Macerata UM et al.; A total of 51 samples from 32 different kinds of mineral water were examined to investigate their microbial facies in view of a possible relationship with ionic composition . No difference was found between mineral and oligomineral waters . The genera most represented were Pseudomonas, Flavobacterium and Alcaligenes; only rarely could members of these genera be assigned precisely to a defined species, because many strains showed intermediate patterns . The authors suggest widening the microbiological examination of mineral waters and improving ecological and taxonomic knowledge of the autochthonous species in all processing phases in order to evaluate not only their compliance with the law, but also the variations imposed on microflora by different conditions related to container materials, time and temperature of storage.

Prep Biochem, 1988, 18(3), 351 - 60
Two quick methods for isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase; Jakob R; Methods are described which allow the isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (rubisco) in a very short time . Source of the material was highly impure commercial enzyme in the case of spinach rubisco or bacteria grown from a fermentor in the case of Alcaligenes eutrophus rubisco . Purity of the enzymes is demonstrated by gel electrophoreses . Enzyme isolated from fresh cells gave crystals of excellent diffraction, suitable for X-ray structure analyisis.

Arch Microbiol, 1988, 150(3), 230 - 6
Chlorobenzoate catabolism and interactions between Alcaligenes and Pseudomonas species from Bloody Run Creek; Wyndham RC et al.; A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate . Alcaligenes and Pseudomonas species were dominant in the community . Alcaligenes sp . BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism . Metabolites detected in culture supernatants included chlorocatechol and chloro-cis, cismuconic acid . Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol . The isolate grew very slowly on benzoate . Alcaligenes sp . BR60 was isolated in co-culture with Pseudomonas fluorescens NR52 . The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture . Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation . Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp . BR60 . Under these conditions the growth of Alcaligenes sp . BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites . No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp . BR60 to P . fluorescens NR52.

Ciba Found Symp, 1988, 140, 16 - 31
Microbial hydrolysis of organic nitriles and amides; Ingvorsen K et al.; Nitrile-hydrating enzymes produced by bacteria and fungi catalyse the conversion of a large number of chemically diverse nitriles, including many economically important compounds used industrially for chemical synthesis of amides and acids . This paper presents data on two new, highly different nitrile-hydrolysing enzymes which were isolated in connection with our studies on enzymic nitrile transformations . Particular attention was paid to the enzymes' substrate specificities and sensitivity to substrate/product inhibition . One of our microbial isolates was a Rhodococcus sp . (strain CH5) . This strain produces a constitutive hydratase that has a broad substrate spectrum, including aliphatic and aromatic nitriles, mononitriles and dinitriles, hydroxynitriles and amino-nitriles . It also produces a constitutive amidase of equally low substrate specificity . The hydratase/amidase system catalysed the hydrolysis of D,L-aminonitriles into racemic mixtures of amino acids . Strain CH5 is able to produce high concentrations of malonic acid monoamide from malononitrile and malonamide . The other isolate, Alcaligenes sp . (strain I4), can convert high concentrations of cyanoacetate into malonic acid, presumably by means of an aliphatic nitrilase that is specific for cyanoacetate . Enzyme kinetic experiments have shown that this enzyme is very resistant to both substrate and product inhibition.

Arch Microbiol, 1988, 150(3), 237 - 43
Catabolic instability, plasmid gene deletion and recombination in Alcaligenes sp . BR60; Wyndham RC et al.; An Alcaligenes sp . BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation . The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation . The deletion mutant BR 40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10(-10) cell-1 generation-1 . Transformation or conjugation of pBR60 into cured strains restored catabolic activity . An EcoRI, BglII, HindIII and SalI restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18 . Conjugation of resistance plasmid R68.45 into Alcaligenes sp . BR 60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45 . In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains . Hybridization of deletion region fragments to BglII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants . Hybridization also revealed a repeated sequence flanking the deletion region of pBR60 . Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba+ mutants of Alcaligenes sp . BR60.

Mol Gen Genet, 1988 Jan, 211(1), 113 - 20
Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27; Ghosal D et al.; The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region . We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al . 1985) which encode pyrocatechase and cycloisomerase, respectively . The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC867 (Ghosal et al . 1985a; Frantz and Chakrabarty 1986), was also sequenced . When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences . In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene . The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.

Biochemistry, 1987 Dec 15, 26(25), 8338 - 46
Phase-resolved spectral measurements with several two tryptophan containing proteins; Eftink MR et al.; We have used frequency domain fluorescence techniques to resolve the component emission spectra for several two tryptophan containing proteins (e.g., horse liver alcohol dehydrogenase, sperm whale apomyoglobin, yeast 3-phosphoglycerate kinase, apoazurin from Alcaligenes denitricans) . We have first performed multifrequency phase/modulation measurements and have found the fluorescence of each of these proteins to be described by a double exponential . Then, using phase-sensitive detection and the algorithm of Gratton and Jameson {Gratton, E., & Jameson, D . M . (1985) Anal . Chem . 57, 1694-1697}, we have determined the emission spectrum associated with each decay time for these proteins . We have compared these phase-resolved spectra with the fractional contributions of the component fluorophores determined by selective solute quenching experiments . Reasonably good agreement is seen in most cases, which argues that the individual Trp residues emit independently . In the case of apoazurin, however, a negative amplitude is seen for the phase-resolved spectrum of the short-lifetime component . This pattern is consistent with the occurrence of energy transfer from the internal Trp residue to the surface Trp of this protein . We also present multifrequency lifetime measurements, phase-resolved spectra, and solute quenching data for a few protein-ligand complexes, to illustrate the utility of this approach for the study of changes in the fluorescence of proteins.

Biochim Biophys Acta, 1987 Dec 11, 905(2), 435 - 46
Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain; Podzuweit HG et al.; Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption . For the enzyme of A . eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose . The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45% . During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2 . The hydrogenase of A . eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield . Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0 . They have the same electron acceptor specificity reacting with similar high rates and similar Km values . The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide . Ubiquinones and NAD were not reduced . The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility . For the membrane-bound hydrogenase of A . eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.

Mikrobiologiia, 1987 Nov-Dec, 56(6), 928 - 32
{Purification and study of the properties of a desulfonating enzyme in Pseudomonas alcaligenes}; Stavskaia SS et al.; An enzyme desulfonating anionic alkyl benzene sulfonate (ABS) surfactants was isolated from Pseudomonas alcaligenes TR and purified . The physicochemical and catalytic characteristics of the enzyme were studied . The kinetic constants of ABS desulfonation were determined and shown to depend on the length of a hydrocarbon radical . The molecular mass of the enzyme was found to be close to 60,000.

Appl Environ Microbiol, 1987 Oct, 53(10), 2470 - 5
Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event; Krockel L et al.; Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors . A recombinant strain, P . putida CB1-9, was isolated in less than 1 month . P . alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P . putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P . putida CB1-9 grew on all of these substrates . Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P . alcaligenes C-0 . Chloride was released in stoichiometric amounts when P . putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene . The recombinant strain was related to P . putida R5-3, phenotypically and genetically . Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA . Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates . Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1987 Oct, 169(10), 4463 - 8
Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus; Doyle CM et al.; Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated . Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source . Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h . The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity . This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein . H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase . H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited . Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely . In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.

J Bacteriol, 1987 Oct, 169(10), 4865 - 8
Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34; Nies D et al.; A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34 . After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290 . When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30 . In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.

J Bacteriol, 1987 Oct, 169(10), 4547 - 58
Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit genes and their gene products; Andersen K et al.; The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined . We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites . Cotranscription of the rbcL and rbcS genes has been demonstrated previously . The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively . Both genes exhibited similar codon usage which was highly biased and different from that of other organisms . The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation . No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl . The A . eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants . The A . eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous . The N- and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions . Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A . eutrophus enzyme . The A . eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase . Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P . Y . Chou and G . D . Fasman, Adv . Enzymol . 47:45-148, 1978) revealed striking similarities between A . eutrophus RuBPCase and other hexadecameric enzymes . This suggests that folding of the polypeptide chains is similar . The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the chemoautotroph A . eutrophus and the thus far characterized rbc genes of photosynthetic organisms have a common origin . This suggests that both subunit genes have a very ancient origin . The role of quaternary structure as a determinant of the rate of accepted amino acid substitution was examined . It is proposed that the sequence of the dimeric R . rubrum RuBPCase may be less conserved because there are fewer structural constraints for this RuBPCase than there are for hexadecameric enzymes.

Nature, 1987 Sep 24-30, 329(6137), 354 - 6
Sliding-layer conformational change limited by the quaternary structure of plant RuBisCO; Chapman MS et al.; RuBisCO, D-ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39), converts carbon dioxide to sugar in the first step of photosynthesis . In plants and some bacteria, this enzyme has an L8S8 structure, where L is the large catalytic subunit and S is the small subunit of unknown function . The molecule resembles a keg 105 A along the 4-fold axis and 132 A in diameter at the widest point of the keg . Here we describe the quaternary structure of RuBisCO from N . tabacum, the first L8S8 type known from an X-ray crystallographic study at near-atomic resolution (3 A) . The structure shows that all eight L subunits are elongated along the 4-fold axis so that the molecule cannot be simply described as layers of subunits, as it had been from studies by electron microscopy . The structure, with its elongated and interdigitated L subunits, is evidence against a large, sliding-layer conformational change in plant RuBisCO, as proposed recently in Nature for the same enzyme from Alcaligenes eutrophus.

Eur J Biochem, 1987 Sep 15, 167(3), 541 - 8
Three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity from Alcaligenes eutrophus; Jendrossek D et al.; The existence of three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity was confirmed in Alicaligenes eutrophus . The fermentative alcohol dehydrogenase, which also exhibits acetaldehyde dehydrogenase activity, is one of these proteins . The other two proteins were purified from A . eutrophus N9A mutant AS4 grown on ethanol applying chromatography on DEAE-Sephacel and triazine-dye affinity media . Acetaldehyde dehydrogenase II, which amounts to about 14% of the total soluble protein in cells grown on ethanol, was purified to homogeneity . The relative molecular masses of the native enzyme and of the subunits were 195,000 or 56,000, respectively . This enzyme exhibits a high affinity for acetaldehyde (Km = 4 microM) . Acetaldehyde dehydrogenase I amounts only to less than 1% of the total soluble protein . The relative molecular masses of the native enzyme and of the subunits were 185,000 and 52,000, respectively . This enzyme exhibits a low affinity for acetaldehyde (Km = 2.6 mM) . Antibodies raised against acetaldehyde dehydrogenase II did not react with acetaldehyde dehydrogenase I . Two different strains, A . eutrophus N9A mutant AS1, which represents a different mutant type and can utilize both ethanol or 2,3-butanediol, and the type strain of A . eutrophus (TF93), which can utilize ethanol, form two acetaldehyde dehydrogenases during growth on ethanol, too . As in AS4, one of these enzymes from each strain amounted to a substantial portion of the total soluble protein in the cells . These major acetaldehyde dehydrogenases were purified from both strains; they resemble acetaldehyde dehydrogenase II isolated from AS4 in all relevant properties . Antibodies against the enzyme isolated from AS4 gave identical cross-reactions with the enzymes isolated from AS1 and TF93.

Appl Environ Microbiol, 1987 Aug, 53(8), 1846 - 9
Growth kinetics of Pseudomonas alcaligenes C-0 relative to inoculation and 3-chlorobenzoate metabolism in soil; Focht DD et al.; Pseudomonas alcaligenes C-0 was isolated from activated sewage sludge by enrichment with 3-chlorobenzoate (3CB) as the sole carbon source . The carbon balance from {14C}3CB in pure culture could be accounted for in substrate, biomass, and CO2 from all sampling periods and inoculum densities (0.012, 0.092, 0.20, and 0.92 micrograms of dry cells X ml-1), and inorganic chloride was produced stoichiometrically . Monod parameters as determined in culture were compared with the kinetics of 3CB metabolism in soil with decreasing inoculum densities (1.9 X 10(-1), 1.9 X 10(-3), and 1.9 X 10(-5) micrograms of cells X g-1) . 3CB was refractile to attack in soil by indigenous microflora, but it was completely metabolized upon inoculation with P . alcaligenes C-0 . The saturation constant KS was much higher in soil than in culture, but the yield coefficient Y and the growth rate constant were the same in both systems: mu max = 0.32 h-1; Y = 34 micrograms cells X mumol-1; KS = 0.18 mM in culture and 6.0 mM in soil solution (1.1 mumol X g-1 of soil) . The parameter estimates obtained from the highest inoculum density could be used for the lower inoculum densities with reasonable agreement between predicted and observed 3CB concentrations in soil, although the residual sum of squares was progressively higher . Since the growth rate of P . alcaligenes C-0 in soil was comparable to its growth rate in culture, inoculation should be a viable strategy for biodegradation of 3CB in soil if indigenous microflora are unable to exploit this metabolic niche.

Mol Gen Genet, 1987 Aug, 209(1), 61 - 70
Shuttle transfer (or retrotransfer) of chromosomal markers mediated by plasmid pULB113; Mergeay M et al.; The IncP1 plasmid pULB113 (RP4::miniMu) not only mediates the transfer of chromosomal markers in the classical direction (i.e . from the donor to the recipient cell) but also in the opposite direction (i.e . from the recipient bacterium to the donor) . This phenomenon of retrotransfer was observed in homologous matings with Pseudomonas fluorescens, Alcaligenes eutrophus and Salmonella typhimurium . Retrotransconjugants could be discriminated from direct transconjugants by appropriate chromosomal and plasmid markers used to distinguish the mating partners not bearing pULB113 . Retrotransfer of chromosomal markers occurred at frequencies equal to, or sometimes greater than, those observed for the direct mobilization, thus allowing the recovery of "recipient" recessive markers in the "donor" with linkage values similar to those found in the normal direction . Retrotransfer was also observed in heterospecific matings involving A . eutrophus and pULB113 bearing P . fluorescens: R-primes carrying different selected and unselected markers were recovered in both bacteria . "Retrotransfer" or "shuttle transfer" seems to be a specific trait of IncP1 plasmids.

Hinyokika Kiyo, 1987 Jul, 33(7), 1080 - 91
{Statistical studies on bacteria isolated from urinary tract infections (report 4)}; Okada K et al.; The statistics and drug sensitivity tests of bacterial florae isolated from the urinary tract in 1983 and 1984 were reviewed . Of the 2,222 strains isolated from outpatients, 593 (26.7%) were gram positive cocci, 21.4% were E . coli, 11.3% were Enterococcus, 10.4% were Proteus sp., 10.0% were P . aeruginosa, 5.6% were Alcaligenes sp., 4.2% were S . epidermidis and the rest were others . Of the 507 strains isolated from hospitalized patients, 107 (33.5%) were gram positive cocci, 20.3% were Enterococcus, 16.2% were P . aeruginosa, 9.1% were E . coli, 7.7% were Enterobacter sp . 7.5% were S . epidermidis, 4.9% were Proteus sp., S . marcescence and the rest were others . The percentage of E . coli, K . pneumoniae and S . epidermidis detected in the isolates from the outpatients and that of K . pneumoniae, Proteus sp . and S . epidermidis detected from the inpatients were lower than in previous reports . The percentage of P . aeruginosa and Enterococcus detected in the isolates from both groups of patients were higher than in previous reports . The major isolates (9 species) from the outpatients were more susceptible to the antimicrobial agents tested than those from the inpatients . The susceptibility of gentamicin, tetracycline and nalidixic acid to the major isolates was lower than in previous reports . During the past 2 years, we have been routinely using on inpatients the so-called new generation cefem antibiotics to treat urinary tract infections . This might be why the number of isolates of Enterococcus has increased especially in the isolates from inpatients.

J Bacteriol, 1987 Jul, 169(7), 2950 - 5
Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134; Streber WR et al.; Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) . Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R . H . Don, A . J . Weightman, H.-J . Knackmuss, and K . N . Timmis, J . Bacteriol . 161:85-90, 1985) . Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5 . A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity . The cloned tfdA gene was also transferred to A . eutrophus JMP222, which is a cured derivative of JMP134 . The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy . Pseudomonas sp . strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid . Gene tfdA was subcloned and analyzed by deletions . Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments . A 2-kilobase subfragment containing tfdA has been sequenced . Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.

Mol Gen Genet, 1987 Jun, 208(1-2), 288 - 93
Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli; Jobling MG et al.; A large (greater than 250 kb) conjugative plasmid, pMER610, specifying resistance to tellurium and mercury was isolated from an Alcaligenes strain and transferred by conjugation to Escherichia coli AB1157 . The acquisition of pMER610 by AB1157 increased the resistance to both telurite and tellurate by 100-fold . Expression of tellurite resistance by pMER610 and the cloned Ter determinant was inducible by prior exposure to tellurite at levels sub-toxic to the sensitive AB1157 . Physical analysis of the cloned Ter fragment located the resistance determinant to a 3.55 kb region . Insertion of Tn 1000 (gamma delta) into this region produced two classes of sensitive mutations, fully sensitive and intermediate or hyposensitive, which map in adjacent regions and form two complementation groups . Maxicell analysis identified four polypeptides (15.5, 22, 23 and 41 kDa) expressed by the Ter clone . The 23 kDa polypeptide may not be required for resistance since tellurium-sensitive gamma delta insertion mutations were not detected in the 23 kDa coding region.

J Bacteriol, 1987 May, 169(5), 2079 - 85
Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria; Karst U et al.; In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system . Of these, the B peptide was purified to homogeneity . This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800) . Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis . About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1 . The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200 . The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens . Immunochemical analysis of other species and genera than A . eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens . The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.

Appl Environ Microbiol, 1987 May, 53(5), 1103 - 12
Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850; Bedard DL et al.; Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved . To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp . strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase . H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring . Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone . Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone . The atypical congener selectivity pattern and novel metabolites produced suggest that A . eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.

Appl Environ Microbiol, 1987 May, 53(5), 1094 - 102
Extensive degradation of Aroclors and environmentally transformed polychlorinated biphenyls by Alcaligenes eutrophus H850; Bedard DL et al.; We have isolated and characterized a strain of Alcaligenes eurtrophus, designated H850, that rapidly degrades a broad and unusual spectrum of polychlorinated biphenyls (PCBs) including many tetra- and pentachlorobiphenyls and several hexachlorobiphenyls . This strain, which was isolated from PCB-containing dredge spoils by enrichment on biphenyl, grows well on biphenyl and 2-chlorobiphenyl but poorly on 3- and 4-chlorobiphenyl . Capillary gas-chromatographic analysis showed that biphenyl-grown resting cells of H850 degraded the components of 38 of the 41 largest peaks of Aroclor 1242 and 15 of the 44 largest peaks of Aroclor 1254, resulting in an overall reduction of PCBs by 81% for Aroclor 1242 (10 ppm) and 35% for Aroclor 1254 (10 ppm) in 2 days . Furthermore, H850 metabolized the predominantly ortho-substituted PCB congeners that resulted from the environmental transformation of the more highly chlorinated congeners of Aroclor 1242 by the upper Hudson River anaerobic meta-, para-dechlorination agent system C (J . F . Brown, R . E . Wagner, Jr., D . L . Bedard, M . J . Brennan, J . C . Carnahan, R . J . May, and J . J . Tofflemire, Northeast Environ . Sci . 3:167-179, 1984) . The congener selectivity patterns indicate that a two-step process consisting of anaerobic dechlorination followed by oxidation by H850 can effectively degrade all of the congeners in Aroclor 1242 and possibly all those in Aroclor 1254.

J Bacteriol, 1987 May, 169(5), 1997 - 2004
Genetic and physical mapping and expression in Pseudomonas aeruginosa of the chromosomally encoded ribulose bisphosphate carboxylase genes of Alcaligenes eutrophus; Andersen K et al.; We have previously shown that functional ribulose bisphosphate carboxylase (RuBPCase, rbc) genes in Alcaligenes eutrophus ATCC 17707 are present both on the chromosome and on the indigenous plasmid pAE7 . Here we demonstrate that the chromosomal rbc locus encodes both a large (rbcL)- and a small (rbcS)-subunit gene . A 2.3-kilobase DNA fragment containing both subunit genes was subcloned into the broad-host-range vector pRK310 to yield plasmid pAE312 . This plasmid was transferred into Pseudomonas aeruginosa in which expression of both the rbcL and rbcS genes took place, as demonstrated by Western blot analysis . A high level of RuBPCase activity was observed for P . aeruginosa(pAE312), suggesting that assembly of the subunits took place . Plasmid pAE312 was mutagenized with Tn5 in Escherichia coli . Complementation of A . eutrophus RuBPCase structural gene mutants with pAE312 containing mapped Tn5 insertions allowed functional analysis of the rbc gene region . The polar effect of the Tn5 insertions suggested that the two subunit genes were cotranscribed in A . eutrophus, with rbcL located promoter proximal . Northern blot analysis of total RNA from P . aeruginosa(pAE312) confirmed cotranscription of the two subunit genes . DNA probes containing both the rbcL and rbcS genes, or fragments of each gene, all hybridized to a predominant transcript about 2.1-kilobases long . These observations indicate that the chromosomally encoded rbcL and rbcS genes of A . eutrophus constitute an operon.

