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The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO2-dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.

A novel enzymatic reaction involved in the metabolism of aliphatic epoxides by Xanthobacter strain Py2 is described. Cell extracts catalyzed the CO2-dependent carboxylation of propylene oxide (epoxypropane) to form acetoacetate and beta-hydroxybutyrate. The time courses of acetoacetate and beta-hydroxybutyrate formaton indicate that acetoacetate is the primary product of propylene oxide carboxylation and that beta-hydroxybutyrate is a secondary product formed by the reduction of acetoacetate. Analogous C5 carboxylation products were identified with 1,2-epoxybutane as the substrate. In the absence of CO2, propylene oxide and 1,2-epoxybutane were isomerized to form acetone and methyl ethyl ketone, respectively, as dead-end products. The carboxylation of short-chain epoxides to beta-keto acids is proposed to serve as the physiological reaction for the metabolism of aliphatic epoxides in Xanthobacter strain Py2.

This paper describes the development of an electroporation procedure for Xanthobacter autotrophicus GJ10. Electroporation has not previously been described for this species. The procedure was optimized with respect to field strength, pulse time and DNA and cell concentration. High transformation efficiencies of up to 2×10 6 cells transformed per μg DNA were found. The conditions optimal for the X. autotrophicus GJ10 were used to electrotransform 9 other Xanthobacter strains 4 of which were transformed with high efficiencies. The electrotransformation procedure described here can be of great help for the genetic availability of Xanthobacter species.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene-utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 x 10(-6) mol m(-2) s(-1) at a propene concentration of 9.3 x 10(-2) mol m(-3) in the gas phase. (c) 1995 John Wiley & Sons, Inc.

The epoxyalkane-degrading enzyme of Xanthobacter Py2 was studied in crude extracts. NAD and a low molecular weight fraction which can be replaced by DTT are necessary for the conversion of epoxyalkanes into ketones. The genes responsible for the epoxyalkane-degrading enzyme were cloned and expressed in Xanthobacter autotrophics GJ10.

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the alpha/beta hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.

Azorhizobium caulinodans strain ORS571 is a Gram-negative bacterium, related to Xanthobacter and more distantly to Bradyrhizobium (Dreyfus et al., 1988); it is a symbiont of the tropical legume Sesbania rostrata on which it induces nodules at predetermined sites: at the bases of lateral roots and at the dormant root primordia that are present on the S. rostrata stem as an adaptation to flooding. Stem and root nodulation of S. rostrata follow a similar pattern (Duhoux, 1984; Ndoye, 1991): bacteria infect by crack entry and proliferate intercellularly; they induce the formation of meristematic nodule foci and migrate towards the meristematic tissue through infection threads.

Soil from a pesticide disposal site was used to enrich for microorganisms that degraded the acylanilide herbicide propachlor (2-chloro-N-isopropylacetanilide). After seven transfers of the enrichment, the culture contained about six strains. The highest yield of microbial biomass occurred if just two of these isolates, strains DAK3 and MAB2, were inoculated into a mineral salts medium containing propachlor. When only strain DAK3 was grown on propachlor, a metabolite (2-chloro-N-isopropylacetamide) was released into the medium. Strain MAB2 could grow on this metabolite. The results of morphological and physiological tests suggest that strains DAK3 and MAB2 most closely resemble species belonging to the genera Moraxella and Xanthobacter, respectively. Strain DAK3 can respire and grow on N-substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but is incapable of respiration or growth on acetanilide, aniline, or the acylanilide herbicides alachlor and metolachlor. Strain DAK3 appears to use the aromatic C atoms of propachlor for growth, as suggested by the growth yield on propachlor and the induction of catechol 2,3-oxygenase activity in acylanilide-grown cells.

The structure of an acidic polysaccharide secreted by a Xanthobacter sp. has been investigated by glycosyl-residue and glycosyl-linkage composition analyses, and the characterization of oligoglycosyl fragments of the polysaccharide has been carried out by chemical analyses, 1 H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry, and electron-impact mass spectrometry. The polysaccharide, which contains O -acetyl groups (∼5%) that have not been located, has the tetraglycosyl repeating unit 1 and belongs to a group of structurally related polysaccharides synthesized by both Alcaligenes and Pseudomonas species.

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicusGJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloro- acetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson -Boltzmann equation explained 93% (74% cross- validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.

