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