Biochem Biophys Res Commun, 1987 Jan 30, 142(2), 297 - 301
NAD-dependent hydrogenase from Alcaligenes eutrophus Z1: does it have a regulatory centre?
Utkin IB, Petrov RR, Egorov AM, Popov VO, Berezin IV.
Evidence is presented for the existence of a relatively high-potential regulatory centre in the NAD-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 . Reduction of the hydrogenase to the redox potentials lower than -100 mV converts the enzyme into a catalytically active state that is remarkably stable to oxidants . Once activated, the enzyme does not loose its activity on intensive oxygenation for at least 3 hours . A novel hydrogenase ESR signal with a wide temperature optimum and a approximately -100 mV midpoint redox potential was detected . We suggest that the reduction of this redox centre trigger conformational changes in the inactive oxidized enzyme molecule, thus reorganizing the latter into the active one.

Folia Microbiol (Praha), 1987, 32(5), 376 - 81
Identification of an inducible penicillinase of the lithoautotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus; Sebo P et al.; The growth of Alcaligenes eutrophus in the presence of benzylpenicillin under heterotrophic and autotrophic conditions was studied . The drug induced a penicillinase in the cells, which can be readily released and extracted from the cells after a lysozyme and EDTA treatment in the course of spheroplast formation . The isoelectric point of the enzyme is 8.1 and the molar mass was estimated to be nearly 25 kg/mol . Phenoxypenicillin is hydrolyzed in the presence of the enzyme at a higher relative rate than benzylpenicillin, ampicillin, amoxycillin and azlocillin . The cephalosporins tested, i.e . cephalosporin C, cefalexin, cefotaxime and 7-aminocephalosporanic acid, were hydrolyzed at a substantially lower relative rate than the penicillins, indicating that the enzyme is a penicillinase.

Arch Microbiol, 1987 Jan, 146(4), 390 - 5
Construction of a transposon containing a gene for polygalacturonate trans-eliminase from Klebsiella oxytoca; Walker MJ et al.; A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5 . This new transposon, designated Tn5-Pga+, had a transposition frequency of 1 X 10(-6) . The broad host range plasmid pR751::Tn5-Pga+ was conjugally transferred to a variety of genetic backgrounds . The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.

Appl Environ Microbiol, 1986 Dec, 52(6), 1374 - 81
Degradation of 1,4-dichlorobenzene by Alcaligenes sp . strain A175; Schraa G et al.; An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source . During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released . Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase . cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene . It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.

J Bacteriol, 1986 Nov, 168(2), 636 - 41
Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16; Eberz G et al.; A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli . hox DNA was identified by screening the E . coli gene bank for restoration of hydrogenase activity in A . eutrophus Hox mutants . Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants . An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment . A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins . hox DNA was subcloned into the vector pVK101 . The resulting recombinant plasmids were used in complementation studies . The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A . eutrophus H16.

Biochem J, 1986 Oct 15, 239(2), 469 - 72
Oxygenase properties of the (4-hydroxybenzoyl)methanol-cleavage enzyme from an Alcaligenes sp; Hopper DJ; Studies using H2(18)O and 18O2 demonstrated that, in the cleavage of (4-hydroxybenzoyl)methanol to 4-hydroxybenzoic and formic acids by an enzyme from an Alicaligenes sp., oxygen is incorporated into both products from dioxygen and not from water . The enzyme is, therefore, an oxygenase.

Appl Environ Microbiol, 1986 Oct, 52(4), 677 - 80
Microbial degradation of 1,3-dichlorobenzene; de Bont JA et al.; A gram-negative, peritrichously flagellated rod, tentatively identified as an Alcaligenes sp., was isolated from a mixture of soil and water samples by using 1,3-dichlorobenzene as the sole carbon and energy source . During growth on 1,3-dichlorobenzene, almost stoichiometric amounts of chloride were released . Simultaneous adaptation studies, as well as enzyme studies, indicated that 1,3-dichlorobenzene was metabolized via 3,5-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene to 3,5-dichlorocatechol . Subsequently, the latter product was cleaved, yielding 2,4-dichloromuconate . No initial hydrolytic step yielding 3-chlorophenol was detected in this species.

J Steroid Biochem, 1986 Sep, 25(3), 403 - 10
A one-step enzymatic assay for the measurement of 17 beta-hydroxy- and 17-oxo-steroid profiles in biological samples; Payne DW et al.; We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures . Analysis of the profiles of individual steroids may be achieved following their chromatographic separation . The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity . The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids . In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues . A recently purified 17 beta-HSD from an Alcaligenes species (D . W . Payne and P . Talalay, J . biol . Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17 beta-hydroxy- and 17-oxo-groups of both C18 and C19 steroids . This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD) . When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme . The linear increase in absorbance with time is a measure of the total concentration of 17 beta-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids . The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography . The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g . 3 alpha-HSD for the measurement of 3 alpha-hydroxy- and 3-oxo-steroids) . The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.

Biochemistry, 1986 Jun 3, 25(11), 3363 - 70
Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones; Tollin G et al.; Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins . Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp . azurin, and Alcaligenes sp . nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times) . Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin . The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin . This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved . The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude) . This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.

Biochem Biophys Res Commun, 1986 May 29, 137(1), 114 - 9
Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus; Yagi K et al.; A gene bank of a megaplasmid encoding the hydrogen-oxidizing enzyme system (Hox) in Alcaligenes hydrogenophilus was constructed using a broad host range cosmid vector pVK102, and established in Escherichia coli . Hybrid cosmids containing hox genes were identified by transferring the bank into Pseudomonas oxalaticus OX1 and screening colonies for the ability of H2-dependent autotrophic growth . About 800 colonies were formed under autotrophic conditions . One of the Hox+ transconjugants was isolated and its hydrogenases activities were measured . Although soluble hydrogenase was not detected, the Hox+ transconjugant had four times the membrane-bound hydrogenase activity of A . hydrogenophilus.

Biochem Biophys Res Commun, 1986 May 29, 137(1), 108 - 13
Conjugal transfer of hydrogen-oxidizing ability of Alcaligenes hydrogenophilus to Pseudomonas oxalaticus; Umeda F et al.; Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined . Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C . The plasmid could be transferred from A . hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H2 and CO2 . Plasmid pHG21-a was maintained in P . oxalaticus OX1 and OX4 as stably as in A . hydrogenophilus.

J Bacteriol, 1986 Apr, 166(1), 319 - 27
Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria; Steinbuchel A; The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E . coli tpi gene . These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity . These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300 . After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E . coli pfkA gene is readily expressed in A . eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway . In contrast, transconjugants of the wild type of A . eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300 . Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed . Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria . The pfkA gene was expressed in most of these species . In addition, most transconjugants of these strains and of A . eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.

Appl Environ Microbiol, 1986 Mar, 51(3), 515 - 20
Investigation of cadmium resistance in an Alcaligenes sp; McEntee JD et al.; The mechanisms of metal resistance of a cadmium-resistant Alcaligenes sp . were studied . Growth in a defined medium was unaffected by cadmium at concentrations up to 0.1 mM, while at concentrations up to 2.5 mM, growth occurred after an extended lag phase . The increase in length of the lag phase was abolished by repeated subculturing at these higher concentrations . However, subculture in the absence of cadmium reversed the adaptation process . Plasmid DNA was not detected in adapted cells, suggesting that adaptation is not plasmid mediated . Increased sulfide production in response to cadmium was observed, although the levels were too low to account fully for cadmium resistance . Adaptation of cells to cadmium resulted in the appearance of a major new membrane protein (molecular weight, 34,500) whose presence was not dependent upon the method of membrane preparation . This protein was induced at cadmium concentrations of 0.1 mM and above, but below this level the protein was absent . The onset of growth at concentrations above 0.1 mM was coincident with the appearance of this protein, which was also induced by zinc (0.4 mM) but not by manganese or nickel . The protein was only solubilized by a sodium dodecyl sulfate-2-mercaptoethanol mixture . Similar solubility properties were shown by a second major membrane protein (molecular weight, 33,000) . These two proteins proved to be similar by peptide-mapping experiments and amino acid analysis . The appearance of the 34,500-molecular-weight protein and its possible role in cadmium resistance are discussed.

J Clin Microbiol, 1986 Mar, 23(3), 647 - 9
Comparison of cross-staining reactions by Pseudomonas spp . and fluorescein-labeled polyclonal and monoclonal antibodies directed against Legionella pneumophila; Tenover FC et al.; Commercially prepared polyclonal antisera to Legionella pneumophila are known to cross-react with organisms of the genus Pseudomonas . To determine whether a commercially available monoclonal antibody reagent specific for L . pneumophila would also cross-react with pseudomonads, a two-laboratory study was undertaken to test both monoclonal and polyclonal reagents against 33 isolates of Pseudomonas spp., including 25 Pseudomonas aeruginosa, 4 P . putida, 2 P . maltophilia, 1 P . fluorescens, and 1 P . alcaligenes . Four antisera were tested; polyclonal anti-legionella antisera pools A and B (Centers for Disease Control {CDC}, Atlanta, (Ga.), polyclonal 1-6 antisera (BioDx, Inc., Denville, N.J.), and a monoclonal antibody reagent produced by Genetic Systems Corp., Seattle, Wash . All reagents were labeled with fluorescein . Cross-staining reactions were found with the BioDx L . pneumophila antisera and 10 isolates of Pseudomonas . Four of these isolates demonstrated cross-staining with CDC pool A . When tested with individual serotype-specific reagents (CDC), three of four cross-reacted with L . pneumophila serotype 1 antisera; the fourth cross-reacted with serotype 3 . No cross-staining reactions were noted with the monoclonal reagent and any of the pseudomonads tested, demonstrating that the Genetic Systems Corp . monoclonal reagent is the most specific of the four reagents tested.

Biochimie, 1986 Jan, 68(1), 63 - 8
NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 . Stabilization against temperature and urea induced inactivation; Popov VO et al.; Chemical modification of the NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 results in considerable enzyme stabilization towards urea and temperature induced inactivation . The stabilizing effect was shown to originate from the suppression of hydrogenase tetramer dissociation . The magnitudes of the stabilizing effects (5-fold and more) were in agreement with the values predicted on the basis of the enzyme thermoinactivation mechanism postulated earlier . Hydrophobic interactions are considered to be critical for the stability of the enzyme quaternary structure . Various methods of hydrogenase immobilization were tested . The enzyme was immobilized with a high retention of activity on aminated silochrom via its carboxylic groups.

Biochimie, 1986 Jan, 68(1), 5 - 13
Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16; Schneider K et al.; The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000 . Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS) . Against each of the 4 subunits, polyclonal antibodies were produced . From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography . By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides . Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits . The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical . A . eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000 . Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochimie, 1986 Jan, 68(1), 15 - 24
Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16; Hornhardt S et al.; The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits . From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity . The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits . In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000 . The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels . It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels . Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c . The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule . As shown by ESR studies, the iron is organized in a {4Fe-4S} cluster but is partially present also in the 3Fe-form . No nickel signal could be detected . The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.

Biochimie, 1986 Jan, 68(1), 103 - 11
Investigation of the H2-oxidizing activities of Alcaligenes eutrophus H16 membranes with artificial electron acceptors, respiratory inhibitors and redox-spectroscopic procedures; Podzuweit HG et al.; Membrane particles, prepared from cells of Alcaligenes eutrophus H16 by lysozyme treatment and 100 000 X g centrifugation, catalyzed a H2-dependent reduction of methylene blue, menadione, 2,6-dichlorophenol-indophenol (DCPIP), and O2 . While the reaction with methylene blue was not altered by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), the H2-dependent reductions of menadione, DCPIP and O2 were strongly inhibited, indicating that in these reaction components of the respiratory chain other than the membrane-bound hydrogenase were involved . The effect of pentane extraction of membranes on the H2-dependent reductions of methylene blue and menadione were different from those of DCPIP and O2 . This suggested that ubiquinone might not be involved in the pathway of the electrons from H2 to methylene blue or menadione, while it might be involved in the pathway to DCPIP and O2 . Because the H2-dependent reduction of menadione is sensitive to HQNO, it follows that HQNO might bind to a site upstream of ubiquinone . Further evidence for this hypothesis came from a new technique to record UV and visible redox-difference spectra of membranes under the conditions of a steady-state electron flow . HQNO did not increase the reduction level of ubiquinone relative to the cytochromes . Neither HQNO nor menadione had any influence on the redox difference patterns of the cytochromes as determined with low temperature and room temperature spectroscopy.

Biochimie, 1986 Jan, 68(1), 85 - 91
Activation and active sites of nickel-containing hydrogenases; Cammack R et al.; Hydrogenases that contain nickel and iron-sulphur clusters also have a regulatory mechanism, by which exposure to oxidants such as oxygen prevents their reaction with hydrogen . Treatment with reducing agents then causes reactivation . In some hydrogenases from Desulfovibrio species, there is evidence that there are at least two different deactivated states, which differ in their rates of reductive reactivation . The membrane-bound hydrogenase of D . desulfuricans, Norway strain, the periplasmic hydrogenase of D . gigas and the membrane-bound hydrogenase of Alcaligenes eutrophus can be isolated in a state (termed "Unready") which requires up to several hours for full activation by hydrogen . By contrast the soluble hydrogenases of D . desulfuricans and A . eutrophus can be reactivated relatively rapidly . In all of these enzymes, with the exception of the latter one, the existence of the activated and deactivated states can be correlated with different ESR-detectable forms of nickel . The possible functions of nickel and {Fe-4S} clusters in catalysis are discussed.

Anal Biochem, 1985 Dec, 151(2), 487 - 94
Difference spectroscopy with respiratory membranes of an H2-oxidizing microorganism layered between two gas-permeable, plastic foils: a procedure that allows measurements in the ultraviolet range; Podzuweit HG et al.; A method for recording redox difference spectra of respiratory membranes which minimizes light scattering and results in increased sensitivity and spectral range when compared to the traditional technique of recording spectra of respiratory particles in suspension is described . Membranes were sandwiched between two gas-permeable, plastic foils, placed in a sealed cuvette, and gassed with H2 as reductant or O2 as oxidant . The membranes of the H2-oxidizing microorganism (Alcaligenes eutrophus H16) used in this work contain a hydrogenase which is coupled to the electron transport chain, thereby allowing H2 to serve as a source of electrons . With this procedure, spectra of both cytochromes and quinones were recorded . By using mixtures of H2 and O2, spectra of cytochromes between the fully oxidized and fully reduced states were recorded.

J Biol Chem, 1985 Nov 5, 260(25), 13648 - 55
Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases . Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp; Payne DW et al.; By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases . Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids . Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase . Unlike any of the other isolated organisms, this Alcaligenes sp . contained no 3 alpha-hydroxysteroid dehydrogenase activity . A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies . The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM) . It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis) . The isoelectric point is pH 5.1 . The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions . Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function . 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates . The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.

J Bacteriol, 1985 Nov, 164(2), 749 - 56
Mathematical model for determining the effects of intracytoplasmic inclusions on volume and density of microorganisms; Mas J et al.; Procaryotic microorganisms accumulate several polymers in the form of intracellular inclusions as a strategy to increase survival in a changing environment . Such inclusions avoid osmotic pressure increases by tightly packaging certain macromolecules into the inclusion . In the present paper, a model describing changes in volume and density of the microbial cell as a function of the weight of the macromolecule forming the inclusion is derived from simple theoretical principles . The model is then tested by linear regression with experimental data from glycogen accumulation in Escherichia coli, poly-beta-hydroxybutyrate accumulation in Alcaligenes eutrophus, and sulfur accumulation in Chromatium spp . The model predicts a certain degree of hydration of the polymer in the inclusion and explains both the linear relationship between volume of the cell and weight of the polymer and the hyperbolic relationship between density of the cell and weight of the polymer . Other implications of the model are also discussed.

J Bacteriol, 1985 Nov, 164(2), 954 - 6
Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus; Klintworth R et al.; Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus . The NH2-terminal sequence of the PRK subunit was determined . With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A . eutrophus H16.

Biochim Biophys Acta, 1985 Oct 18, 831(3), 267 - 74
Identification of heme axial ligands of cytochrome c' from Alcaligenes sp . N.C.I.B . 11015; Yoshimura T et al.; The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp . N.C.I.B . 11015 are reported . The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins . In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state . In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH . EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state . Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values . Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced . The heme environment of this low-spin species is also discussed.

J Biol Chem, 1985 Sep 5, 260(19), 10768 - 70
Single and twinned crystals of ribulose-1,5-bisphosphate carboxylase-oxygenase from Alcaligenes eutrophus; Pal GP et al.; Ribulose-1,5-bisphosphate carboxylase-oxygenase (L8S8) from Alcaligenes eutrophus has been crystallized by equilibrium vapor diffusion techniques with ammonium sulfate as precipitant . Crystals thus obtained either as the ternary complex with CO2 and Mg2+ or as the quaternary complex with CO2, Mg2+, and 2-carboxyarabinitol 1,5-bisphosphate, a transition state analogue, diffract at least to 2.8-A resolution . Both are essentially isomorphous to each other, having orthorhombic space group C222(1) with cell dimensions a = 159 A, b = 159 A, and c = 200 A, and there is half a molecule in the asymmetric unit . The crystals of the ternary complex are sometimes twinned about the c axis so that the space group appears to be tetragonal . In this light, our earlier report (Bowien, B., Mayer, F., Spiess, E., Pahler, A., Englisch, U., and Saenger, W . (1982) Eur . J . Biochem . 106, 405-410) on a tetragonal space group P4(2)2(1)2 with crystals obtained from the same enzyme with Mg2+ and CO2 but without 2-carboxyarabinitol 1,5-bisphosphate might be incorrect.

J Antibiot (Tokyo), 1985 Sep, 38(9), 1117 - 27
Formadicins, new monocyclic beta-lactam antibiotics of bacterial origin . I . Taxonomy, fermentation and biological activities; Katayama N et al.; A Gram-negative bacterium produces new monocyclic beta-lactam antibiotics with a formylamino substituent, named formadicins A, B, C and D . The producing bacterium was taxonomically characterized and designated as Flexibacter alginoliquefaciens sp . nov . YK-49 . Formadicins have narrow antibacterial spectra . They are highly active against some species of Pseudomonas, Proteus and Alcaligenes . Of the four, formadicin C shows the most potent antibacterial activity . Several amino acids such as glycine, D-alanine and D-leucine were antagonistic against formadicins . Formadicins, especially formadicins A and C having the formylamino substituent bound to the 3-position of a beta-lactam nucleus, were highly resistant to hydrolysis by various types of beta-lactamases . Formadicins A and C showed affinity for penicillin-binding proteins (PBPs) 1A and 1B in Pseudomonas aeruginosa IFO 3080, but formadicin B and nocardicin A showed affinity only for PBP 1B . Formadicins A and C did not lyse Escherichia coli LD-2 solely at their MICs, but when combined with mecillinam each induced a rapid lysis of this organism.

J Antimicrob Chemother, 1985 Sep, 16(3), 297 - 304
Purification and properties of a beta-lactamase from Alcaligenes dentrificans subsp . xylosoxydans; Fujii T et al.; A penicillin beta-lactamase was purified from a strain of Alcaligenes dentrificans subsp . xylosoxydans resistant to beta-lactam antibiotics . The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 18,000 from sodium dodecylsulphate-acrylamide gel electrophoresis and gel filtration . Its isoelectric point was 9.8, the optimal pH was 8.5 and the optimal temperature was 35 degrees C . The enzyme hydrolyzed penicillin G and ampicillin more rapidly than cephalosporins . Relative rates, with penicillin G as 100, were: ampicillin, 102; carbenicillin, 15; cloxacillin, less than 1; piperacillin, 9; cephaloridine, 41; cefoperazone, 36; cefpiramide, 36 and cefmenoxime, 14 . Clavulanic acid, sulbactam, imipenem, and cephamycins had low affinities for the enzyme . The enzyme activity was inhibited by iodine, Hg2+, clavulanic acid and sulbactam.

J Bacteriol, 1985 Jul, 163(1), 15 - 20
Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii; Arp DJ et al.; The membrane-bound hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii were purified extensively and compared . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons . The migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except A . vinelandii hydrogenase, which migrated further than the other three . The amino acid composition of each hydrogenase was determined, revealing substantial similarity among these enzymes . This was confirmed by calculation of S delta Q values, which ranged from 8.0 to 26.7 S delta Q units . S delta Q is defined as sigma j(Xi,j-Xk,j)2, where i and k identify the proteins compared and Xj is the content (residues per 100) of a given amino acid of type j . The hydrogenases of this study were also compared with an enzyme-linked immunosorbent assay . Antibody raised against B . japonicum hydrogenase cross-reacted with all four hydrogenases, but to various degrees and in the order B . japonicum greater than A . latus greater than A . eutrophus greater than A . vinelandii . Antibody raised against A . eutrophus hydrogenase also cross-reacted with all four hydrogenases, following the pattern of cross-reaction A . eutrophus greater than A . latus = B . japonicum greater than A . vinelandii . Antibody raised against B . japonicum hydrogenase inhibited B . japonicum hydrogenase activity to a greater extent than the A . eutrophus and A . latus activities; no inhibition of A . vinelandii hydrogenase activity was detected . The results of these experiments indicated remarkable homology of the hydrogenases from these four microorganisms.