The isoprenoid compounds in gram-negative methanol-, methane-, and methylamine-utilizing bacteria were investigated. All strains tested contained ubiquinone, but none contained menaquinone. The ubiquinone types were Q-8, Q-9, or Q-10. The so-called obligate methylotrophs and methanotrophs (genus Methylobacillus, Methylophaga, Methylomonas, Methylococcus, and Methylovibrio) contained ubiquinone Q-8. The Hyphomicrobium strains contained Q-9. The other facultative methylotrophs and methylamine-utilizing bacteria contained Q-10. A large amount of squalene occurred in the Methylobacillus, Methylophaga, Methylomonas, and Methylococcus strains which utilize one-carbon compounds via the ribulose monophosphate pathway. The Protomonas extorquens and Methylobacterium organophilum strains contained a large amount of sterols (Hop-22(29)-ene and Hopan-22-ol), carotenoid pigments, and a small amount of squalene. The Hyphomicrobium strains contained a small amount of squalene and Hop-22(29)-ene. The Xanthobacter strains contained a large amount of carotenoid pigments (zeaxanthin, zeaxanthin monorhamnoside, and zeaxanthin dirhamnoside). The Protomonas and Methylobacterium strains were unique in the existence of sterols and large amounts of total isoprenoid compounds, 4.68 to 7.97mg/g of dry cell. The distribution of squalene, sterols, quinones, and carotenoid pigments conforms with the morphological, physiological, and other chemo- taxonomic characteristics in gram-negative methanol-, methane-, and methylamine-utilizing bacteria.

Yellow-pigmented bacteria showing typical characteristics of Xanthobacter spp. were isolated from enrichments with propene and 1-butene, using classical techniques. The generation time for growth on propene and 1-butene of these bacteria ranged from 5 to 7h. A NADH-dependent mono-oxygenase was identified in cell-free extract of Xanthobacter Py2. This mono-oxygenase was not influenced by potential inhibitors tested indicating that propene mono-oxygenase is different from other hydrocarbon mono-oxygenases described until now. Nitrogenase activity could be measured using the acetylene reduction assay with propene as energy source, because acetylene did not inhibit the mono-oxygenase activity.

DNA base composition was determined in 73 strains of aerobic, gram negative, methanol-utilizing bacteria, which are divided into nine groups on the basis of chemotaxonomic characteristics and utilization of carbon compounds. Guanine plus cytosine (G+C) contents in their DNAs ranged from 50.0 to 68.8mol% G+C. Group 1 bacteria (obligate methanol -utilizing bacteria) and group 4 bacteria (Hyphomicrobium strains) showed rather wide distributions in DNA base composition, 50.0 to 56.0 mol% G+C and 59.3 to 65.6mol% G+C, respectively. In contrast, bacteria of group 2 (Protomonas strains), group 3 (Microcyclus strains), group 5 (Xanthobacter strains), group 6, group 7, and group 8 (Paracoccus denitrificans strains) had narrow ranges in DNA base composition. Group 9 (Thiobacillus novellus) had 67.7mol% G+C. DNA-DNA homologies among the strains selected from the above-mentioned groups indicated a clear separation of the group 1 from the other groups. Further, group 1 was divided into six subgroups but not enough phenotypic characteristics were found to separate them from each other. Therefore, despite the heterogeneity of group 1 suggested by DNA base composition, DNA-DNA homology relatedness, and other chemotaxonomic characteristics, this group of bacteria are considered to be a single species from the practical point of view.

The ability of three species of Xanthobacter to metabolize cyclohexane and its derivatives has been compared. Xanthobacter flavus was unable to utilize any of the cycloalkanes under investigation. X. autotrophicus was unable to utilize cyclohexane but was able to grow with a limited range of substituted cycloalkanes, including cyclohexanol and cyclohexanone. Comparison of a previously isolated cyclohexane growing Xanthobacter sp. with X. flavus and X. autotrophicus indicated it to be closely related to X. autotrophicus. Studies with cell-free extracts have indicated that the route of metabolism for cyclohexanol by X. autotrophicus is the same as that shown for the cyclohexane growing Xanthobacter sp., proceeding via cyclohexanol→cyclohexanone→ ε-caprolactone→→ adipic acid. A comparison of the cyclohexanol dehydrogenase found in X. autotrophicus with that found in the cyclohexane-growing Xanthobacter sp. indicated these enzymes to be distinctly different from one another on the basis of substrate specificity, molecular weight, and pH optima. The cyclohexanone monooxygenase enzymes found in the two bacteria were also found to be different when the pH optima and cofactor specificity of the two enzymes were compared. Preliminary genetic studies on the cyclohexane-growing Xanthobacter sp. have indicated that there are no plasmids present in this bacterium. The presence of RP4 in the Xanthobacter sp. can be detected following its conjugation with an RP4-carrying Escherichia coli strain.