J Bacteriol, 1985 Jul, 163(1), 291 - 5
Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene; Carlson CA et al.; Unlike enteric bacteria, Pseudomonas spp . generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo . To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010 . From transformed E . coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes . Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous {2-14C}thymidine into their DNA . Thymidine incorporation into P . stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity . These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.

Appl Environ Microbiol, 1985 May, 49(5), 1237 - 45
Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid; Amy PS et al.; Water samples from rivers, streams, ponds, and activated sewage were tested for the presence of bacteria which utilize 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole source of carbon . Seventy percent of the attempted enrichments yielded pure cultures of 2,4-D-metabolizing bacteria . All but 1 of the 30 isolates were gram-negative rods, all but 2 were motile, and all were nonfermentative and oxidase and catalase positive . Nine isolates had DNA guanine-plus-cytosine values of 61.1 to 65 mol% . One isolate had a 67 mol% guanine-plus-cytosine value . The results suggest that these 2,4-D-metabolizing bacteria belong to the genus Alcaligenes . Fourteen of 23 isolates contained one or more detectable plasmids of about 20, 60, or 100 megadaltons . HindIII restriction fragment patterns showed these plasmids to be different from each other with one exception . Very similar restriction fragment patterns were revealed with a plasmid isolated from an Alcaligenes eutrophus strain obtained from Australia (pJMP397) and in an Alcaligenes sp . isolated in Oregon (pEML159) . These two plasmids were about 56 megadaltons, had the same guanine-plus-cytosine value, were transmissable, and coded for 2,4-D metabolism and resistance to HgCl2 . Hybridization of these two plasmids was demonstrated by using nick-translated 32P-labeled pJMP397 . The vector pBR325 was used to clone HindIII fragments from pEML159 . One cloned fragment of 14.8 megaldaltons expressed in Escherichia coli the ability to release 14CO2 from 2,4-D labeled in the acetate portion.

J Bacteriol, 1985 Apr, 162(1), 328 - 34
Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals; Mergeay M et al.; Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A . eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose . Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance . The plasmids are self-transmissible in homologous matings, but at low frequencies . The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids . The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6 . Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

Arch Biochem Biophys, 1985 Mar, 237(2), 504 - 12
Rhizobium japonicum hydrogenase: purification to homogeneity from soybean nodules, and molecular characterization; Arp DJ; Rhizobium japonicum hydrogenase was purified to homogeneity from soybean root nodules by four column chromatography steps after solubilization from membranes by treatment with a nonionic detergent . The specific activity was from 40 to 65 mumol H2 oxidized min-1 mg protein-1 and was increased 450-fold relative to that in bacteroids . The yield of activity was from 7 to 12% . The molecular weight of the native enzyme was 104,000 as determined by sucrose density gradient centrifugation . Electrophoresis in the presence of sodium dodecyl sulfate revealed two subunits with molecular weights of 64,000 and 35,000, indicating an alpha beta subunit structure . The amino acid content of the protein indicated 20 cysteine residues . Analysis of the metal content indicated 0.59 +/- 0.06 mol Ni/mol hydrogenase and 6.5 +/- 1.2 mol Fe/mol hydrogenase . Antisera prepared to the hydrogenase cross-reacted with the enzyme in bacteroid extracts at all stages of the purification but did not cross-react with extracts of Alcaligenes eutrophus grown under chemolithotrophic conditions . The similarity of rhizobial hydrogenase to the particulate hydrogenases of A . eutrophus and A . latus is discussed.

Proc Natl Acad Sci U S A, 1985 Mar, 82(6), 1638 - 42
Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4; Ghosal D et al.; All of the structural genes for 3-chlorobenzoate degradation are clustered in a 4.2-kilobase (kb) region of plasmid pAC25 (or pAC27) in Pseudomonas putida . An approximate 10-kb DNA segment containing three structural genes for chlorocatechol metabolism present on plasmid pJP4 in Alcaligenes eutrophus shows homology with the above 4.2-kb region of pAC27 . In spite of the detectable sequence homology in the structural genes present on both plasmids, the regulation of their expression seems quite different; unlike pAC27, structural rearrangements are prerequisite for efficient expression of the 3-chlorobenzoate genes on plasmid pJP4 . Structural features such as stem-loop structures present on plasmid pJP4 are most likely the starting materials for such rearrangements.

Vopr Pitan, 1985 Jan-Feb, (1), 55 - 7
{Biochemical quality assessment of broiler production using a hydrogen bacteria biomass in the diet}; Trubachev IN et al.; The authors studied the biochemical composition of the meat of broilers, eggs, liver and muscles of laying hens of 3 generations on a 5, 10, 25, 50 and 100% (broilers), 10 and 20% (laying hens) replacement of the animal protein quota in the diet by protein obtained from the hydrogen bacteria Alcaligenes eutrophus L-1 . No deterioration of the quality of the produce was found from the standpoint of the main biochemical parameters.

J Bacteriol, 1985 Jan, 161(1), 466 - 8
Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4; Don RH et al.; Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate . By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.

Chemotherapy, 1985, 31(3), 181 - 90
The activity of ceftazidime compared with those of aztreonam, newer cephalosporins and Sch 29482 against nonfermentative gram-negative bacilli; Schell RF et al.; The activity of ceftazidime was compared with those of aztreonam, newer cephalosporins and Sch 29482 against nonfermentative gram-negative bacilli . Ceftazidime was consistently more active (MIC less than or equal to 8 micrograms) against the nonfermenters . Only Flavobacterium odoratum, F . spp., Pseudomonas alcaligenes, P . maltophilia and P . stutzeri demonstrated substantial resistance (MIC90 greater than or equal to 64 micrograms) to ceftazidime . Sch 29482 and ceftriaxone also exhibited good activity (MIC90 less than or equal to 8 micrograms) against many of the nonfermenters . The broad activity of ceftazidime, however, makes it a potentially more useful therapeutic agent against these microorganisms.

J Bacteriol, 1985 Jan, 161(1), 85 - 90
Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4); Don RH et al.; Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R . H . Don and J . M . Pemberton, J . Bacteriol . 145:681-686, 1981) . Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways . Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively . No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase . Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols . Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.

Gene, 1984 Dec, 32(1-2), 235 - 41
A portable DNA sequence carrying the cohesive site (cos) of bacteriophage lambda and the mob (mobilization) region of the broad-host-range plasmid RK2: a module for the construction of new cosmids; Selvaraj G et al.; A polylinker DNA sequence carrying the cos site of bacteriophage lambda and the mob (oriT) region of the IncP group plasmid RK2 was constructed . This composite polylinker has EcoRI sites at both termini and also unique sites for ClaI, HindIII, PstI and XbaI . The cos-mob region is portable with the use of EcoRI or a combination of EcoRI with ClaI, HindIII or XbaI . Another cos-mob cassette was also constructed from which the cos-mob region can be lifted with HindIII, ClaI or either of these enzymes in combination with others . These cos-mob cassettes can be used in constructing new cosmids that can be mobilized into a variety of Gram-negative bacteria . Using one of these cassettes we have constructed a small IncW group cosmid (11.1 kb) that was mobilizable into Escherichia coli, Rhizobium spp . and Alcaligenes eutrophus at high frequency.

J Bacteriol, 1984 Sep, 159(3), 973 - 8
Construction and use of a gene bank of Alcaligenes eutrophus in the analysis of ribulose bisphosphate carboxylase genes; Andersen K et al.; A gene bank of the DNA from the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was constructed in the broad host range cosmid vector pVK102 and established in Escherichia coli . A triparental replica plating procedure was developed to allow rapid screening of large numbers of isolated E . coli gene bank clones for complementation of A . eutrophus mutants . This procedure was used to identify hybrid cosmids that complemented CO2 fixation-negative (Cfx-), H2 uptake-negative (Hup-), and auxotrophic A . eutrophus mutants . The average insert DNA size in these hybrid cosmids was 22 kilobases . Nine hybrid cosmids that complemented ribulose bisphosphate carboxylase-negative (RuBPCase-) mutants were characterized . They fell into two distinct groups with respect to their restriction patterns . Complementing subclones from the two groups contained no common restriction fragments, but hybridization experiments indicated a high degree of sequence homology . Restriction fragments corresponding to one of the subclones were absent in total DNA from a cured strain that had lost plasmid pAE7, indigenous to the wild type . It is concluded that functional CO2 fixation genes in the A . eutrophus ATCC 17707 chromosome are reiterated on plasmid pAE7.

J Bacteriol, 1984 Aug, 159(2), 633 - 9
Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions; Kuhn M et al.; When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved . Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h . The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution . Formate is not the source of H2 evolution . The rates of H2 evolution with formate as the substrate were lower than those with gluconate . The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts . Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors . This conclusion is based on the following experimental results . H2 is formed only by cells which had synthesized the hydrogenases during growth . Mutants lacking the membrane-bound hydrogenase were still able to evolve H2 . Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.

J Bacteriol, 1984 Jul, 159(1), 167 - 72
Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus; Schobert P et al.; Phosphoenolpyruvate (PEP) carboxykinase was identified to be the only C3-carboxylating enzyme in Alcaligenes eutrophus . The enzyme requires GDP or inosine diphosphate (GTP or inosine triphosphate) for activity . Pyruvate- and other PEP-dependent CO2-fixing enzyme activities were not detected, regardless of whether the cells were grown autotrophically or heterotrophically . It is suggested that two pathways are present in the organism for the formation of PEP from C4 dicarboxylic acids . Besides decarboxylation of oxaloacetate by PEP carboxykinase, the consecutive action of NADP+-malic enzyme and PEP synthetase can also accomplish this synthesis . An oxaloacetate decarboxylase activity observed in the cell extracts may also contribute to the latter route . The properties of a mutant deficient in PEP synthetase supported the biochemical data . This mutant was unable to grow on pyruvate or lactate and grew slower than the wild type on direct or indirect metabolites of the tricarboxylic acid cycle such as succinate, glutamate, or acetate . Growth on fructose and autotrophic growth were not affected by the enzyme defect . The findings suggest that, depending on the growth substrate utilized, PEP carboxykinase can serve a dual physiological function in A . eutrophus, an anaplerotic function in oxaloacetate synthesis from PEP, or a gluconeogenic function in PEP synthesis from oxaloacetate.

J Bacteriol, 1984 Jul, 159(1), 138 - 44
Regulation by molecular oxygen and organic substrates of hydrogenase synthesis in Alcaligenes eutrophus; Cangelosi GA et al.; Chemoautotrophic growth of Alcaligenes eutrophus 17707 is inhibited by 20% oxygen in the gas phase . Lowering the oxygen concentration to 4% results in chloramphenicol-sensitive derepression of soluble and membrane-bound hydrogenase activity (and of soluble hydrogenase antigen), showing that oxygen inhibition is due at least in part to repression of hydrogenase synthesis . Mutations resulting in derepression of hydrogenase activity (and antigen) under 25% oxygen (Ose-) mobilized with a self-transmissable plasmid which is already known to carry genes necessary for hydrogenase expression . Plasmid-borne mutations resulting in loss of soluble hydrogenase activity have no effect on the Ose phenotype, but chromosomal mutations resulting in reduction or loss of both hydrogenase activities cannot be made Ose- . The Ose- mutation does not alter the thermostability of either hydrogenase, and soluble hydrogenase in the mutant reacts with complete identity with that of the wild type, indicating that the Ose- phenotype does not result from structural alterations in either enzyme . Ose- mutants are also relieved of normal hydrogenase repression by organic substrates, which aggravates hydrogenase-mediated inhibition of heterotrophic growth by hydrogen . Regulation of hydrogenase in Ose- strains of A . eutrophus 17707 is nearly identical to that of wild-type A . eutrophus strains H1 and H16.

Eur J Biochem, 1984 Jun 15, 141(3), 555 - 64
A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties; Steinbuchel A et al.; A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A . Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times . The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose . The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde . During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant . Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation) . The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits . The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme . The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol . This reaction was studied in detail, and its possible involvement in acetate formation is discussed . Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.

Appl Environ Microbiol, 1984 May, 47(5), 993 - 7
Characterization of pseudomonads isolated from diseased fleece; London CJ et al.; A total of 59 Pseudomonas isolates was obtained from 11 samples of diseased fleece taken from live sheep . All but four of the isolates could be assigned to one of nine Pseudomonas species, of which P . aeruginosa, P . alcaligenes, P . mendocina , and P . putida were the most common . P . aeruginosa was found in four of the fleece samples and, when present, appeared to predominate . Although several of the isolates of P . aeruginosa lacked the ability to produce pyocyanin (and some produced neither pyocyanin nor fluorescein), nearly all produced several virulence factors . Of the other pseudomonads, many produced proteinase, esterase, and catalase, several were able to grow at 42 degrees C and reduce nitrate, and some also produced lipase and hemolysin and, like P . aeruginosa, might serve to initiate (or sustain) the dermatitis frequently associated with fleece rot in sheep.

J Biol Chem, 1984 Apr 10, 259(7), 4463 - 5
Small angle x-ray study on the structure of active and inactive ribulose bisphosphate carboxylase from Alcaligenes eutrophus . Evidence for a configurational change; Meisenberger O et al.; Two small angle x-ray scattering curves have been obtained from active and inactive ribulose 1,5-bisphosphate carboxylase from Alcaligenes eutrophus . The radius of gyration was calculated to be R = 47.8 +/- 0.1 nm for the active enzyme and R = 49.2 +/- 0.1 nm for the inactive enzyme . The maximum particle dimension amounts to 13.5 +/- 0.5 nm for the active and 15.7 +/- 0.5 nm for the inactive enzyme . A model of the active carboxylase is presented . It is in good agreement with models derived from electron microscopical data . Model calculations for the inactive enzyme show some evidence for a configurational change.

J Clin Microbiol, 1984 Apr, 19(4), 477 - 81
Differentiation of Alcaligenes-like bacteria of avian origin and comparison with Alcaligenes spp . reference strains; Berkhoff HA et al.; Although standard biochemical tests used for the identification of Alcaligenes spp . revealed only minor differences, the oxidative low-peptone technique clearly differentiated between Alcaligenes-like bacteria of avian origin and Alcaligenes spp . reference strains . Based on their colonial morphology, biochemical profiles, and hemagglutination, the Alcaligenes-like bacteria of avian origin were further divided into two subgroups, C1-T1 and C2-T2 . Colonies of subgroup C1-T1 were nondescript, round, raised, glistening, translucent, greyish, and about 2 mm in diameter . Colonies of subgroup C2-T2 were off-white, flat, dry and wrinkled, generally round, and resembled tiny lily pads . Biochemical profiles by the oxidative low-peptone method showed the C1-T1 subgroup alkalinizing only three substrates (citrate, acetate, and succinate), whereas the C2-T2 subgroup alkalinized eight substrates (citrate, acetate, butyrate, itaconate, malonate, saccharate, succinate, and M-tartrate) . Subgroup C1-T1 agglutinated human, chicken, and turkey erythrocytes, whereas subgroup C2-T2 did not . The recognition of these two subgroups within the Alcaligenes-like bacteria of avian origin is important, since it may explain the differences seen in pathogenicity among isolates.

J Bacteriol, 1984 Apr, 158(1), 79 - 83
Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus; Hughes EJ et al.; A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways . Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway . The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates . Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions . Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes . Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.

J Bacteriol, 1984 Apr, 158(1), 43 - 8
Alcaligenes eutrophus hydrogenase genes (Hox); Hogrefe C et al.; Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen . Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen . The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor . This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1 . The Hox function of one class of Hox- mutants could not be restored by conjugation . These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-) . Nitrate was scarcely utilized as electron acceptor or as nitrogen source . Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character . Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome . Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes . We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics . Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.

J Bacteriol, 1984 Apr, 158(1), 331 - 3
Expression of hydrogenase in Alcaligenes spp . is altered by interspecific plasmid exchange; Friedrich B et al.; Alcaligenes hydrogenophilus was found to contain a soluble and a particulate hydrogenase whose control and structure differed in part from that in Alcaligenes eutrophus . One of at least two plasmids indigenous to A . hydrogenophilus determines hydrogenase genes (Hox) . The interspecific exchange of Hox-encoding plasmids generated transconjugants which expressed the structural and regulatory Hox phenotype of the donor.

J Bacteriol, 1984 Apr, 158(1), 73 - 8
Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway; Hughes EJ et al.; Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway . DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000 . HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO . In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000 . On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000.

Eur J Biochem, 1984 Feb 1, 138(3), 533 - 41
Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1 b; Schneider K et al.; The NAD-reducing hydrogenase of Nocardia opaca 1 b was found to be a soluble, cytoplasmic enzyme . N . opaca 1 b does not contain an additional membrane-bound hydrogenase . The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 mumol H2 oxidized min-1 mg protein-1 . NAD reduction with H2 was completely dependent on the presence of divalent metal ions (Ni2+, Co2+, Mg2+, Mn2+) or of high salt concentrations (0.5-1.5 M) . The most specific effect was caused by NiCl2, whose optimal concentration turned out to be 1 mM . The stimulation of activity by salts was the greater the less chaotrophic the anion . Maximal activity was achieved in 0.5 M potassium phosphate . Hydrogenase was also activated by protons . The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl2 was 7.8-8.0 . In the absence of Ni2+, hydrogenase was only active at pH values below 7.0 . The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5-fold stimulation of the reactions by 0.1-10 microM NiCl2 was observed . With the most effective electron acceptor, benzyl viologen, a 50-fold higher specific activity was determined than with NAD . The total molecular weight of hydrogenase has been estimated to be 200 000 (gel filtration) and 178 000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively . The enzyme is a tetramer consisting of non-identical subunits with molecular weights of 64 000, 56 000, 31 000 and 27 000 . It was demonstrated by electrophoretic analyses that in the absence of NiCl2 and at alkaline pH values the native hydrogenase dissociates into two subunit dimers . The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64 000 and 31 000 . The second dimer was light yellow, inactive with NAD but still active with methyl viologen . It was composed of subunits with molecular weights of 56 000 and 27 000 . Immunological comparison of the hydrogenase of N . opaca 1 b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD-linked hydrogenases are partially identical proteins.

Cell, 1984 Jan, 36(1), 27 - 33
Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil; Horwitz MA; Phagocytosis of Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in human mononuclear phagocytes and causes Legionnaires' disease, occurs by a novel mechanism . A phagocyte pseudopod coils around the bacterium as the organism is internalized . Human monocytes, alveolar macrophages, and polymorphonuclear leukocytes all phagocytize L . pneumophila by this unusual process, termed "coiling phagocytosis," and these leukocytes phagocytize not only live L . pneumophila in this way, but also formalin-killed, glutaraldehyde-killed, and heat-killed L . pneumophila . In contrast, under the same experimental conditions, monocytes phagocytize Streptococcus pneumoniae, encapsulated and unencapsulated E . coli, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Neisseria gonorrhoeae, and Neisseria meningitidis by conventional phagocytosis . Treatment of L . pneumophila with high-titer anti-L . pneumophila antibody abolishes coiling phagocytosis; such bacteria are internalized by conventional phagocytosis . These experiments raise the possibility that a surface component of L . pneumophila mediates the unusual response by the phagocyte . Such a component, if elaborated in vivo, might be responsible for extrapulmonary manifestations of Legionnaires' disease suspected of being toxin-mediated.

Basic Life Sci, 1984, 28, 47 - 80
The identification and cloning of genes encoding haloaromatic catabolic enzymes and the construction of hybrid pathways for substrate mineralization; Weightman AJ et al.; This paper reviews the genetic basis of haloaromatic biodegradation by bacteria, with a focus on the genetic analysis of Alcaligenes eutrophus JMP134, an organism which can utilize 3-chlorobenzoate, 2,4-dichlorophenoxyacetate (2,4-D) and related compounds as sole carbon and energy sources, and Pseudomonas sp . B13, a chlorobenzoate degrader . The involvement of transmissible plasmids pJP4 and pWR1, isolated from strains JMP134 and B13, respectively, in chloroaromatic mineralization has been examined, and restriction fragments of both plasmids have been cloned on the broad host range plasmid vector pKT231 . Transposon Tn5 mutagenesis of these and other soil isolates enriched in and purified from mixed cultures utilizing 2,4,5-trichlorophenoxyacetate (2,4,5-T) as sole carbon and energy source, has been carried out using a "suicide" transposon donor, pLG221 (Co1Ibdrd-1::Tn5) . Mapping of Tn5 insertions in mutants which accumulate pathway intermediates has facilitated the identification and cloning of genes encoding chlorocatechol 1,2-dioxygenase, and other key enzymes in haloaromatic catabolism . There are good prospects for the genetic construction of hybrid haloaromatic catabolic pathways by combining genes encoding broad specificity enzymes, capable of transforming halogenated analogues of their natural substrates, with genes for halocatechol degradation.

J Bacteriol, 1984 Jan, 157(1), 95 - 9
Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase; Leadbeater L et al.; Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy . A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth . The extent of inactivation depended on the added substrate . Pyruvate or lactate caused the strongest inactivation among the tested substrates . Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates . This loss of enzyme activity was not the result of degradation of enzyme protein . Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells . Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium . Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur . We suggest that the pyruvate metabolism of A . eutrophus is involved in these regulatory processes which act on phosphoribulokinase . They appear to contribute to the control of autotrophic CO2 assimilation in this organism.

Antonie Van Leeuwenhoek, 1984, 50(5-6), 473 - 87
Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria; Dijkhuizen L et al.; The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide . The product of this cycle can be used as a precursor for the synthesis of all components of cell material . Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control . Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression . In this control phosphoenolpyruvate most likely has an alarmone function . Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity . In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenolpyruvate are effective inhibitors . Recent studies of phosphoribulokinase in Alcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.

Appl Environ Microbiol, 1983 Nov, 46(5), 1038 - 44
Degradation of chlorophenols by a defined mixed microbial community; Schmidt E et al.; Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp . strain B13 as a chlorocatechol-dissimilating member of the community . Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents . During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II . In the fully acclimated culture, hybrid strains such as Alcaligenes sp . strain A7-2 were detected, which are more competitive than Pseudomonas sp . strain B13 with respect to chlorophenol degradation.

J Antibiot (Tokyo), 1983 Nov, 36(11), 1425 - 30
Fermentation, isolation, characterization and structure of nitrosofungin; Dolak LA et al.; The new antifungal agent nitrosofungin was isolated in high yields from a mixed culture of two organisms consisting of a bacterium of the genus Alcaligenes (UC 9152) and Streptomyces plicatus UC 8272 . The bacterium produces the agent, the streptomycete enhances the production by providing a precursor or an inducer . Nitrosofungin in high concentrations inhibits a broad variety of pathogenic fungi in vitro . The agent is relatively non-toxic in small laboratory animals and high blood levels are obtained after either oral or systemic administration . Nitrosofungin is only the second N-nitrosohydroxylamine isolated from microbial sources to date . It has been identified as 2-N-nitrosohydroxylamino-1-propanol, an acidic and highly water-soluble compound.

Arch Microbiol, 1983 Nov, 136(2), 140 - 6
Occurrence of deletion plasmids at high rates after conjugative transfer of the plasmids RP4 and RK2 from Escherichia coli to Alcaligenes eutrophus H16; Schwab H et al.; The broad host-range IncP-1 plasmids RP4 and RK2 were transferred by conjugation from Escherichia coli to Alcaligenes eutrophus H16 . Among the transconjugants selected on media containing tetracycline, a considerable number did not express kanamycin resistance . By comparing restriction patterns of plasmids isolated from a large number of transconjugants a variety of different deletion derivatives were found . All of these possess more or less extended deletions always including parts of the tra 1-region . The plasmids RP4 and RK2, once established in A . eutrophus H16 showed a high stability and it can be concluded that deletion formation is connected with the conjugation process . Evidence is given that degradation of DNA entering an A . eutrophus recipient cell during the conjugative transfer process may be involved in deletion formation . Furthermore, the finding of a small deletion derivative of RP4 lacking the transacting replication function trfB and the entire kil-kor-system may allow the assumption that these gene functions are not essential for replication and maintenance of RP4 in A . eutrophus hosts.

Arch Microbiol, 1983 Nov, 136(3), 163 - 8
Evolution of L-phenylalanine biosynthesis in rRNA homology group I of Pseudomonas; Byng GS et al.; Group I pseudomonads exhibit diversity for L-phenylalanine biosynthesis that is a basis for separation of two subgroups . Subgroup Ib (fluorescent species such as Pseudomonas aeruginosa, P . fluorescens, or P . putida) possesses an unregulated overflow pathway to L-phenylalanine, together with a second, regulated pathway . Subgroup Ia (non-fluorescent species such as P . stutzeri, P . mendocina, or P . alcaligenes) possess only the regulated pathway to L-phenylalanine . Thus, subgroup Ia species lack an unregulated isozyme of chorismate mutase and arogenate dehydratase, enzymes which are thought to divert chorismate to L-phenylalanine under conditions of high carbon input into aromatic biosynthesis . A priori the overflow pathway could have been either lost in subgroup Ia or gained in subgroup Ib . Since Group V pseudomonads (mainly Xanthomonas) are known to branch off from the Group I lineage at a deeper phylogenetic level than the point of divergence for subgroups Ia and Ib, the presence of the overflow pathway in Group V pseudomonads reveals that the overflow pathway must have been lost in the evolution of subgroup Ia . All Group I species possess a bifunctional protein (P-protein) which catalyzes both chorismate mutase and prephenate dehydratase reactions . In subgroup Ia species this highly conserved protein must be the sole source of prephenate to be used for tyrosine biosynthesis . Thus, the channeling action of the P-protein whereby chorismate is committed towards L-phenylalanine formation can be negated by selective feedback inhibition exerted by L-phenylalanine upon the prephenate dehydratase component of the P-protein . Diversion of prephenate molecules under the latter conditions towards L-tyrosine comprises a channel-shuttle mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1983 Oct, 156(1), 455 - 7
Microbial distribution of selenocysteine lyase; Chocat P et al.; We studied the distribution of selenocysteine lyase, a novel enzyme catalyzing the conversion of selenocysteine into alanine and H2Se, which we first demonstrated in various mammalian tissues (Esaki et al., J . Biol . Chem . 257:4386-4391, 1982) . Enzyme activity was found in various bacteria such as Alcaligenes viscolactis and Pseudomonas alkanolytica . No significant activity was found in yeasts and fungi . Selenocysteine lyases from A . viscolactis and P . alkanolytica acted specifically on L-selenocysteine and required pyridoxal 5'-phosphate as a cofactor.

Appl Environ Microbiol, 1983 Sep, 46(3), 605 - 10
Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures; Shimao M et al.; From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied . Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain . All of the type I strains were taxonomically identical and assigned as Pseudomonas sp . In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp . and Alcaligenes sp . PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp . VM15C . The growth rates of these substituted, mixed cultures differed from each other.

J Biochem (Tokyo), 1983 Sep, 94(3), 879 - 92
Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin; Ohta M et al.; Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp . choline oxidase in the presence of EDTA . Further irradiation slowly converted the semiquinone form into the fully reduced state . The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state . This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen . This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase . The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase . The dissociation constant for this complex was found to be 17 mM by spectroscopic titration . Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form . This species was found to comprise two flavin species . One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state . The other is converted slowly to the oxidized form upon aerobic standing in the dark . We tentatively assigned this latter species as a C(4a)-adduct . Formaldehyde was detected as a product of this photoreaction . The amount of formaldehyde formed coincided with that of the fully reduced enzyme . On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed . Betaine also affects the dithionite reduction . In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.

J Bacteriol, 1983 Sep, 155(3), 1015 - 26
Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2; Lejeune P et al.; Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34 . Homologous gene transfer in P . fluorescens 6.2 and A . eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization . Loci involved in autotrophic growth were among those transferred in A . eutrophus . In heterospecific matings, markers were transferred from P . fluorescens to A . eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A . eutrophus to P . fluorescens, and from Erwinia carotovora subsp . chrysanthemi to A . eutrophus . Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid . When S . typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113 . R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu . R-prime plasmids could be transferred easily into different recipients and were used in complementation studies . PstI restriction digests of four R-prime plasmids carrying P . fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.

Biochem J, 1983 Aug 1, 213(2), 391 - 8
Purification of hydrogenases by affinity chromatography on Procion Red-agarose; Schneider K et al.; The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum . In the case of the soluble hydrogenase of A . eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity . For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined . Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes . For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A . eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.

Appl Environ Microbiol, 1983 Aug, 46(2), 475 - 9
Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1; Sheela S et al.; Fensulfothion (O,O-diethyl O-{4-(methylsulfinyl)phenyl}phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide . Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp . strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon . P . alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid . The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules . However, utilization of the phosphoric acid ester and ethanol by P . alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester . The pathway of degradation of fensulfothion by P . alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.

Biochim Biophys Acta, 1983 Jun 29, 745(3), 267 - 78
Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus; Pinkwart M et al.; The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source . The hydrogenase is membrane-bound and relatively oxygen-sensitive . The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase . The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction) . SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000 . The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors . The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.

J Bacteriol, 1983 Jun, 154(3), 1363 - 70
Control of catechol meta-cleavage pathway in Alcaligenes eutrophus; Hughes EJ et al.; Alcaligenes eutrophus 335 (ATCC 17697) metabolizes phenol and p-cresol via a catechol meta-cleavage pathway . Studies with mutant strains, each defective in an enzyme of the pathway, showed that the six enzymes assayed are induced by the primary substrate . Studies with a putative polarity mutant defective in the expression of aldehyde dehydrogenase suggested that the structural genes encoding this and subsequent enzymes of the pathway exist in the same operon . From studies with mutant strains that constitutively synthesize catechol 2,3-oxygenase and subsequent enzymes and from the coordination of repression of these enzymes by p-toluate, benzoate, and acetate, it is proposed the catechol 2,3-oxygenase structural gene is situated in this operon (2,3-oxygenase operon) . Studies with regulatory mutant strains suggest that the 2,3-oxygenase operon is under negative control.

J Bacteriol, 1983 May, 154(2), 803 - 8
Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus; Im DS et al.; Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose . Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity . With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity . Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87% . Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity . Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed . From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation . Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity . Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate . Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A . eutrophus.

Eur J Biochem, 1983 Feb 1, 130(2), 329 - 34
NAD-linked L(+)-lactate dehydrogenase from the strict aerobe alcaligenes eutrophus . 2 . Kinetic properties and inhibition by oxaloacetate; Steinbuchel A et al.; The L(+)-lactate dehydrogenase (EC 1.1.1.27) of Alcaligenes eutrophus catalyzes the NADH-dependent reduction of pyruvate and a few other 2-oxoacids . The Km values for NADH, NAD, pyruvate and L(+)-lactate are 0.075 mM, 0.130 mM, 0.820 mM and 7.10 mM, respectively . The reaction follows a rapid equilibrium ordered bi-bi mechanism and involves the formation of a dead-end EBQ complex . The competitive inhibition of pyruvate reduction caused by NAD (with respect to NADH) is regarded to be of physiological importance . The enzyme is strongly inhibited by oxaloacetate, oxalate and to a less extent by oxamate . Oxaloacetate was found to be the most powerful inhibitor of the enzyme and exerts an almost complete inhibition of the reduction of pyruvate and some 2-oxoacids at concentrations of 1 microM and less . At 0.1 microM oxaloacetate the inhibition of pyruvate reduction is about 90% . The kinetics of pyruvate reduction in the presence of oxaloacetate is characterized by a burst phase followed by a decreased steady-state velocity . During the burst phase, which lasts from several seconds to some minutes, the enzyme undergoes transition to a less active enzyme form . The inhibition studies revealed the lactate dehydrogenase to be a hysteretic enzyme, due to its slow response to the ligand . The characteristics of the transient were examined . The inhibition of lactate dehydrogenase from A . eutrophus by oxaloacetate is considered to be of great physiological importance, allowing its function only at a low oxaloacetate concentration and consequently at high NADH/NAD ratios.

Am J Clin Pathol, 1983 Feb, 79(2), 245 - 7
Pseudomonas alcaligenes endocarditis; Valenstein P et al.; Pseudomonas alcaligenes is a common soil and water inhabitant that has rarely been proven a human pathogen . We describe a fatal case of Pseudomonas alcaligenes endocarditis . The need for accurate identification of unusual organisms isolated in a clinical setting are discussed.

J Clin Microbiol, 1983 Feb, 17(2), 332 - 7
Determination of antigenic relationships among legionellae and non-legionellae by direct fluorescent-antibody and immunodiffusion tests; Orrison LH et al.; Six isolates, five from water samples and one from a human tracheal swab taken at autopsy, reacted strongly with working dilutions of Legionella fluorescent-antibody conjugates . Of these, two isolates of Pseudomonas fluorescens (EB and CDC93), one isolate of the Flavobacterium-Xanthomonas group (CDC65), and one isolate of P . alcaligenes (CDC11) reacted with Legionella pneumophila serogroup 1 conjugate . P . alcaligenes ABB 50 reacted with an L . pneumophila serogroup 3 conjugate and of P . maltophilia reacted with the L . micdadei conjugate . Antisera and labeled conjugates were prepared for these new cross-reacting isolates, and their relationships to the legionellae were examined by direct fluorescent-antibody and immunodiffusion tests . A nonreciprocal cross-reaction existed between L . micdadei and P . maltophilia and also between serogroups 3 of L . pneumophila and P . alcaligenes ABB50 . Of the four isolates that reacted with serogroup 1 of L . pneumophila, P . fluorescens CDC93 had the strongest relationship, and the other three had only minor relationships . Although cross-reactivity among non-legionellae and legionellae has not been a major problem, these findings are relevant to the interpretation of direct fluorescent-antibody tests for detecting these bacteria.

Arch Microbiol, 1983 Feb, 134(2), 92 - 7
Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria; Friedrich B et al.; Plasmid pJP4 encoding the ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (Tfd+) was transferred by conjugation from Escherichia coli JMP397 to various lithoautotrophic strains of Alcaligenes eutrophus and to the autotrophic bacterium Pseudomonas oxalaticus . The herbicide-degrading function of the plasmid was phenotypically expressed in all of the recipients . The majority of Tfd+ transconjugants also exhibited additional plasmid-encoded properties such as 3-chlorobenzoate degradation, resistance to mercuric ions, and sensitivity to the male-specific bacteriophage PR11 . Furthermore, Tfd+ transconjugants were able to act as donors of plasmid pJP4 . Physical evidence is presented by agarose gel electrophoresis showing that plasmid pJP4 coexisted with the resident plasmids widely distributed in this group of bacteria . However, in some of the hosts plasmids pJP4 was not stably maintained, had a reduced size and tended to form multimers.

Biochem Biophys Res Commun, 1983 Jan 27, 110(2), 412 - 6
Purification of a bacterial organophosphate-hydrolysing phosphatase by Cibacron 3GA-Sepharose affinity chromatography; Pai SB; A phosphatase catalysing the hydrolysis of organophosphorus pesticides was purified to homogeneity using Cibacron 3GA-Sepharose CL 6B affinity chromatography . The enzyme which is localized in the periplasm of the bacterium Alcaligenes NC5 was extracted by treating with 0.2M MgCl2, pH 8.4 . The enzyme was adsorbed to the Cibacron-Sepharose at pH 7.0 and eluted with Tris-HCl buffer at pH 8.0, with 47 per cent recovery . The enzyme thus obtained was electrophoretically homogeneous . This simple affinity purification procedure enhances the potential for its use in large scale detoxification systems.

J Mol Evol, 1983, 19(3-4), 272 - 82
The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria; Byng GS et al.; Pseudomonad bacteria are a phylogenetically diverse assemblage of species named within contemporary genera that include Pseudomonas, Xanthomonas and Alcaligenes . Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization . A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups . We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning . The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American type Culture Collection . Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels . Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning . Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case . It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.

J Gen Microbiol, 1983 Jan, 129 (Pt 2), 1 - 5
The effects of nalidixic acid on respiratory activity of asynchronous and synchronous cultures of Alcaligenes eutrophus; Edwards C et al.; Nalidixic acid inhibited DNA synthesis and cell division during asynchronous growth of Alcaligenes eutrophus but treated cells continued to grow as monitored by A550, respiratory activity and cell volume . Differential effects on cell division were seen when the antibiotic was added at different times during synchronous growth . The earlier in the cell cycle the time of addition the greater the inhibition of cell division . These results suggests that chromosome replication is confined to the first half of the cell cycle in A . eutrophus grown at 30 degrees C . Respiration rates of cells selected by continuous-flow centrifugation and immediately treated with nalidixic acid increased in a stepwise fashion . These steps were similar to those observed in untreated control cells and imply that the DNA-division cycle is not the causal factor for the periodicities in respiration rates and ATPase activity that have been previously reported in this bacterium.

J Bacteriol, 1983 Jan, 153(1), 93 - 9
Pseudomonas stutzeri and related species undergo natural transformation; Carlson CA et al.; Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent . This capacity to undergo natural transformation was found to be shared by the closely related species P . mendocina, P . alcaligenes, and P . pseudoalcaligenes, but was not detectable in strains of P . aeruginosa, P . perfectomarinus, P . putida, P . fluorescens, or P . syringae . P . stutzeri could be transformed either on plates or in liquid medium . Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not . DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments . The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating . The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase . A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance . Other systems for genetic exchange in P . stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.

J Bacteriol, 1983 Jan, 153(1), 176 - 81
Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus; Friedrich CG et al.; Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts) . At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism . The soluble hydrogenase activity was stable at 37 degrees C . The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr) . Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments . Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive . No cross-reactivity was detected above temperatures of 34 degrees C . The genetic information of Hox resides on a self-transmissible plasmid in A . eutrophus . Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant . This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.

J Biol Chem, 1982 Oct 25, 257(20), 11845 - 7
Influence of the activation state on the sedimentation properties of ribulose bisphosphate carboxylase from Alcaligenes eutrophus; Bowien B et al.; Ribulose-1,5-bisphosphate carboxylase from the chemolithotrophic hydrogen bacterium Alcaligenes eutrophus was maximally active in the presence of 50 mM HCO3- plus 10 mM Mg2+ . Deactivation occurred upon removal of these ions . Reactivation was achieved by incubation of the enzymes with HCO3- plus Mg2+ . The concentration of HCO3- (CO2) required for half-maximal activation was 1.84 nM (0.064 mM) . Sedimentation velocity studies revealed that activation/deactivation is associated with drastic changes in the sedimentation properties of the enzyme . While the inactive form had a sedimentation coefficient, s20,w, of 17.5 S, the s20,w gradually decreased as the enzyme was reactivated and the fully reactivated form exhibited an s20,w of 14.3 S . A structural analogue of ribulose 1,5-bisphosphate, xylulose 1,5-bisphosphate, caused a deactivation of the enzyme concomitant with an increase in the sedimentation velocity . It is suggested that the alterations in the hydrodynamic properties accompanying the activation/deactivation process are due to considerable conformational changes that affect the molecular volume and/or the shape of the enzyme . Dissociation/association events were not involved in the changes . The s20,w of about 18 S, generally reported for the large hexadecameric ribulose bisphosphate carboxylases, appears to be characteristic of the inactive form.

J Bacteriol, 1982 Oct, 152(1), 42 - 8
Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus; Friedrich CG et al.; Nickel is a constituent of soluble and particulate hydrogenase of Alcaligenes eutrophus . Incorporation of 63Ni2+ revealed that almost the total nickel taken up by the cells was bound to the protein . Chromatography of a crude extract on diethylaminoethyl cellulose demonstrated an association of 63Ni2+ with soluble and particulate hydrogenase, supported by further analysis like polyacrylamide gel electrophoresis . Unspecific binding of 63Ni2+ to the protein was excluded by comparison with a mutant extract free of hydrogenase protein . X-ray fluorescence analysis of the homogeneous soluble hydrogenase indicated the presence of 2 mol of nickel per mol of enzyme, whereas the amount of nickel determined by incorporation of 63Ni2+ was calculated to be approximately 1 mol/mol of enzyme . Cells grown under nickel limitation contained catalytically inactive, but serologically active, soluble and particulate hydrogenase . The immunochemical reactions were only partially identical with the enzyme from nickel-cultivated cells indicating a structural modification of the proteins in the absence of nickel . It is concluded that nickel is essential for the catalytic activity of hydrogenase and not involved as a regulatory component in the synthesis of this enzyme.

J Gen Microbiol, 1982 Aug, 128 (Pt 8), 1667 - 78
The bacterial biogenesis of isobutyraldoxime O-methyl ether, a novel volatile secondary metabolite; Harper DB et al.; Production of the volatile metabolite, isobutyraldoxime O-methyl ether (IBME) by a Moraxella-like organism NCIB 11650 was investigated under a variety of environmental conditions using gas chromatography . Under aerobic conditions up to 10 micrograms IBME ml-1 was produced on mineral salts media containing 0.5% (w/v) glucose or succinate as sole C source with 0.1% (w/v) NH4Cl as sole N source . Exogenous L-valine further stimulated IBME formation up to 25 micrograms ml-1 but supplementation of the medium with D-isomer or other amino acids had little effect on IBME production and did not lead to the appearance of analogues of IBME . Trapping experiments using {14C}valine confirmed that IBME was derived from this amino acid . Several other bacterial species examined, e.g . Alcaligenes sp . NCIB 11652, another Moraxella-like organism NCIB 11651 and Pseudomonas sp . NCIB 11653 also produced IBME under similar conditions . The Alcaligenes strain synthesized up to 20 micrograms ml-1 in the absence of valine and up to 90 micrograms ml-1 in its presence . The product of IBME exhibited many features characteristic of the formation of a secondary metabolite . Thus biosynthesis was confined to a narrower range of temperature than cell division, was almost completely suppressed by 300 mM-phosphate and was inhibited by high concentrations of readily utilizable C sources . Although IBME synthesis in the Moraxella-like organism NCIB 11650 appeared to be growth-related, its formation by both the Alcaligenes sp . and the Moraxella-like organism NCIB 11651 was delayed until the late-exponential and early-stationary phases of growth . The biological significance of this novel class of secondary metabolite is discussed and a possible biosynthetic route proposed.