Styrene oxide and 2-phenylethanol metabolism in the styrene-degrading Xanthobacter sp. strain 124X was shown to proceed via phenylacetaldehyde and phenylacetic acid. In cell extracts 2-phenylethanol was oxidized by a phenazine methosulfate-dependent enzyme, probably a pyrroloquinoline quinone enzyme. Xanthobacter sp. strain 124X also contains a novel enzymatic activity designated as styrene oxide isomerase. Styrene oxide isomerase catalyzes the isomerization of styrene oxide to phenylacetaldehyde. The enzyme was partially purified and shown to have a very high substrate specificity. Of the epoxides tested, styrene oxide was the only substrate transformed. The initial step in styrene metabolism in Xanthobacter sp. strain 124X is oxygen dependent and probably involves oxidation of the aromatic nucleus.

Summary: Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AMI. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AMI contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AMI.

An unusual Xanthobacter sp., capable of independent growth on cyclohexane as the sole source of carbon and energy, has been isolated from soil by using classical enrichment techniques. The mean generation time for growth on cyclohexane was 6 h. The microorganism showed a limited ability to utilize hydrocarbons, with only alicyclic hydrocarbons closely related to cyclohexane supporting growth. Ultrastructural studies indicated the presence of electron-transparent vesicles in the cyclohexane-grown Xanthobacter sp., but the presence of complex intracytoplasmic membranes could not be identified. A soluble inducible enzyme capable of oxidizing cyclohexane was identified in cell extracts. This enzyme had a pH optimum of 6.5, an absolute specificity for NADPH, and a stoichiometric requirement for molecular O 2 which was consistent with the formation of cyclohexanol. The enzyme showed no activity towards straight chain alkanes and only a limited activity towards unsaturated ring compounds. Enzymatic studies with cell extracts have indicated the main route of metabolism of cyclohexane by this Xanthobacter sp. to proceed via cyclohexane → cyclohexanol → cyclohexanone → 1-oxa-2-oxocycloheptane (ε-caprolactone) → 6-hydroxyhexanoate (6-hydroxycaproate) → → adipic acid. Alternative routes involving initial double hydroxylation of the cyclohexane ring may operate fortuituously but are unlikely to represent major pathways for the dissimilation of cyclohexane by this microorganism.

Summary: Two methylotrophic bacteria having similar cell morphologies have been isolated from cultures of “Methylobacterium ethanolicum” grown on methane. One of these, strain POC, is an obligate methanotroph containing the serine pathway and Type II intracytoplasmic membranes, which appears to be a “Methylocystis” species. The second organism, strain H4-14, can fix N2 and grows on a variety of substrates, including methanol, formate, ethanol, succinate, fructose and H2 + CO2; during growth on methanol, carbon is assimilated via the Calvin-Benson cycle. Strain H4-14 appears to be a Xanthobacter species. Mixtures of the two organisms formed stable associations during growth on methane, consisting of approximately 5-20% H4-14 and 80-95% POC. Strain POC excreted biotin, which was required by strain H4-14. Our results suggest that “Methylobacterium ethanolicum” cultures grown on methane consist of a syntrophic association of two methylotrophs.

Summary The taxonomic relationships among strains described as or previously tentatively assigned to the genus Xanthobacter were investigated using numerical classification, DNA base composition and DNA-DNA hybridization. Both numerical analysis and DNA-DNA hybridization studies gave similar groupings. Three main clusters were identified: the first can be equated with Xanthobacter autotrophicus , the type species of the genus. The second cluster contains the type strain of Xanthobacter flavus and several other biotin-requiring strains, leading to an emendation of the original description of X. flavus . The third cluster comprises motile strains which probably represent another species of the genus Xanthobacter .

Bacteria isolated from soil, canal water, and sewage utilised short-chain (C1–C4) primary alkyl sulphate esters as sources of carbon and energy. Butyl and propyl sulphates, but not the C1 and C2 homologues, were degraded by a soil isolate (coryneform rod, strain B1a) which liberated SO2−4 stoicheiometrically from growth-supporting esters. Cell-extracts contained a sulphatase active towards C3–C7 primary alkyl sulphates, and this enzyme was presumed responsible for initiating the degradation. A sewage isolate (Xanthobacter sp. E5a) utilised exclusively ethyl sulphate, and a canal water isolate (strain M3C, tentatively assigned to the genus Agrobacterium) used only methyl sulphate. Strains E5a and M3c liberated SO2−4 stoicheiometrically from ethyl and methyl sulphates, respectively, but efforts to detect corresponding sulphatases in cell extracts failed, suggesting that metabolism of the C1 and C2 esters was initiated by an alternative, possibly oxidative, step. A Hyphomicrobium strain isolated from soil grew either aerobically or as a denitrifier in methyl sulphate minimal medium.