J Bacteriol, 1982 Jul, 151(1), 8 - 14
Effect of phosphoglycerate mutase deficiency on heterotrophic and autotrophic carbon metabolism of Alcaligenes eutrophus; Reutz I et al.; Mutants of Alcaligenes eutrophus were isolated on the basis of their inability to grow on succinate as the sole source of carbon and energy . The mutants also failed to grow on other gluconeogenic substrates, including pyruvate, acetate, and citrate . Simultaneously, they had lost their capability for autotrophic growth . The mutants grew, but slower than the wild type, on fructose or gluconate . Growth retardation on gluconate was more pronounced . The mutants lacked phosphoglycerate mutase activity, and spontaneous revertants of normal growth phenotype had regained the activity . The physiological characteristics of the mutants indicate the role of phosphoglycerate mutase in heterotrophic and autotrophic carbon metabolism of A . eutrophus . Although the enzyme is necessary for gluconeogenesis during heterotrophic growth on three- or four-carbon substrates, its glycolytic function is not essential for the catabolism of fructose or gluconate via the Entner-Doudoroff pathway . The enzyme is required during autotrophic growth as a catalyst in the biosynthetic route leading from glycerate 3-phosphate to pyruvate . It is suggested that the mutants accomplish the complete degradation of fructose and gluconate mutase lesion . The catabolically produced triose phosphates are converted to fructose 6-phosphate which is rechanneled into the Entner-Doudoroff pathway . This carbon recycling mechanism operates less effectively in mutant cells growing on gluconate.

Hoppe Seylers Z Physiol Chem, 1982 Jun, 363(6), 627 - 33
Rhein as an electron acceptor for various flavoproteins and for electron transport particles; Egerer P et al.; Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) which has been previously employed as an inhibitor for electron transport particles, NADH dehydrogenase, and other flavoproteins is reducible under physiological conditions . Soluble hydrogenase from Alcaligenes eutrophus H 16, several flavoproteins, and electron transport particles from baker's yeast and from beef heart were found to catalyse NADH oxidation with 9 micrometers to 2mM rhein as the electron acceptor . Dithionite or enzymatically reduced rhein (lambda max = 408 nm) is immediately reoxidized to rhein lambda max = 437 nm) by oxygen . Cyclovoltagrams reveal the midpoint redox potentials --0.240 V, -0.270 V, -0.280 V, -0.335 V at pH 6.0, 7.0, 7.7, 9.2, respectively . Due to its redox behaviour, caution should be exercised using rhein as a flavin-site-directed inhibitor for biological electron transfer systems.

Arch Microbiol, 1982 May, 131(3), 203 - 7
Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5; Srivastava S et al.; Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16 . The vehicle for introducing Tn5 into A . eutrophus was plasmid pJB4JI harboured by Escherichia coli . Kanamycin-resistant transconjugants occurred at a frequency of approximately 5 x 10(-8) . One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin . However, the latter markers were not stably maintained in the new host . Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate or isoleucine . Out of eleven auxotrophic mutants examined eight reverted to prototrophy . However, none of the revertants was kanamycin-sensitive . (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10% . (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1% . Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.

Vet Med (Praha), 1982 Mar, 27(3), 185 - 90
{Proteolytic microorganisms in liver products}; Lukasova J; Liver products were studied for the content of proteolytic microorganisms during production and storage . Their number was at the lowest level in products of thermal processing (10(5) per g on the average) . Storage at a refrigerator and room temperature increased this level to the average values of 10(6) to 8.10(6) per g . Isolated colonies of proteolytic microorganisms were included in the following genera: Pseudomonas, Aeromonas, Escherichia, Alcaligenes, Bacillus and Staphylococcus . The content of ammonia was studied at the same time; ammonia was found to be in correlation with the numbers of proteolytes in the raw material and in the stored products.

J Bacteriol, 1982 Jan, 149(1), 203 - 10
Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus; Friedrich CG; Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures . During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed . With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D . An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen . During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low . These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells . RuBPC activities were not correlated with the hydrogenase activities . During succinate-limited growth, RuBPC appeared at intermediate activities . During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed . RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate . From these data I concluded that RuBPC formation in A . eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.

Antonie Van Leeuwenhoek, 1981 Dec, 47(5), 423 - 48
Numerical taxonomy of Pseudomonas based on published records of substrate utilization; Sneath PH et al.; Data published by R . Y . Stanier, N . J . Palleroni, M . Doudoroff and their colleagues on Pseudomonas have been analysed by numerical taxonomy . Records on 401 strains were used, representing 155 characters, mostly utilization of substrates as carbon-energy sources . Twenty-nine phenons were recognized, which included 394 strains: the remaining 7 remained unclustered . The results were in very good accord with the conclusions of these authors . Almost all phenons were well separated with very little overlap . Many of them corresponded to distinct species, and others corresponded to recognized biotypes . Some small groups may represent unnamed new species . Analyses by Gower's Coefficient showed five major groupings: A) the fluorescent pseudomonads; B) biochemically active species (Pseudomonas cepacia, P . pseudomallei and allies); C) moderately active free-living species (P . acidovorans, P . alcaligenes and allies); D) P . solanacearum and allies; and #) P . mallei P . diminuta does not appear to be clearly distinct from P . vesicularis, nor does P alcaligenes appear clearly distinct from P . pseudoalcaligenes . There may, however, be some difference between P . multivorans and P . cepacia . Analyses using the Pattern Coefficient differed mainly in the relationships shown by a few of the metabolically active species . Of the two coefficients, the Pattern Coefficient gave results that were in somewhat better agreement with evidence from nucleic acids, but it showed an unexpectedly close relationship between P . solanacearum and P . cepacia.

Jpn J Antibiot, 1981 Oct, 34(10), 1366 - 86
{Comparison of antibacterial activity of cefmenoxime with other cephalosporins against clinically isolated bacteria (author's transl)}; Kosakai N et al.; We examined the antibacterial activity of MX in comparison with those of other CEPs, using aerobic Gram-positive cocci, aerobic Gram-negative bacilli and anaerobic bacteria, 870 strains in total, all isolated from clinical specimens, in 1979 and 1980 . Against Streptococcus, CMX showed superior antibacterial activity than those of CFX, CMZ, CXM and CTM . Against H . influenzae, CMX also showed superior antibacterial activity than those of CFX, CMX, CXM, CTM and CEZ . ABPC-and PIPC-resistant strains were sensitive to CMX . CTX, CPZ and CZX also showed antibacterial activities equivalent to that of CMX . Against enteric bacteria, E . coli, Klebsiella, E . cloacae, Serratia, C . freundii and Proteus, CMX showed superior antibacterial activity than those of CFX, CMZ, CXM, CTM and CEZ . Especially, against E . coli, Klebsiella, P . mirabilis, P . rettgeri and P . inconstans, CMX showed strong antibacterial activity . As to non-fermentation bacteria, CMX's antibacterial activity was relatively weak except P . putrefaciens, Alcaligenes and Comamonas . However, it was superior than that of CEZ . In comparison with other CEPs, the strength of CMX varied according to the kinds of bacteria . As to anaerobic bacteria, CMX showed strong antibacterial activity against Peptococcus, Peptostreptococcus, Lactobacillus, Propionibacterium, C . perfringens, Veillonella and Fusobacterium . However, its antibacterial activity against Bacteroides was similar to those of other CEPs.

Mikrobiologiia, 1981 Sep-Oct, 50(5), 864 - 8
{Effect of an electrical field on Alcaligenes eutropha cells}; Ksenzhek OS et al.; The electrophoretic mobility and electrokinetic potential of the hydrogen-oxidizing bacterium Alcaligenes eutropha were measured using microelectrophoresis . The cells were shown to bear a negative charge of ca . 2 x 10(-8) coulomb/cm2 . The electrokinetic potential causing electrophoresis of the bacterium changes from negative values in an alkaline or neutral medium to positive values in an acid medium, and the isoelectric point is at pH approximately 6.0 . The electrophoretic mobility of the bacterium rises with an increase of the ionic strength of solutions . The electric field of a low voltage (up to 5 V/cm) does not induce a noticeable change in the behaviour and growth of the bacterium under the autotrophic conditions.

Mikrobiologiia, 1981 Jul-Aug, 50(4), 645 - 9
{Coincidence of the process of hydrogen-oxidizing bacterial culture and nutrient medium electrolysis}; Ksenzhek OS et al.; The main factors limiting the possibility of organizing the cultivation of the hydrogen-oxidizing bacterium Alcaligenes eutropha Z-1 in combination with electrolysis were determined: accumulation of the oxidizing agents (hypochlorite, persulfate ions, hydrogen peroxide) in the process of electrolysis and deposition of microelements contained in the growth medium on the cathode . The limits for the susceptibility of the bacterium to the oxidizing agents were established . The rate of accumulation of the oxidizing agents and formation of the cathode deposit were found to depend on the current density, the pH and composition of a solution, and several other factors . The concentration of hypochlorite decreased if the content of chloride ions in the growth medium was reduced to approximately 10(-5) g/l; the concentrations of persulfate and hydrogen peroxide is decreased using a certain electrolysis regime and adding ferrous ions which reduce the oxidizing agents . The cathode deposit is dissolved by periodically reversing the current in the course of electrolysis.

J Bacteriol, 1981 Jul, 147(1), 198 - 205
Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus; Friedrich B et al.; Mutants defective in chemolithoautotrophic growth (Aut-) have been isolated from Alcaligenes eutrophus strains H16, N9A, G27, and TF93 . Spontaneous Aut- mutants were obtained only with strain TF93 . Mutants of the other strains were selected after conventional mutagenesis or treatment with mitomycin . Most of the mutants, including the spontaneous Aut- strains, lacked hydrogenase activity (Hox-) but possessed the ability to fix carbon dioxide (Cfx+) . Agar mating of A . eutrophus H16 with Hox- mutants of the various strains resulted in transconjugants which had recovered the ability to grow autotrophically and to express activity of hydrogenase as examined by enzymatic and immunochemical analysis . Transfer of hydrogen-oxidizing ability occurred in the absence of a mobilizing plasmid such as Rp4 . The transfer frequency was particularly high (ca . 10(-2) per donor) when the spontaneous Hox- mutants of strain TF93 were used as recipients . These strains proved to be plasmid free, whereas donors, transconjugants, and the mutagen-treated Hox- mutants contained a large plasmid (molecular weight, 270 +/- 10 X 10(6) revealed by agarose gel electrophoresis . The results allow the conclusion that A . eutrophus H16 harbors a self-transmissible plasmid designated pHG1, which carries information for hydrogen-oxidizing ability.

Vet Rec, 1981 May 2, 108(18), 393 - 5
Incidence of otitis externa in dogs and cats in Japan; Baba E et al.; The incidence of otitis externa in dogs and cats admitted to the animal hospital of the University of Osaka Prefecture was investigated and the bacteria isolated were tested for antibiotic susceptibility . Of the various breeds examined, the incidence of otitis externa was highest in miniature poodles and cocker spaniels and Himalayan and Persian cats . The organisms most commonly associated with otitis externa were coagulase-negative staphylococci, followed by coagulase-positive staphylococci, streptococci and Escherichia coli . Most staphylococci were susceptible to the antibiotics tested, but 15 per cent of staphylococci were resistant to more than three antibiotics . Pseudomonas and Alcaligenes species were resistant to almost all antibiotics except gentamicin and colistin.

Nord Vet Med, 1981 Apr-May, 33(4-5), 206 - 9
{Alteromonas putrefaciens . Taxonomy and cultivation (author's transl)}; Gregersen T; The morphology and biochemical activity of pseudomonas/Alteromonas putrefaciens is described . Features indicating the uncertain taxonomic position of the organism are discussed, and it is pointed out why A . putrefaciens does not fit into neither Pseudomonas nor Alteromonas, but should be considered as a separate unit or group . Table I shows criteria which can be used for differentiation between A . putrefaciens and the genera pseudomonas, Alcaligenes and Alteromonas . Essential characteristics are it's ability to decarboxylate ornithine, it's production of a typical pink pigment and the production of H2S in suitable media . Methods for isolation and quantitation of the organism are generally based on the latter two characteristics.

Arch Microbiol, 1981 Apr, 129(2), 150 - 3
In vivo inactivation of soluble hydrogenase of Alcaligenes eutrophus; Schlesier M et al.; The soluble, NAD+-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available . The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme . Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide . The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30 degrees C was 1.5h . The process of inactivation did not require ATP-synthesis . There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein . The possibility is discussed that the inactivation is due to superoxide radical anions (O2-) produced by the hydrogenase itself.

J Bacteriol, 1981 Mar, 145(3), 1144 - 9
Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus; Friedrich B et al.; The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation . Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level . The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly . In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected . The addition of nickel allowed active hydrogenase formation even when EDTA was present . When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found . This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein . The formation of the membrane-bound hydrogenase also appeared to be nickel dependent . Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.

Zentralbl Bakteriol A, 1981 Feb, 248(4), 579 - 83
Alcaligenes odorans, var . viridans as a hospital infectant; Brzin B; The hazards of acquiring a nosocomial infection are significant even in modern hospitals in spite of aseptic procedures and antibiotics . In order to prevent or stop a hospital epidemic it is necessary to know or to recognize the infective agent, its source and its way of spreading . This is not always easy, esp . with some potential pathogens which are frequently found in the pathological materials as well as in the hospital environment . In the described series of hospital spread of Alcaligenes odorans, var . viridans, it was not difficult to identify the bacterium, it was easy to state the source and not very hard to eradicate it . The way of spreading, however, remained unclear . Hospital infections occur everywhere, even if sometimes unrecognized . With modern treatments facultatively pathogenic bacteria are increasingly becoming hospital infectants . Our aim was to look for such ones, esp . if not described yet as hospital infections.

J Bacteriol, 1981 Feb, 145(2), 752 - 9
Comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase as an indicator of taxonomic relatedness in pseudomonad genera; Whitaker RJ et al.; Recently, an analysis of the enzymological patterning of L-tyrosine biosynthesis was shown to distinguish five taxonomic groupings among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J . Bacteriol . 144:247--257, 1980) . These groupings paralleled with striking consistency those previously defined by ribosomal ribonucleic acid-deoxyribonucleic acid homology relationships . The comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase has previously been shown to be a useful indicator of taxonomic relationship at about the level of genus . The comparative allostery of DAHP synthetase was evaluated in relationship to data available from the same pseudomonad species previously studied . Species of Xanthomonas and some named species of Pseudomonas, e.g., P . maltophilia, were unmistakably recognized as belonging to group V, having a DAHP synthetase sensitive to sequential feedback inhibition by chorismate . This control pattern is thus far unique to group V pseudomonads among microorganisms . Group V organisms were also unique in their possession of DAHP synthetase enzymes that were unstimulated by divalent cations . Group IV pseudomonads (P . diminuta) were readily distinguished by the retro-tryptophan pattern of control for DAHP synthetase . Activity for DAHP synthetase was not always recovered in group IV species, e.g., P . vesicularis . The remaining three groups exhibited overlapping patterns of DAHP synthetase sensitivity to both L-phenylalanine and L-tyrosine . Individual species cannot be reliably keyed to group I . II, or III without other data . However, each group overall exhibited a different trend of relative sensitivity to L-tyrosine and L-phenylalanine . Thus, although enzymological patterning of L-tyrosine biosynthesis alone can be used to separate the five pseudomonad groups, the independent assay of DAHP synthetase control pattern can be used to confirm assignments . The latter approach is, in fact, the easiest and most definitive method for recognition of group V (and often of group IV) species.

Poult Sci, 1981 Jan, 60(1), 107 - 13
Bacterial coryza in turkeys in Texas; Panigrahy B et al.; A motile, gram-negative, short bacillus was isolated from the tracheas of turkey poults with coryza . An Escherichia coli also was isolated from the tracheas of poults . The former bacterium possessed characteristics similar or identical to those isolated from coryza outbreaks in other states . The characteristics were similar to those described for Alcaligenes fecalis . Cultures of the turkey coryza isolate produced coryza when inoculated intranasally in 1 to 3-day-old poults . The bacterium was reisolated consistently from the tracheas of the affected poults . In one experiment, poults inoculated with the coryza bacterium and the E . coli isolate had an apparent increased incidence of air sacculitis . No viruses were isolated from the tracheas of coryza-affected poults . Blood serums were negative for precipitating and hemagglutination-inhibition antibodies to avian influenza and Newcastle disease viruses, respectively . The serum neutralizing antibody titers to infectious bursal disease virus in noninoculated poults, and poults inoculated with the coryza bacterium, or E . coli or both, were undetectable or low . Serum agglutination was not a reliable method for determining infection by the coryza bacterium.

Mikrobiologiia, 1981 Jan-Feb, 50(1), 41 - 5
{Arsenic oxidation by the heterotrophic bacteria Pseudomonas putida and Alcaligenes eutrophus}; Abdrashitova SA et al.; Two heterotrophic bacteria, Pseudomonas putida 18 and Alcaligenes eutrophus 280, were isolated from gold-arsenic deposits . The bacteria oxidize As(III) to As(V) at pH 6-9 and temperatures 4-28 and 28 degrees C respectively . The oxidation is accompanied by a decrease in the pH of the medium . The rate of the oxidation directly depends on the number of cells in the inoculum.

Biochem J, 1981 Jan 1, 193(1), 99 - 107
Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16; Schneider K et al.; The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent . The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration . The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively . Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite . The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation . During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly . In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.

J Bacteriol, 1980 Oct, 144(1), 247 - 57
Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae; Byng GS et al.; Enzymes of tyrosine biosynthesis (prephenate dehydrogenase and arogenate dehydrogenase) were characterized in 90 species currently classified within the genera Pseudomonas, Xanthomonas, and Alcaligenes . Variation in cofactor specificity and regulatory properties of the dehydrogenase proteins allowed the separation of five groups . Taxa defined by enzymological patterning corresponded strikingly with the five ribosomal ribonucleic acid (rRNA) homology groups established via rRNA-deoxyribonucleic acid hybridization . rRNA homology groups I, IV, and V all lack activity for arogenate/nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase and separated on this criterion from groups II and III, which have the activity . Group II species possess arogenate dehydrogenase enzyme (reactive with either NAD or NADP) sensitive to feedback inhibition by tyrosine, thereby separating from group III species whose corresponding enzyme was totally insensitive to feedback inhibition . The presence of prephenate/NADP dehydrogenase in group IV defined its separation from groups I and V, which lack this enzyme activity . Group I species possess an arogenate/NAD dehydrogenase that was highly sensitive to inhibition by tyrosine and a prephenate/NAD dehydrogenase of relative insensitivity to tyrosine inhibition . The opposite pattern of sensitivity/insensitivity was seen in group V species . These dehydrogenase characterizations are highly reliable for the keying of a given species to one of the five rRNA homology groups . If necessary, other confirmatory assays can be included using other aromatic pathway enzymes . These results further document the validity and utility of the approach of comparative enzymology and allostery for classification of microorganisms.

Biotechnol Bioeng, 1980 Sep, 22(9), 1877 - 94
Oxygen supply to bacterial suspensions of high cell densities by hydrogen peroxide; Ibrahim M et al.; The supply of heterotrophically growing suspensions of Alcaligenes eutrophus PHB-4 with oxygen formed by the continuous addition of H2O2 in the presence of bovine liver catalase was found to be restricted to well-defined conditions . The catalase-H2O2 system proved to be suitable during the growth at low cell densities equivalent to 2 g dry weight/liter . When under these conditions the oxygen concentration was held constant at 1.8 mg O2/liter, the cells grew for 6-8 hr at a rate almost identical to that observed with conventional aeration . However, aeration with H2O2 for longer durations (10-20 hr) and at higher cell densities (5-20 g dry weight/liter) led invariably to cell damage and retardation of growth . The impairment of growth observed during the oxygen supply by the catalase-H2O2 system was traced back to the formation of gradually increasing steady-state concentrations of H2O2 in the medium . Possible sites of cell damage by H2O2 such as membrane function, excretion and function of siderophores, and synthesis of cell polymers have been studied, and the cytotoxic mechanism of low concentrations of H2O2 was discussed.

Biokhimiia, 1980 Aug, 45(8), 1405 - 11
{Reversible oxidation-reduction of NAD by hydrogen, catalyzed by soluble hydrogenase from Alcaligenes eutrophus Z-1}; Pinchukova EE et al.; The kinetics of NAD reduction by hydrogen, catalyzed by soluble hydrogenase from the hydrogen bacterium A . eutrophus Z-1 within a wide range of NAD substrate concentrations and pH were studied under aerobic and anaerobic conditions . The autocatalytic type of the reaction (with an induction period) and positive kinetic cooperativity with respect to NAD substrate at pH values greater than 8.0 were observed . A steady hydrogen release in a two-enzyme system involving hydrogenase, formiate dehydrogenase, formiate and NAD was demonstrated . A multistep pattern of the reaction mechanism of NAD reduction allowing to explain the autocatalytic type of NAD reduction by hydrogen as well as insensitivity of the reaction to air oxygen were proposed . Possible types of regulation of the soluble hydrogenase activity in the cell are discussed.

Mikrobiologiia, 1980 Jul-Aug, 49(4), 465 - 71
{Growth of hydrogen bacteria inhibited by carbon monoxide}; Volova TG et al.; The growth of the hydrogen bacterium Alcaligenes eutrophys Z-1 was studied during inhibition by carbon monoxide in turbidostat . The specific growth rate of the bacterium was shown to depend on the concentration of CO in the gaseous phase . The deceleration of the cultural growth during inhibition by CO was accompanied with a decrease of economic coefficients with respect to oxygen and hydrogen . At a CO concentration of 10%, the specific growth rate of the bacterium was 0.25 hr-1 (mu) and the coefficient for utilizing the energy of hydrogen oxidation was 19.6% (eta); at a CO concentration of 20%, mu was 0.18 hr-1 and eta was 14.2%; at a CO concentration of 30%, mu was 0.07 hr-1 and eta was 10.2% . The deceleration of the cultural growth caused by inhibition with CO was not accompanied by changes in the synthesis of nitrogen containing and reserve substances in A . eutrophys Z-1 . This strain of hydrogen bacteria was found to be resistant against extraneous microflora during inhibition with CO.

J Biochem (Tokyo), 1980 Jul, 88(1), 197 - 203
Identification and properties of the prosthetic group of choline oxidase from Alcaligenes sp; Ohta-Fukuyama M et al.; Choline oxidase from Alcaligenes sp . catalyzed the oxidation of choline and betaine aldehyde to betaine with concomitant consumption of oxygen and production of hydrogen peroxide . The values of Km for choline and betaine aldehyde were 0.87 and 6.2 mM, respectively . The molecular weight of the enzyme was estimated to be 66,000 by SDS-gel electrophoresis and 72,000 by gel-filtration using a high performance liquid chromatograph . The prosthetic group of the enzyme was identified as 8 alpha-{N(3)-histidyl}-FAD from the electrophoretic mobility at pH 6.25 of the hydrolysate of the methylated histidylflavin . The visible absorption spectrum of the enzyme showed peaks at 358 and 453 nm and a shoulder at about 480 nm . The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration . The enzyme was found to contain 1 mol of FAD per mol enzyme . Amino acid analysis of a purified flavin peptide gave the following molar ratios of amino acids to flavin: pro(1), Asp + Asn(3), Ser(1), His(1), and Arg(1) . Aspartic acid was the N-terminal amino acid . The partial sequence of amino acids in the flavin peptide was as follows: Formula (See Text).

J Bacteriol, 1980 Jul, 143(1), 59 - 69
Evidence for isofunctional enzymes used in m-cresol and 2,5-xylenol degradation via the gentisate pathway in Pseudomonas alcaligenes; Poh CL et al.; Study of the reaction sequence by which Pseudomonas alcaligenes (P25X1) and derived mutants degrade m-cresol, 2,5-xylenol, and their catabolites has provided indirect evidence for the existence of two or more isofunctional enzymes at three different steps . Maleylpyruvate hydrolase activity appears to reside in two different proteins with different specificity ranges, one of which (MPH1) is expressed constitutively; the other (MPH11) is strictly inducible . Two gentisate 1,2-dioxygenase activities were found, one of which is constitutively expressed and possesses a broader specificity range than the other, which is inducible . From oxidation studies with intact cells, there appear to be two activities responsible for the 6-hydroxylation of 3-hydroxybenzoate, and again a broadly specific activity is present regardless of growth conditions; the other is inducible by 3-hydroxybenzoate . Three other enzyme activities are also detected in uninduced cells, viz., xylenol methylhydroxylase, benzylalcohol dehydrogenase, and benzaldehyde dehydrogenase . All apparently possess broad specificity . Fumarylpyruvate hydrolase was also detected but only in cells grown with m-cresol, 3-hydroxybenzoate, or gentisate . Mutants, derived either spontaneously or after treatment with mitomycin C, are described, certain of which have lost the ability to grow with m-cresol and 2,5-xylenol and some of which have also lost the ability to form the constitutive xylenol methylhydroxylase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase, 3-hydroxybenzoate 6-hydroxylase, and gentisate 1,2-dioxygenase . Such mutants, however, retain ability to synthesize inducibly a second 3-hydroxybenzoate 6-hydroxylase and gentisate 1,2-dioxygenase, as well as maleylpyruvate hydrolase (MPH11) and fumarylpyruvate hydrolase; MPH1 was still synthesized . These findings suggest the presence of a plasmid for 2,5-xylenol degradation which codes for synthesis of early degradative enzymes . Other enzymes, such as the second 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, maleylpyruvate hydrolase (MPH1 and MPH11), and fumarylpyruvate hydrolase, appear to be chromosomally encoded and, with the exception of MPH1, strictly inducible.

J Biochem (Tokyo), 1980 Jul, 88(1), 205 - 9
Circular dichroism studies on flavoproteins containing covalently bound coenzymes; Ohta-Fukuyama M et al.; Absorption and circular dichroism spectra of cholesterol oxidase from Schizophyllum commune and choline oxidase from Alcaligenes sp . were measured and compared . The prosthetic group of cholesterol oxidase is 8 alpha-{N(1)-histidyl}-FAD (1, 2), while that of choline oxidase is 8 alpha-{N(3)-histidyl}-FAD (3) . In the CD spectra of the two enzymes in either the oxidized or reduced state, the corresponding bands in the visible region are of approximately the same intensity and shape but of opposite sign . A notable feature in the CD spectra of the two enzymes after light irradiation is the appearance of a CD band in the longer wavelength region (550-650 nm) and the opposite signs of the CD band in this region in the two enzymes . The similarity of the shape and intensity of the CD spectra of the two enzymes suggests that the environments surrounding the flavin moieties are very similar, and the sign reversal of the CD bands suggests that the mutual orientations between the transition moment of flavin and that of its environment differ in the two enzymes.

J Clin Microbiol, 1980 Jun, 11(6), 664 - 8
Cellular fatty acid composition of group IVe, a nonsaccharolytic organism from clinical sources; Dees S et al.; The cellular fatty acid composition of a group of gram-negative nonfermentative organisms designated group IVe was studied by gas-liquid chromatography . Strains of this group are isolated most frequently from urine and most closely resemble the Alcaligenes in conventional biochemical tests . On the basis of cellular fatty acids, however, we found these organisms to be strikingly different from Alcaligenes and other gram-negative species with similar phenotypic characteristics . The gas-liquid chromatography procedure offers an additional diagnostic test for rapid identification of unclassified bacteria like group IVe.

Eur J Biochem, 1980 May, 106(2), 405 - 10
On the structure of crystalline ribulosebisphosphate carboxylase from Alcaligenes eutrophus; Bowien B et al.; Ribulosebisphosphate carboxylase from the hydrogen bacterium Alcaligenes eutrophus having a molecular weight of 534000 and consisting of eight large and eight small subunits has been crystallized by microdialysis using inorganic as well as organic precipitating agents . Crystals have tetragonal space group P42212, a = b = 11.27 nm, c = 20.14 nm, and contain one quarter molecule per asymmetric unit . X-rays are diffracted to 0.35-nm resolution on still photographs . Light optical diffractions of electron micrographs of thin sectioned crystals displayed patterns which could be interpreted on the basis of the unit cell determined by X-rays . Packing considerations are in accord with our earlier proposal regarding the subunit arrangement of this enzyme which differs from that reported for tobacco ribulosebisphosphate carboxylase.

Rev Argent Microbiol, 1980 May-Aug, 12(2), 44 - 51
{Changes in the bacteriological, chemical and organoleptic characteristics of the Antartic krill (Euphausia superba) during storage at 0-2 degrees C}; Locati GA et al.; A study was performed on the bacteriological, chemical and organoleptic characteristics of antartic krill (Euphausia superba) stored at 0-2 degrees C . After 6-8 hours of storage a dark color started in the head and legs and spread slowly to the tail . Within 24 hours 17% of the total nitrogen was lost by hepatopancreas autolisis . After 72 hours the krill became inedible due to strong amoniacal odor and flavor . These changes were associated with the multiplication of aerobic psychrophilic bacteria . The bacterial counts of freshly caught krill ranged between 3,7 X 10(2)/g and 2,5 X 10(5)/g at 21 degrees C . During storage at 0-2 degrees C the counts gradually increased and off-odors were produced when they reached values of 10(6)/g at 21 degrees C . The total volatile bases content of freshly caught krill, 0.018 to 0.038%, increased considerably during storage reaching values of approximately 0.100% when off-odors became noticeable and 0.200% or more when the odor was clearly ammoniacal . Pseudomonas spp Gp . II (Shewan) were predominant in the bacterial flora of the freshly caught krill along with Moraxella spp Alcaligenes spp, Vibrio spp, Micrococcus spp and coryneforms . The spoilage flora developed during cold storage consisted mainly of Pseudomonas spp G . II (96-100%) . The results were related to the saline composition of medium; however, Pseudomonas spp Gp . II were predominant with both media used.

J Biochem (Tokyo), 1980 Apr, 87(4), 1177 - 84
Components of the cytochrome system of Alcaligenes sp . N.C.I.B., 11015, with special reference to particulate bound c-type cytochromes; Shidara S; 1 . The cytochrome type of the particulate fraction from cells of Alcaligenes sp . was examined . The particulate fraction of aerobically-grown cells contained b-, c-, a-type and o-type like cytochromes, whereas that from nitrate-grown cells contained b-, c-type and o-type like cytochromes . 2 . Two kinds of c-type cytochromes were extracted and purified from the particulate fraction of nitrate-grown cells of Alcaligenes sp . One of them showed absorption maxima at 415.5, 522, and 551 nm is the reduced form, and at 410.5 nm in the oxidized form; the other at 417.5, 523, and 556 nm in the reduced form, and at 412 nm in the oxidized form; these cytochromes were designated cytochrome c-551 and cytochrome c-556, respectively . 3 . Cytochrome c-551 had a molecular weight of 28,800 and had two heme groups per molcule . The midpoint oxidation reduction potential at pH 7.0 (Em, 7) was +262 mV . Cytochrome c-556 had a molecular weight of 18,200 and contained one heme group per molecule . The Em, 7 value was +291 mV . These cytochromes were not autoxidizable, did not react with carbon monoxide or KCN at pH 7.0, and were reduced by ascorbate . 4 . Each cytochrome acts to some degree as electron carrier in the dissimilatory nitrite reduction reaction of Alcaligenes sp.

Vet Rec, 1980 Jan 12, 106(2), 25 - 8
Clinical bacteriological and epidemiological observations on infectious atrophic rhinitis of pigs in southern England; Giles CJ et al.; Three distinct patterns of infection with Bordetella (Alcaligenes) bronchiseptica were found in groups of 12 to 24 pigs born in 1977-78 in 12 herds in southern England . In five of these, heavy bordetella infection of a substantial proportion of unweaned piglets persisted to a variable extent until slaughter . Clinical disease and severely atrophied turbinates were most marked in these groups . In three other herds the infection first appeared soon after weaning and occasionally persisted until slaughter . Clinical disease occurred in only one of these other groups and conchal atrophy at slaughter was moderate . In the groups of the four remaining herds there was no clinical disease and conchal atrophy at slaughter was slight, infection appearing only late in the weaning, or even the fattening, stages . These varying patterns suggest that immunological phenomena were involved in the infection in the least affected herds and that such responses might, if reproducible artificially, provide a better means of control of this disease in badly affected herds than the available forms of chemotherapy.

Antonie Van Leeuwenhoek, 1980, 46(1), 1 - 14
The membrane-bound hydrogenase of Alcaligenes eutrophus: II . Localization and immunological comparison with other hydrogenase systems; Schink B et al.; Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing . Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane . The membrane-bound hydrogenases of A . eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas . Alcaligenes and Aquaspirillum . Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotraphicus GZ 29 was found only in a few hydrogen-oxidizing bacteria . Material cross-reacting to the soluble hydrogenase of A . eutrophus was detected in strains of A . eutrophus and A . ruhlandii only . Comparison of the membrane-bound hydrogenase of A . eutrophus, P . pseudoflava and X . autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only . The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.

J Antibiot (Tokyo), 1979 Nov, 32(11), 1096 - 103
G1549, a new cyclic hydroxamic acid antibiotic, isolated from culture broth of Pseudomonas alcaligenes; Barker WR et al.; Antibiotic G1549, isolated from culture broth of Pseudomonas alcaligenes, is a new cyclic hydroxamic acid with a 1-hydroxy-2(1H)-pyridinone structure that complexes with metals . The structure of G1549 is suggested to be 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone . In vitro, G1549 and its copper and ferric complexes show moderate activity against Gram-positive bacteria, fungi and Trichomonas vaginalis . Topical application of G1549 and its copper and ferric complexes protect guinea pigs against cutaneous infection with Microsporum canis . The compounds, however, have some systemic toxicity in mice.

Appl Environ Microbiol, 1979 Nov, 38(5), 800 - 5
Cationic composition of 22 species of bacteria grown in seawater medium; Jones GE et al.; Twenty-two species of bacteria of marine, estuarine, and terrestrial origin were analyzed for cationic content by atomic absorption spectrophotometry after growth in a basal seawater medium . Alcaligenes marinus was analyzed from eight separate but replicate determinations yielding the following cationic concentrations: Na, 5,600 +/- 2,260; Mg 1,580 +/- 740; K, 700 +/- 360; Ca, 790 +/- 390; Mn, 1.7 +/- 0.5; Fe, 256 +/- 57; Ni, 1.7 +/- 0.7; Cu, 14 +/- 4; Zn, 122 +/- 27; Cd, 2.8 +/- 0.7; and Pb, 10 +/- 3 ppm/(dry weight) . Washing A . marinus cells before analyses was necessary due to interstitial medium within the cell pellets after centrifugation and loose cationic retention by the cells . The principal source of error in the procedure was ascribed to variability due to washing cells with 0.5 M ammonium formate . The mean cationic concentrations for trace elements in the 22 bacterial cultures grown in the basal seawater medium to constant optical density and washed three times with 0.5 M ammonium formate were: Mn, 2.4 +/- 3.8; Fe, 262 +/- 112; Ni, 2.3 +/- 1.8; Cu, 24 +/- 17; Zn, 146 +/- 72; Cd, 3.8 +/- 2.5; and Pb, 17 +/- 21 ppm (dry weight) . Major ions were concentrated only occasionally by the cells after washing, whereas Mn, Fe, Ni, Cu, Zn, Cd, and Pb were concentrated from the medium by the following factors on the average: 180, 1,600, 140, 1,200, 750, 1,900, and 900, respectively.

Appl Environ Microbiol, 1979 Sep, 38(3), 494 - 8
Production of arsine and methylarsines in soil and in culture; Cheng CN et al.; Arsenate, arsenite, monomethylarsonate, and dimethylarsinate were added to different soils, and evolution of gaseous arsenical products was determined over 3 weeks . Arsine was produced in all three soils from all substrates, whereas methylarsine and dimethylarsine were produced only from methylarsonate and dimethylarsinate, respectively . At least three times more arsine than dimethylarsine was produced in soil incubated with dimethylarsinate . Resting cell suspensions of Pseudomonas and Alcaligenes produced arsine as the sole product when incubated anaerobically in the presence of arsenate or arsenite . In all instances, no trimethylarsine was observed, nor could any evidence be shown for the methylation of any arsenical substrate in soil or in culture . It was concluded that reduction to arsine, not methylation to trimethylarsine, was the primary mechanism for gaseous loss of arsenicals from soil.

Biochim Biophys Acta, 1979 Jun 19, 578(2), 445 - 61
The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus; Schneider K et al.; The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step . The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification . After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively . p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2 . Chelating agents, CO and O2 failed to inhibit enzyme activity . The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02 . On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed . The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the {2Fe-2S} and {4Fe-4S} types are present in the enzyme . Quantitation of the EPR signals indicated the existence of two identical centres each of the {4Fe-4S} and of the {2Fe-2S} type . The midpoint redox potentials of the {4Fe-4S} and the {2Fe-2S} centres were determined to be -445 mV and -325 mV, respectively . Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies . On reduction of the {Fe-4S} centres, the electron spin relaxation rate of the {2Fe-2S} centres was considerably increased . Treatment of hydrogenase with CO caused no change in EPR spectra.

Biochim Biophys Acta, 1979 Jun 1, 585(1), 1 - 11
Mutations altering the catalytic activity of a plant-type ribulose biphosphate carboxylase/oxygenase in Alcaligenes eutrophus; Andersen K; Use of a bacterial system has allowed the isolation of several mutationally altered species of ribulose-1,5-bisphosphate carboxylase/oxygenase having a wide range of catalytic activities . Biochemical and serological techniques were used to classify the mutant strains into three groups: (1) no detectable ribulose-1,5-bisphosphate carboxylase/oxygenase protein present (both subunits missing); (2) mutant strains having ribulose-1,5-bisphosphate carboxylase/oxygenases with lowered specific activities; and (3) mutant strains producing large quantities of catalytically inactive ribulose-1,5-bisphosphate carboxylase/oxygenase.

Biochim Biophys Acta, 1979 Apr 12, 567(2), 315 - 24
The membrane-bound hydrogenase of Alcaligenes eutrophus . I . Solubilization, purification, and biochemical properties; Schink B et al.; The membrane-bound hydrogenase of Alcaligenes eutrophus was solubilized from washed membranes of autotrophically grown cells . The enzyme consists of two types of subunits and is an iron-sulfur protein . A flavin compound was not detected . The enzyme reacts only with few artificial electron acceptors.

Appl Environ Microbiol, 1979 Apr, 37(4), 680 - 5
Degradation of polychlorinated biphenyls by mixed microbial cultures; Clark RR et al.; Three different enriched mixed cultures capable of degrading polychlorinated biphenylas were isolated from two soil samples and a river sediment, respectively . The predominant organisms found in all three mixed cultures were Alcaligenes odorans, Alcaligenes dentrificans, and an unidentified bacterium . The polychlorinated biphenyl isomers that were more water soluble and had lower chlorination were not only degraded at a faster rate than those that were less water soluble and had higher chlorination, but were also more completely utilized by these mixed cultures . This resulted in the presence in the environment of polychlorinated biphenyl residues consisting mainly of higher-chlorinated isomers . A form of cometabolism of polychlorinated biphenyls was also found with these cultures in the presence of acetate as the cosubstrate.

Biokhimiia, 1979 Apr, 44(4), 605 - 15
{Isolation, purification and study of the stability of the soluble hydrogenase of Alcaligenes eutrophus Z-1}; Pinchukova EE et al.; Soluble hydrogenase was isolated from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-1 and purified to electrophoretical homogeneity . The purification procedure included fractionation by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose and gelfiltration through Ultragel AcA-34 . The resulting preparation had a specific activity of 25 mkmoles H2.min-1.mg of protein as measured by the rate of hydrogen evolution from sodium dithionite-reduced methyl viologen . The enzyme has a molecular weight of 200,000 and is made up of two subunits with mol . weights of 30,000 and two subunits with mol . weights of 65,000 . The effects of pH, oxidants and reducers, as well as aerobic and anaerobic conditions on the hydrogenase preparations inactivation kinetics in intact cells and in a highly purified state were studied . The kinetic data suggest a possible existence of two enzyme forms differing in their activities and stabilities to denaturating influences.

Mikrobiologiia, 1979 Mar-Apr, 48(2), 360 - 2
{Relationship of hydrogen bacteria to carbon monoxide}; Savel'eva ND; The purpose of this work was to study the ability of various cultures of hydrogen bacteria to grow in a gaseous mixture containing, apart from H2, O2 and CO2, carbon monoxide at a concentration of 10% (v/v) and higher . In contrast to CO-oxidizing bacteria, these organisms could not use CO as a sole source of energy . Among 17 studied strains of hydrogen bacteria, only those belonging to the group Alcaligenes eutrophus could withstand 10% of CO (v/v) in a gaseous mixture.

Biochem J, 1979 Mar 1, 177(3), 819 - 23
The amino acid sequence of cytochrome c' from the purple sulphur bacterium Chromatium vinosum; Ambler RP et al.; An amino acid sequence is proposed for the cytochrome c' from the photosynthetic purple sulphur bacterium Chromatium vinosum strain D . It is single polypeptide chain of 131 residues, with haem-attachment cysteine residues at positions 121 and 124 . The results discredit an earlier report {Dus, Bartsch & Kamen (1962) J . Biol . Chem 237, 3083--3093} of a di-haem peptide sequence from this protein . The sequence belongs to the same class as the published Alcaligenes and Rhodospirillum rubrum cytochrome c' squences, but the resemblance is not close . Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50,093 (15 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem . J . (1978) 169, 5.

Biochim Biophys Acta, 1979 Feb 26, 576(2), 471 - 8
An oxygen-binding flavohemoprotein from Alcaligenes eutrophus; Probst I et al.; A procedure is described for the purification of a soluble flavohemoprotein from the hydrogen bacterium Alcaligenes eutrophus . The isolated protein exists as a monomer with a molecular weight of approx . 43,000 . The molecule contains two prosthetic groups, 1 mol each of noncovalently bound FAD and protoheme per monomer . The absorption spectra of the protein in its ferric, ferrous-deoxy and ferrous-carboxy forms are similar to those of hemoglobins, with the exception of the flavin contribution (absorption maxima--ferric form: 395, 456, 483, 645 nm; ferrous-deoxy form: 436, 560 nm; ferrour-CO form: 423, 539, 569 nm) . The flavohemoprotein when reduced by NADH in aerobic solution is capable of binding oxygen reversibly . The stable oxygenated complex exhibits absorption maxima at 414, 541, and 576 nm . The protein catalyzes the reduction of various dyes and cytochrome c by NADH.

J Gen Virol, 1978 Nov, 41(2), 239 - 54
Isolation and characterization of polysheaths, phage tail-like defective bacteriophages of Alcaligenes eutrophus H 16; Walther-Mauruschat A et al.; Polysheaths were spontaneously formed inside cells of the hydrogen bacterium Alcaligenes eutrophus H 16 . These particles are long tube-like structures of 24 nm diam . belonging to the phage tail-like defective bacteriophages (Lotz, 1976) . In mid log-phase fermenter-grown cells, polysheaths were observed in about 20% of all cells sectioned . Evidence is provided for an inhibition of cell fission by polysheaths . Polysheaths were isolated by differential centrifugation and precipitation techniques using PEG and antibodies . The morphology of polysheaths was investigated electron microscopically by negative staining, ultrathin sectioning and metal shadowing . A surface lattice of the polysheath was derived from light optical diffraction data . The particles were also characterized by their biochemical and biophysical features: mol . wt . of the subunit determined by SDS-gel electrophoresis (58 000), amino acid composition, isoelectric point (4.4), u.v . absorbance spectrum indicating the absence of nucleic acid, buoyant density (1.258), and stability against denaturants and proteolytic enzymes.

Biochim Biophys Acta, 1978 Sep 6, 542(3), 424 - 9
Chemical structure and biodegradability of halogenated aromatic compounds . Substituent effects on dehydrogenation of 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid; Reineke W et al.; The dehydrogenation of substituted 3,5-cyclohexadiene-1,2-diol-1-carboxylic acids by dihydrodihydroxybenzoic acid dehydrogenases from benzoate grown cells of Alcaligenes eutrophus and Pseudomonas sp . B 13 and 3-chlorobenzoate grown cells of the latter organism was examined . No significant differences (Km and Vrel values) were detected for the enzymes from both organisms . The same dihydrodihydroxybenzoic acid dehydrogenase is formed in Pseudomonas sp . B13 during growth on benzoate as well as on 3-chlorobenzoate . The lower turnover rates of 3- and 5-chlorodrodihydroxybenzoic acid compared to dihydrodihydroxybenzoic acid are counterbalanced by an increase in specific activity . With the exception of 4-substituted dihydrodihydroxybenzoic acids exhibiting relative high Km values, only slight sterical and electronic substituent effects are evident . Reaction rates were never reduced to a critical level.

Biochim Biophys Acta, 1978 Sep 6, 542(3), 412 - 23
Chemical structure and biodegradability of halogenate aromatic compounds . Substituent effects on 1,2-dioxygenation of benzoic acid; Reineke W et al.; Dioxygenation of substituted benzoic acids by whole cells of 3-chlorobenzoate-utilizing Pseudomonas sp . B 13, benzoate-induced cells of Alcaligenes eutrophus B 9 and toluate-grown cells of Pseudomonas putida mt-2 was examined . Electron-attracting substituents like halogen decreased the reaction rates of benzoate 1,2-dioxygenation . Dioxygenation of substituted benzoic acids by P . putida mt-2 was mostly undisturbed by steric effects of the substituents . Good correlation resulted between the log Vrel values and the Hammett substituent constant sigma . In contrast the reaction rates of dioxygenation by Pseudomonas sp . B 13 and A . eutrophus were decreased predominantly by steric effects of substituents . A non-polar reaction mechanism of benzoate 1,2-dioxygenation is discussed . Results from inhibition studies demonstrate high stereospecificities for the 1,2-dioxygenation by Pseudomonas sp . B 13 of benzoic acids with substituents in ortho- or para-position . In the case of P . putida mt-2 steric handrance by substituents was observed only with orth-substituted benzoic acids . Stereospecificities of the benzoate 1,2-dioxygenation by Pseudomonas sp . B 13 and P . putida mt-2 are illustrated schematically.

J Bacteriol, 1978 Sep, 135(3), 798 - 804
Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus; Fisher PR et al.; A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid . The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1.

Eur J Biochem, 1978 Jul 17, 88(1), 97 - 107
Further studies on the quaternary structure of D-ribulose-1, 5-bisphosphate carboxylase from Alcaligenes eutrophus; Bowien B et al.; Homogeneous D-ribulose-1,5-bisphosphate carboxylase, isolated from the hydrogen bacterium Alcaligenes eutrophus, has been studied by analytical ultracentrifugation . Sedimentation equilibrium experiments showed the enzyme to have a molecular weight M c =0 r =534000 . The sedimentation coefficient was S0(20); w = 14.1S . The two types of subunits constituting the ribulosebisphosphate carboxylase were separated by gel filtration in the presence of sodium dodecylsulphate and the amino acid compositions of the isolated large and small subunits were determined . Rabbit antibodies were developed against the ribulosebisphosphate carboxylase and its isolated subunits . The specific reactivity of the respective antibodies with their homologous antigens was proven by double immunodiffusion and quantitative immunoprecipitation analyses . Antibodies elicited against the whole enzyme also reacted with both the isolated large and small subunits as did the subunit-specific antibodies with the whole enzyme . There was no immunological correspondence between the large and the small subunits . The specific inhibition of the enzyme activity by antibodies directed against sites on the large subunit suggests that the catalytic function resides in the large subunit . Electron microscopic examination of antibody . carboxylase complexes formed upon mixing of the specific immunoglobulins G with the enzyme was used to verify the arrangement of the large and small subunits in our recently proposed structural model of the enzyme molecule . The results confirmed that the large subunits are located in the central two layers of the four-layered enzyme molecule, whereas the two outer layers consist of small subunits . The observations are discussed with respect to an alternative model for the quaternary structure of ribulosebisphosphate carboxylase from tobacco.

Biochem J, 1978 Jul 15, 174(1), 85 - 94
Chemical structure and biodegradability of halogenated aromatic compounds . Substituent effects on 1,2-dioxygenation of catechol; Dorn E et al.; 1 . The influence of halogen substituents on the 1,2-dioxygenation of catechols was investigated . The results obtained with the two isoenzymes pyrocatechase I and pyrocatechase II from the haloarene-utilizing Pseudomonas sp . B 13 and the pyrocatechase from benzoate-induced cells of Alcaligenes eutrophus B.9 were compared . 2 . Substituents on catechol were found to interfere with O2 binding by the two isoenzymes from Pseudomonas sp . B 13, whereas the Km value for catechol kept constant at different O2 concentrations . 3 . Electron-attracting substituents decreased the Km values for catechols . 4 . Results from binding studies with substituted catechols demonstrated narrow stereospecificities of pyrocatechase I from pseudomonas sp . B 13 and the pyrocatechase from alcaligenes eutrophus B.9 . In contrast, a low steric hindrance by substituents in the binding of catechols with pyrocatechase II was observed . 5 . Low pK'1 values of substituted catechols resulted in low Michaelis constants . 6 . Electron-attracting substituents such as halogen decreased the reaction rates of catechol 1,2-dioxygenation . The correlation of the Vmax . values observed with pyrocatechase II from Pseudomonas sp . B 13 with the substituent constant sigma+ (Okamoto--Brown equation) was distinctly greater than with Hammett's sigma values . The corresponding logVmax . against sigma+ correlation for pyrocatechase I was considerably disturbed by steric influences of the substituents.

Arch Microbiol, 1978 May 30, 117(2), 123 - 9
Mutants of Alcaligenes eutrophus defective in autotrophic metabolism; Schink B et al.; Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions . Four types were characterized with respect to their defects and their physiological properties . One mutant lacked both enzymes specific for autotrophic CO2 fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase . Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate . When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone . Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell . Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO2 . The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.

Anesth Analg, 1978 Mar-Apr, 57(2), 191 - 6
Prevalence and survival of microbial contaminants in heated nebulizers; Spaepen MS et al.; Over a 2-year period, 600 cultures of fluid in heated nebulizers in use by patients in the Peter Bent Brigham Hospital were performed . The most commonly isolated microorganisms were Pseudomonas and Alcaligenes species . Pseudomonas aeruginosa was never isolated . Three types of heated nebulizers were in use, and their contamination rate was significantly different (45 percent, 21 percent, and 8 percent, respectively; p less than 0.001) . In the course of the study, the overall contamination rate decreased from 47 percent to 10 percent . This was mainly due to elmination of the type of heated nebulizer that was most prone to contamination . Five types of currently available large-reservoir nebulizers were inculated with various organisms and growth patterns studied . The various nebulizing equiment differed in its ability to inhibit or eliminate microbial growth; 1 of the heated nebulizers appeared to enhance growth of some of the inoculated bacteria.

Zh Mikrobiol Epidemiol Immunobiol, 1978, (12), 46 - 9
{L-asparaginase activity of microorganisms}; Orovere MIa et al.; L-asparaginase content was studied in 67 different microorganisms, bacterial strains of E . coli, Pseudomonas, Erwinia, Proteus, Alcaligenes, Bacillus, Bacteria . Xanthomonas, and yeasts and fungi-Saccharomyces, Candida, Aspergillus, Epidermophyton, Trichophyton . All the bacterial strains studied possessed L-asparaginase activity, which varied greatly depending on the representative (10-1200 IU/g dry weight of cells) . Yeasts and fungi displayed low L-asparaginase activity.

Vet Med Nauki, 1978, 15(1), 91 - 4
{Effect of drugs on the pyrogenic reactivity of sheep}; Planski B; Investigated was the effect of coffeine, bromine, atropine, and pilocarpine on the course of experimentally induced (by means of an Alcaligenes falcalis lipopolysaccharide) pyrogenic response in sheep . It was noted that coffeine aggravated, and bromine and partly atropine made more feasible the manifestation of the pyrogenic response in these animals . Pilocarpine enhanced some of the functions of the body while this reaction tookplace . Some arguments are given to elucidate the mechanism of the pyrogenic response.

Biochimie, 1978, 60(3), 297 - 305
Hydrogen metabolism in aerobic hydrogen-oxidizing bacteria; Schink B et al.; A survey on organisms able to use molecular hydrogen as electron donor in the energy-yielding process is presented . In the group of the aerobic hydrogen-oxidizing bacteria so far two types of hydrogenases have been encountered, a NAD-reducing, soluble enzyme (H2 : NAD oxidoreductase) and a membrane-bound enzyme unable to reduce pyridine nucleotides . With respect to the distribution of both types of hydrogenases three groups of hydrogen-oxidizing bacteria can be diffentiated containing (i) both types (Alcaligenes eutrophus), (ii) a soluble enzyme only (Nocardia opaca lb), and (iii) a membrane-bound hydrogenase only (majority of genera and species) . The results of studies on the NAD-specific hydrogenase of A . eutrophus are summarized . Results on the solubilization and purification of the membrane-bound hydrogenase of A . eutrophus are presented in detail . The enzyme was solubilized from purified membranes by Triton X-100 and sodium desoxycholate or phospholipase D . The crude membrane extract was fractionated by ammonium sulfate precipitation and chromatography on carboxymethylcellulose at pH 5.5 . The enzyme was stable in potassium phosphate buffer; it resembles the soluble enzyme with respect to stability under oxidizing conditions . Further biochemical and immunological data indicate, however, that both enzymes are different with respect to their native structure.

Appl Environ Microbiol, 1978 Jan, 35(1), 51 - 3
Structural factors influencing the biodegradation of imides; Ennis DM et al.; Comparative studies on the biodegradability of amides and imides are presented . Low-molecular-weight imides of varying chain lengths (4, 6, 7, 8, 18, and 20 carbons) were biodegrable . N-alkyl substitution of amides and imides resulted in non-biodegrable derivatives when the amide portion was greater than two carbons in length . N-alkyl-substituted derivatives of acetamide or diacetamide, however, were biodegrable . Several soil isolates, including Aspergillus niger and species of Flavobacterium and Alcaligenes, were capable of growth with imides as sole N or C sources.

Arch Microbiol, 1977 Dec 15, 115(3), 237 - 47
Isolation and partial characterization of normal and defective bacteriophages of gram-negative hydrogen bacteria; Auling G et al.; Widespread defective lysogeny was detected in Alcaligenes eutrophus by electron microscopic analysis of cultures . Mitomycin C treatment of the cultures resulted in the production of defective (inco-) particles . Polysheaths were produced both with and without induction . With the simultaneous isolation technique six phages were isolated for hydrogen-oxidizing strains of the new species Pseudomonas pseudoflava . The phages were able to replicate under autotrophic conditions and were found to have a very restricted host range . Electron microscopic analysis allowed classification into two structural groups . Group I contained phages with contractile tails; group II contained phages with flexible, noncontractile tails . All but one (gb) of the new phages were shown to be temperate by isolation of lysogens and induction with mitomycin C.

Arch Microbiol, 1977 Sep 28, 114(3), 203 - 10
Alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16 . III . Endproduct inhibition and its relief by valine and isoleucine; Wiegel J et al.; The alpha-isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by L-leucine and alpha-ketoisocaproate . The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH . Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects . The maximal Hill coefficient was found to be two . At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate alpha-ketoisovalerate . The inhibition was specifically relieved by the addition of valine or isoleucine . The anomalous effect of temperature on enzyme activity was diminished by leucine . The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine . The further addition of valine reversed this effect . The physiological relevance of the alpha-ketoisocaproate-mediated inhibition is discussed.

Surgery, 1977 Sep, 82(3), 382 - 5
Antibiotic resistance transfer from nonpathogenic to pathogenic bacteria; Hummel RP et al.; Alcaligenes species is a common contaminant of "wet" environmental areas on the surgical ward . Although thought to be a nonpathogenic organism, recent clinical experience on the burn and trauma service has led us to believe that antibiotic resistance transfer may occur between Alcaligenes and Pseudomonas aeruginosa . To evaluate this possibility, germ-free mice were contaminated with Alcaligenes species, which quickly established in the animals' gastrointestinal tracts . These animals then were burned and the wound was seeded with additional Alcaligenes . After 72 hours the average bacterial count was 4.5 X 10(6) cells/gm of tissue, and all animals survived . Ten additional germ-free mice were contaminated with a resistant (Amikacin, tobramycin, gentamicin, and Sisomicin) Alcaligenes species . When a Pseudomonas aeruginosa strain sensitive to these antibiotics was introduced into the environment, it rapidly overgrew the Alcaligenes species but developed resistance to those four antibiotics to which it had been sensitive previously . These animals were then subjected to a 10 second immersion burn, and the wound was seeded with the same strain of Alcaligenes . The Pseudomonas quickly overgrew the Alcaligens on the burn wound and became established, with an average count being 5.2 X 10(8) cells/gm of tissue . When this experiment was repeated, establishing antibiotic sensitive Pseudomonas in the germ-free animals prior to inoculation of resistant Alcaligenes, the R-transfer again occurred but required a longer time.

Can J Microbiol, 1977 Jun, 23(6), 733 - 50
Numerical taxonomy and ecology of oligotrophic bacteria isolated from the estuarine environment; Mallory LM et al.; Slow-growing bacteria, isolated on nutrient-rich and nutrient-limited media, from Chesapeake Bay water and sediment samples, were examined for 119 biochemical, cultural, morphological, nutritional, and physiological characters . Those bacteria which grow on low nutrient media, termed oligotrophs, a total of 162 strains, were subjected to taxonomic analysis, as a preliminary step in determining their ecological significance . The data for all strains included in the study were examined by computer and the simple matching (S SM) and Jaccard (SJ) coefficients calculated . Clustering was achieved by the unweighted average-linkage method . From sorted similarity matrices and dendrograms, 148 strains, 90% of the total, were recovered in 24 phenetic groups defined at the 80 to 85% similarity level . Only 12 phena could be presumptively identified and these included representatives of Alcaligenes, Corynebacterium, Hyphomicrobium, Hyphomonas polymorpha, Listeria, Nocardia marina, Pedomicrobium, Planococcus citreus, Sphaerotilus, Streptothrix, and Streptomyces . Of the remaining organisms, 10% were unidentified sheathed bacteria . It is concluded that slow-growing bacteria are distributed throughout the estuarine environment and can account for a large proportion of the colonies observed on media after prolonged periods of incubation . The oligotrophic bacteria appear to predominate in areas where the concentration of available nutrients is low and are more characteristic of non-eutrophic aquatic systems.

Arch Microbiol, 1977 May 13, 113(1-2), 145 - 51
{Energy-dependent 63Ni-uptake by Alcaligenes eutrophus strains H1 and H16 (author's transl)}; Tabillion R et al.; Kinetic studies of the uptake of 63Ni were undertaken with two strains of Alcaligenes eutrophus, known to require nickel ions for chemolithotrophic growth . Using carbon dioxide as sole carbon source, growth is stimulated by low concentrations of nickel with optimum concentration for growth stimulation at about 0.3 micron nickel . Higher nickel concentrations were inhibitory . Heterotrophic growth on fructose was not stimulated by nickel ions.--Upon transfer into phosphate buffer freed of heavy metal ions, autotrophically grown cells exhibited rapid uptake of 63Ni which was dependent upon the presence of hydrogen, oxygen and carbon dioxide . Within 60 min nickel was accumulated from the medium, reaching 280-fold concentration in the cells . The observed uptake exhibited a temperature optimum at about 29 degrees C and was markedly inhibited by metabolic inhibitors such as arsenite, iodoacetate, methylene-blue, sodium azide and sodium cyanide . Other heavy metal ions (Zn, Co, Mn and Cu) only slightly inhibited 63Ni-uptake . The efflux of 63Ni from the cells was stimulated by 58NiCl2 and by toluene . These data indicate that nickel ions are accumulated by an energy dependent mechanism in chemolithotrophically grown cells of these strains.

Arch Microbiol, 1977 Apr 1, 112(3), 247 - 54
alpha-Isopropylmalate synthase from Alcaligenes eutrophus H 16 . II . Substrate specificity and kinetics; Wiegel J et al.; The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following alpha-keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): alpha-ketoisovalerate, alpha-keto-n-valerate, alpha-ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, valeryl-CoA, and crotonyl0CoA . alpha-Ketoisocaproate, however, is a strong inhibitor of the enzyme . All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct L-leucine . The substrate saturation curves of alpha-ketoisovalerate or other alpha-keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between nH-values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates . The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (alpha-ketoisovalerate and acetyl-CoA) . Free coenzyme A (1 micronM) inactivated the enzyme irreversibly . The 3'-phosphate of coenzyme A and the free carboxyl group of alpha-ketoisovalerate were involved in optimal binding of these substrates, but 3'-dephospho-acetyl-coenzyme A and the methylester of alpha-keto-isovalerate were also converted by this enzyme . A CH3--CH2-grouping of the alpha-keto acids seemed to be necessary for binding this substrate.

Arch Microbiol, 1977 Apr 1, 112(3), 239 - 46
Alpha-Isopropylmalate synthase from Alcaligenes eutrophus H 16 I . Purification and general properties; Wiegel J et al.; alpha-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of L-leucine, was purified to a specific activity of 12 micronmole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H16 . The activity in crude extracts of derepressed cells was 0.106 micronmoles of isopropylmalate formed per min and per mg protein . Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate alpha-ketoisovalerate, feedback inhibitor leucine or other effectors . The isoelectric points of the enzyme protein was between 3.9 and 4.0 . The molecular weight was 114 500 daltons and 100 000 respectively in the absence and presence of the feedback inhibitor leucine . The enzyme activity depended strongly on the pH, the optimum is at pH 8.2 . The enzyme was could labile and exhibits temperature anomalies.

Arch Microbiol, 1977 Mar 1, 112(2), 169 - 72
Pathways of D-fructose and D-glucose catabolism in marine species of Alcaligenes, Pseudomonas marina, and Alteromonas communis; Sawyer MH et al.; Cell-free extracts of D-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of D-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P2 aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase . The presence of these enzyme activites would allow D-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway . In cell-free extracts of D-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of D-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in D-fructose grown cells . Radiolabeling experiments suggested that both D-fructose and D-glucose were utilized primarily via the Entner-Doudoroff pathway . Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activites necessary for the catabolism of D-fructose and D-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consitent with the results of the radiolabeling experiments.

Folia Microbiol (Praha), 1977, 22(5), 410 - 4
Production of L-aspartic acid from fumaric acid by Alcaligenes metalcaligenes CCEB 312; Plachy J et al.; Among 23 microbial strains, Alcaligenes metalcaligenes CCEB 312 was found to be most suitable for the conversion of fumaric acid to L-aspartic acid . In a growth medium containing 4% peptone, the strain produced as much as 50 g L-aspartic acid per litre.

Biochim Biophys Acta, 1976 Nov 8, 452(1), 66 - 80
Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16; Schneider K et al.; The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein . The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis . Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively . The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive . Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable . The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen . In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity . The reversibility of hydrogenase function (i.e . H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated . With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4 . The apparent Km value for H2 was found to be 0.037 mM . The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm . A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.

Arch Microbiol, 1976 Nov 2, 110(23), 157 - 66
Purification, some properties and quaternary structure of the D-ribulose 1,5-diphosphate carboxylase of Alcaligenes eutrophus; Bowien B et al.; D-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacterium Alcaligenes eutrophus . The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis . The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained . It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000 . Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits . The most probable symmetry of the enzyme molecule is 4:2:2 . Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2+, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air . Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity . The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.

Arch Microbiol, 1976 Nov 2, 110(23), 167 - 71
Glycollate production and excretion by Alcaligenes eutrophus; Codd GA et al.; Autotrophic cultures of the facultative chemolithotroph Alcaligenes eutrophus have been found to excrete glycollate . This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing . The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO2 to the gassing mixture . Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen . No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells . The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction of D-ribulose 1,5-diphosphate carboxylase under the same conditions.

J Clin Microbiol, 1976 Nov, 4(5), 443 - 9
Fluorescent pseudomonads capable of growth at 41 degrees C but distinct from Pseudomonas aeruginosa; Ajello GW et al.; One hundred and twenty-seven apyocyanogenic fluorescent Pseudomonas strains capable of growth at 41 degrees C, but differing from Pseudomonas aeruginosa, were typed serologically and tested for pyocin production, antibiotic susceptibility, selected biochemical reactions, and utilization of selected substrates . Results were compared with those from 40 apyocyanogenic and 14 pyocyanin-producing strains of P . aeruginosa . Unidentified fluorescent Pseudomonas (UFP) strains generally were not agglutinated by P . aeruginosa antisera and showed little or no pyocin activity . In contrast to P . aeruginosa strains, UFP strains usually failed to oxidize D-gluconate or reduce nitrate to nitrogen gas . They could not use D-gluconate or D-mannitol as sole carbon source and were susceptible to kanamycin . The cellular fatty acid compositions of major UFP groups resembled those of the alcaligenes-stutzeri groups.

Arch Microbiol, 1976 Oct 11, 110(1), 129 - 34
Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa; Enouf J et al.; The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa . 0.1 mM of this molecule added to the culture, inhibits the growth of S . typhimurium . The inhibition is specifically reversible by 0.1 mM L-methionine . In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P . aeruginosa and only during the first two generations . The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida . The growth rate of P . testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P . aeruginosa but the other member of the acidovorans group P . acidovorans is much more affected by the smae concentration of the inhibitor . --P . multivorans is inhibited by MAMP like P . acidovorans but with a somewhat higher yield at the end of the culture . --MAMP has no effect on P . alcaligenes . The possible reasons for the weak bacteriostatic effect of MAMP on P . aeruginosa were investigated . It was established that the inhibitor enters the cells and is not used as a carbon and energy source . The intracellular methionine concentration in S . typhimurium and in P . aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP . The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent . Furthermore the tRNAmet from P . aeruginosa are fully acylated after 3 to 4 generations with this compound . Nevertheless MAMP elicits higher MTS activity in P . aeruginosa and in P . acidovorans after 1 h of incubation . The most striking difference between S . typhimurium and P . aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

Ann Microbiol (Paris), 1976 Oct, 127B(3), 373 - 91
{Isolation and study of a new marine bacterium growing on hydrocarbons . I . Physiological study (author's transl)}; Bertrand JC et al.; The bacterial strain L.16.1 isolated from coastal waters polluted by oil-waste, close to the genus Alcaligenes, utilizes preferentially alkanes with a carbon number greater than 9 . Sugars and amino-acids cannot serve as carbon source to this bacterium . Cells grown on hydrocarbon the chain length of which ranges from C10 to C18 exhibit very high yield (98%) with a growth rate of 0.47 . From our studies it appears that strain L.16.1 is strictly dependent on the presence of the Na+ ion and that this Na+ dependence can be seen at each level of the physiological activity . Alkane-grown cells show morphological features namely disc shaped cytoplasmic vesicles (6-8 per cell) . Such vesicles are to be regarded as a consequence of the very high lipid content (twice the standard) which characterizes these cells . Additional lipids belong essentially to the nonsaponifiable fraction (20 times more in hexadecane grown cells); on the contrary, the phospholipid content at both qualitative and quantitative points of view does not depend on the nature of the growth substrate.

Appl Environ Microbiol, 1976 Oct, 32(4), 585 - 91
Quantitative requirements for exponential growth of Alcaligenes eutrophus; Repaske R et al.; Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined . Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively . Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals . Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth . Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.

Appl Environ Microbiol, 1976 Oct, 32(4), 592 - 7
Dense autotrophic cultures of Alcaligenes eutrophus; Repaske R et al.; Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere . It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting . As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h . Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h . The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.

Biochim Biophys Acta, 1976 Aug 13, 440(2), 412 - 28
Respiratory components and oxidase activities in Alcaligenes eutrophus; Probst I et al.; 1 . Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock . By this method, 93% of the in vivo activity of the H2 oxidase is recovered and the ATPase remains particle bound . In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H2 oxidase by 50% and solubilizes the bulk of the ATPase . 2 . The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum) . In addition to cytochrome aa3, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm) . 3 . The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2--3-fold higher than membranes obtained from cells disrupted in a French pressure cell . With the exception of the H2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20-100% stimulation of oxygen uptake) . 4 . The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem . In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs . The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.

J Gen Microbiol, 1976 Aug, 96(2), 287 - 96
Facultative wood-digesting bacteria from the hind-gut of the termite Reticulitermes hesperus; Thayer DW; Among the facultative bacteria capable of growth on mesquite wood which were isolated from the asceptically dissected hind-gut of the termite Reticulitermes hesperus were two strains of Bacillus cereus, one strain each of Arthrobacter, Alcaligenes and Serratia, and a very small Gram-negative fermentative rod . The B . cereus strains, the Serratia marcescens strain and the Arthrobacter sp . grew well on a mineral salts alpha-cellusose agar . One of the Bacillus cereus strains and Serratia marcescens hydrolysed gels of carboxymethylcellulose . All isolates grew well with mesquite wood as the carbon source . The Serratia marcescens isolate produced prodigiosin but differed from a typed strain both in size and in some physiological characteristics.

J Bacteriol, 1976 May, 126(2), 712 - 22
Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus; Friedrich B et al.; Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24% . During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association . The ratio of their specific activities remained almost constant . The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used . Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits . The isoelectric point of the bifunctional enzyme was 5.8 . Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy . Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10% . The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis . Its isoelectric point was pH 6.6 . In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture . The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.

J Bacteriol, 1976 May, 126(2), 723 - 32
Regulation of Chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from alcaligenes eutrophus; Friedrich CG et al.; Highly purified enzymes from Alcaligenes eutrophus H 16 were used for kinetic studies . Chorismate mutase was feedback inhibited by phenylalanine . In the absence of the inhibitor, the double-reciprocal plot was linear, yielding a Km for chorismate of 0.2 mM . When phenylalanine was present, a pronounced deviation from the Michaelis-Menten hyperbola occurred . The Hill coefficient (n) was 1.7, and Hill plots of velocity versus inhibitor concentrations resulted in a value of n' = 2.3, indicating positive cooperativity . Chorismate mutase was also inhibited by prephenate, which caused downward double-reciprocal plots and a Hill coefficient of n = 0.7, evidence for negative cooperativity . The pH optimum of chorismate mutase ranged from 7.8 to 8.2; its temperature optimum was 47 C . Prephenate dehydratase was competitively inhibited by phenylalanine and activated by tyrosine . Tyrosine stimulated its activity up to 10-fold and decreased the Km for prephenate, which was 0.67 mM without effectors . Tryptophan inhibited the enzyme competitively . Its inhibition constant (Ki = 23 muM) was almost 10-fold higher than that determined for phenylalanine (Ki = 2.6 muM) . The pH optimum of prephenate dehydratase was pH 5.7; the temperature optimum was 48 C . Prephenate dehydrogenase was feedback inhibited by tyrosine . Inhibition was competitive with prephenate (Ki = 0.06 mM) and noncompetitive with nicotinamide adenine dinucleotide . The enzyme was further subject to product inhibition by p-hydroxyphenylpyruvate (Ki = 0.13 mM) . Its Km for prephenate was 0.045 mM, and that for nicotinamide adenine dinucleotide was 0.14 mM . The pH optimum ranged between 7.0 and 7.6; the temperature optimum was 38 C . It is shown how the sensitive regulation of the entire enzyme system leads to a well-balanced amino acid production.

Arch Microbiol, 1976 Mar 19, 107(2), 125 - 31
Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16 III . Properites and regulation of anthranilate synthase; Friedrich CG et al.; Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated . Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements . During the purification procedure the enzyme was stabilized by 50 mM L-glutamine or during chromatography on DEAE- cellulose and Sephadex G-200 with 30% glucerol, respectively.

Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1976, 131(8), 678 - 91
{Adenosine-dependent death of Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16 (author's transl)}; Kaltwasser H et al.; Heterotrophic growth with fructose and autotrophic growth with hydrogen and carbon dioxide was entirely inhibited by adenosine at a concentration of 0.6 mg/ml in Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16 . Growth inhibition was not accompanied by a detectable uptake of the nucleoside . Adenosine caused a rapid inhibition of protein synthesis, followed by a decrease in nucleic acid formation . Enzyme synthesis was also impared, whilst cell respiration remained uneffected . Adenosin also caused a drastic but temporary decrease in viable cell counts . Cells incubated in presence of adenosine and fructose for several days, however, were no longer susceptable to this nucleoside . Adenosine-dependent growth inhibition turned out to be contingent upon the nature of the organic substrate . Cells growing with succinate, glutamate or peptone were less effected than cells, growing autotrophically or with fructose . No inhibition was observed in fructose-growing cells, if amino acids were also present in the medium . Several other nucleosides tested, did not show such growth inhibition, except desoxyadenosine, which, however, did not effect viable cell counts.

Biochimie, 1976, 58(7), 843 - 54
{Hydrocarbon metabolism in a marine bacterium}; Bertrand JC et al.; The marine bacterium L.16.1 (Alcaligenes sp.) grows preferentially on alkanes (C10 to C18) with a very high growth yield (98 per cent); optimal growth depends strictly on the presence of a well-defined NaCl concentration (100 mM) . Our strain is constitutive for the enzymatic systems responsible for the oxidation of alkanes to fatty acids, i.e . NADH-dependent hydroxylase, alcohol and aldehyde dehydrogenases, the latter of which located at the cytoplasmic membrane level . The aerobic oxidation of primary alcohols by particulate extracts prepared in the presence of 400 mM NaCl is NAD+-dependent (Km = 0.082 mM, Vmax = 238 with decanol) . With extracts prepared in the absence of NaCl, Vmax undergoes a very strong decrease . On the contrary , the NAD+ (P)+-dependent oxidation of aldehydes is carried out anaerobically by the same extracts irrespective of the presence or the absence of added Na+ in the solutions used for the preparation of these extracts . A possible explanation for our results could be that Na+ acts on the enzymatic systems for which the maintenance of the membrane integrity is essential . This interpretation is consistent with the slowing down of the growth speed accompanying the decrease of NaCl concentration in the growth medium . With regard to alcohol and aldehyde-dehydrogenases, it is noteworthy that these enzymes behave like similar enzymatic activities induced by alkanes in other microorganisms.

J Biol Chem, 1975 Nov 10, 250(21), 8416 - 21
Amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1; Meyer TE et al.; The amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1 has been determined and is consistent with homology to cytochrome c' from the nonphotosynthetic bacterium Alcaligenes sp . NCIB 11015 . There is 29% identity in the chosen alignment of these two proteins . R . rubrum cytochrome c' is composed of a single peptide chain of 126 amino acid residues with a single heme covalently bound near the COOH terminus . There is no sequence similarity to mitochondrial cytochrome c, except at the heme binding site.

Zentralbl Bakteriol {Orig A}, 1975 Nov, 233(3), 342 - 6
{About the decomposition of acetamide as taxonomic marker for some species of the genus pseudomonas (author's transl)}; Schubert RH et al.; The comparative examination of the acetamide-decompostition and of the acetamide-utilisation as sole source of carbon demonstrates that various groups of the Pseudomonas species with the same kind of reaction have to be distinguished . The acetamide-decomposition-test according to BUHLMANN et al . and the methylene-blue-reduction-test show homogeneous results . As a positive result of these tests the formation of ammonia has always been shown . An acetamide decomposition of this kind has been demonstrable with P . aeruginosa, P . acidovorans and many P . cepacia strains . On liquid mineral media containing acetamide P . fluorescens, P . putida and P . pseudomallei show growth although ammonia is not produced and the test according to BUHLMANN et al . as well as the methylene-blue-reduction-test result negatively . In all tests P . alcaligenes, P . marginata, P . caryophilli and with certain exceptions P . sutzeri and P . putrefaciens react negatively.

Arch Microbiol, 1975 Oct 27, 105(2), 109 - 15
Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo; Wilke D et al.; The biosynthesis of the enzyme pyruvate kinase (E.C . 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source . After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 mumoles/min-g protein, respectively) . The pyruvate kinase from autotrophically grown cells was purified 110-fold . The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose 5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate . In addition to phosphate ATP caused inhibition but in this case nonsigmoidal kinetics was obtained . The half maximal substrate saturation constant S0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM . The corresponding Hill values were 0.96, 1.1 and 2.75 . The ADP saturation curve was hyperbolic even in the presence of the effectors, the Km value was 0.14 mM ADP . When the known intracellular metabolite concentrations in A . eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.

Zhonghua Min Guo Wei Sheng Wu Xue Za Zhi, 1975 Sep, 8(3), 213 - 7
Extracellular deoxyribonuclease activity of Pseudomonas aeruginosa; Tseng CC et al.; Pseudomonas aeruginosa cultured on deoxyribonucleic acid (DNA) media containing Nissan's heart extract or chicken heart extract could produce extracellular deoxyribonuclease, while on other DNA media with heart extracts from cow, pig and mouse could not . Variation in the kind of peptones in the DNA media did not make significant difference in this activity, although some peptones caused cloudiness of the media . P . aeruginosa did not need cation activators of chloride compounds to produce extracellular deoxyribonuclease . On the modified Eiken's DNA medium, gram negative bacilli other than P . aeruginosa and Serratia marcescens, i.e . Klebsiella, Salmonella, Shigella, Escherichia coli and Alcaligenes did not produce deoxyribonuclease . The production of extracellular deoxyribonuclease activity on the modified Eiken's DNA medium can be used as a supporting test for biochemical identification of P . aeruginosa.

Arch Microbiol, 1975 Apr 7, 103(2), 141 - 9
Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16 . II . The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine; Friedrich B et al.; 1 . Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method . Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase . Both activities were regained by reversion to prototrophy . The auxotrophic strains accumulated chorismic acid . (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion . Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold . Mutants grown in minimal medium excreted prephenic acid . (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate . The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present . Some of them were even more active than in the wild-type . 2 . Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough . The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth . 3 . A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16 . Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.

Arch Microbiol, 1975 Apr 7, 103(2), 133 - 40
Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16 . I . Properties and regulation of 3-deoxy-d-arabino heptulosonate 7-phosphate synthase; Friedrich CG et al.; Properties and regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP-synthase), EC4.1.2.15, from Alcaligenes eutrophus H16 were investigated . DAHP synthase was unstable during manipulations such as dialysis, dilution, ammonium sulfate fractionation, chromatography on DEAE-cellulose or Sephadex G-200 . For kinetic measurements Sephadex G-25 treated crude extracts were used . The enzyme was not affected by thiol reagents, EDTA or divalent metal ions . The activation energy, deltaH, amounted to 16100 cal/mole . Between pH 7.2 and pH 8.2 there was little change of enzyme activity . The Km-values for the two substrates were found to be 0.043 mM phosphoenolpyruvate and 0.055 mM erythrose-4-phosphate . DAHP-synthase was inhibited by 0.5 mM phenylalanine for 60% and by 0.5 mM tyrosine for 20% . In the presence of both amino acids cumulative inhibition occurred amounting to about 70% . No other amino acid exerted inhibitory effects . A repression of DAHP-synthase by the aromatic amino acids was not observed . Some other strains of hydrogen bacteria were included in this study . The DAHP synthase from strain 12/60/X and Corynebacterium autotrophicum 7C was unregulated . The enzyme from strain 33/X was subject to retro-tyrosine inhibition and from strain 3/2, H1 and H20 were subject to cumulative inhibition.

Arch Microbiol, 1975 Mar 12, 103(1), 21 - 30
Kinetics and properties of beta-ketothiolase from Clostridium pasteurianum; Berndt H et al.; 1 . Beta-Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite . Subjected to gel electrophoresis beta-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated . As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000 . 2 . The condensation reaction was measured by a coupled optical test using beta-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates . Beta-Ketothiolase from C . pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25% . When the regenerating system was added the condensation reaction continued . The enzyme from C . pasteurianum was inactivated by free coenzyme A, while the enzyme from A . eutrophus was inhibited . When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity . With acetyl-CoA as the substrate the Km-value was found to be 2.5 mM acetyl-CoA . If free CoASH (or acetyl-CoA) plus regenerating system was added the Km was 0.44 mM (0.42 mM) acetyl-CoA . 3 . The beta-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm . The activity was maximal at 24 nM MgCl2 . The apparent Km values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively . The Km-values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively . The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inihibition was partially relieved by CoASH . In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed . The Km for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively . High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction . With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the beta-ketothiolases of C . pasteurianum and A . eutrophus were rather similar.

Natl Inst Anim Health Q (Tokyo), 1975 Spring, 15(1), 29 - 37
Nasal infection of Alcaligenes bronchisepticus (Bordetella bronchiseptica) and lesions in newborn rabbits; Maeda M et al.; The mode of experimental infection with Alcaligenes bronchisepticus (Bordetella bronchiseptica) of pig and rabbit origin, and lesions in the respiratory organs were examined in three groups, A, B and C, of newborn rabbits . Groups A and C were free from the organism and agglutinating antibody and inoculated with the organisms of pig and rabbit origin, respectively . Group B had maternal antibody and was inoculated with the organism of pig origin . Establishment and persistence of infection with the organism were certified in the nasal cavities, trachea, or lungs of groups A and C 3 days after inoculation or later . Late establishment of infection occurred in trachea and lungs of group B . Agglutinating antibody was detected in groups A and B mostly 14 days after inoculation or later . Ventral turbinate atrophy occurred in groups A and B mostly 10 days after inoculation or later . Histologically, it was hypo-osteogenesis caused by degeneration of osteoblasts and proliferation of fibroblast-like cells in the osseous tissue . Catarrhal inflammation in the nasal and tracheal mucous membranes, and bronchopneumonia with peribronchiolitis developed commonly in all the groups . The fluorescent antibody technique revealed antigen of the organism of pig origin on the nasal mucosa, mostly of the dorsal and ventral meatus, and on the tracheal and bronchiolar mucosa in groups A and B.

Antonie Van Leeuwenhoek, 1975, 41(4), 465 - 77
Oxalate and formate in Alcaligenes and Pseudomonas species; Chandra TS et al.; Oxalate is metabolized by the glycerate pathway involving glyoxylate carboligase in Alcaligenes LOx and Pseudomonas KOx, and by the serine pathway involving hydroxypyruvate reductase in Ps.MOx and Ps.AM1 (var . 470) . Although A.LOx does not grow on formate, stimulation of growth was observed in the presence of amino acids and a few Kreb's cycle intermediates . A.LOx possesses two different mechanisms for the oxidation of formate: (1) the constitutive formate oxidase which is present in the particulate fraction of oxalate-grown and succinate-plus-formate-grown cells; (2) the inducible NAD-linked formate dehydrogenase present in the 100 000 x g supernatant fraction of the cell-free extracts of oxalate-grown cells alone . The two systems occur simultaneously in oxalate-grown cells . The effect of inhibitors on formate oxidase activity and the other enzyme activities of the particulate formate-oxidizing fraction indicate that the oxidation of formate is linked to the respiratory chain.

Antonie Van Leeuwenhoek, 1975, 41(1), 101 - 11
Isolation and characterization of some new oxalate-decomposing bacteria; Chandra TS et al.; Forty-one cultures degrading and assimilating oxalate were isolated from chicken dung . Characterizarion indicated six different types . One of these belonged to thhe genus Alcaligenes hitherto never reported to degrade oxalte . Three groups of Pseudomonas strains differed physiologically from strains already known.

N Y State J Med, 1968 Nov 1, 68(21), 2769 - 70
Semen analysis in unilateral epididymitis; Tozzo PJ; PIP: Semen analyses were done on 16 men between 18 and 45 years of age who had unilateral nongonorrheal epididymitis, to evaluate its effect on the sperm . 8 men were black and 8 were white; average age was 28 years . 5 had had children before the onset of the disease but none had impregnated their wives since becoming ill . Patients whose past history or physical examination revealed factors that might contribute to abnormal semen analysis were eliminated from the study . Semen specimens were taken at intervals of from 5 days-1 1/2 years after onset of acute epididymitis . Chronic prostatitis with Bacteriaceae alcaligenes urinary tract infection was seen in 2 cases . Average ejaculate volume was below 2.4 cc in 9 of the 16 subjects, 4 had average volumes between 2.4-3.3 cc, and the remaining 3 had volumes above 3.3 cc . 1 case was azoospermic and 6 had average counts below 20 million/cubic cm . Abnormal motility (less than 40% active sperm/field) was seen in 3 of 16 cases . At no time were fewer than 60% oval forms seen . It has been estimated that in normal men, after 3 days of continence, sperm count should be at least 20 million/cubic cm . 40% of the sperm should be motile, and at least 60% should be normal in structure . In this series a man with a count of 5 million/cubic cm had 2 children by his 1st wife before the onset of epididymitis but was unable to impregnate his 2nd wife in 6 months of follow-up . Sterility work-up by the wife's gynecologist was negative .

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