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

SUMMARY: Seven mutants of Xanthobacter H4-14, unable to grow on methanol but capable of growth on formate, were isolated and complemented with a chromosomal clone bank constructed in the broad-host-range cosmid pVK100. One mutant could not be complemented but the others fell into four distinct complementation groups that involved three different recombinant clones. All of the complementing regions were separated by at least 10 kbp. The five complementation classes had different phenotypic characteristics and were defective in different aspects of methanol and formaldehyde oxidation. Class I mutants were defective in methanol oxidation, class II mutants were impaired in formaldehyde oxidation, class III mutants appeared to be defective in a regulatory element involving the methanol oxidation system, and class IV mutants appeared to be defective in a regulatory element involving formaldehyde oxidation. Class V mutants exhibited a methanol-sensitive phenotype, which was correlated with an imbalance between methanol and formaldehyde dehydrogenase activities. Analysis of this class suggested it was defective in a repressor that regulated methanol dissimilation functions.

Summary Production of (α-amylase, cellulase, protease, pectinase and lipase by 17 N 2 -fixing microorganisms isolated from different sources was studied. Cellulase and pectinase activities were found to be inducible. Azospirillum brasilense and Xanthobacter flavus were the most active α-amylase and pectinase producers. Protease was produced in appreciable quantities by all the microorganisms. All microorganisms also produced cellulase and lipase in varying degrees. The N 2 -fixing strains of Klebsiella -KUP 4 , KUPBR 2 , and KUPM as well as the 2 unidentified strains KUP 6 D and KUP 7 D isolated from leaf surfaces of different plants, and which had proven to be beneficial on the leaf surface for growth and nitrogen nutrition of crop plants, also produced hydrolases actively.

Eleven strains of alkene-utilizing bacteria belonging to the genera Mycobacterium, Nocardia, and Xanthobacter were tested for their ability to grow with C 1 to C 6 alkanes, C 2 to C 6 alkenes, alkadienes, and monoterpenes furnished individually as sole sources of carbon and energy in a mineral salts medium. A limited number of alkenes and alkanes supported growth of the bacteria; some bacteria were unable to grow on any of the saturated hydrocarbons tested. Monoterpenes were frequently used as carbon and energy sources by alkene-utilizing bacteria belonging to the genera Mycobacterium and Nocardia. Washed cell suspensions of alkene-grown bacteria attacked the whole range of alkenes tested, whereas only three strains were able to oxidize alkanes as well. The alkenes tested were oxidized either to water and carbon dioxide or to epoxyalkanes. Few epoxides accumulated in stoichiometric amounts from the corresponding alkenes, because most epoxides formed were further converted to other compounds like alkanediols.

Mycobacterium flavum strain 301 (= Deutsche Sammlung von Mikroorganismen 338), a hydrogen-utilizing bacterium, is capable of fixing molecular nitrogen and resembles other nitrogen-fixing hydrogen bacteria. However, it is clearly different in many characters from other strains of M. flavum (Orla-Jensen) Jensen (syn.: Microbacterium flavum Orla-Jensen). It does resemble strains of Xanthobacter Wiegel et al. with respect to cell wall composition, production of carotenoid pigments, carbon source utilization pattern, and deoxyribonucleic acid (DNA) base composition (69 mol% guanine + cytosine). Strain 301 is here regarded as belonging to a new and distinct species, for which the name Xanthobacter flavus is proposed. Strain 301 is the type strain of this species. X. flavus differs from Xanthobacter autotrophicus, the only other species in this genus to date, in several respects, and the DNA-DNA hybridization between X. flavus and X. autotrophicus is only 25%.

Thirty-five nitrogen-fixing, hydrogen-oxidizing bacteria, all members of Cory-nebacterium autotrophicum Baumgarten, Reh, and Schlegel 1974, and including the type strain of this species, were compared with 28 strains of coryneform bacteria by numerical taxonomy methods. The nitrogen-fixing hydrogen bacteria formed a well defined cluster; their similarity to the other strains tested was low, however. Furthermore, the chemotaxonomic characters of these strains excluded them from the coryneform bacteria. Therefore C. autotrophicum was reclassified, following the keys of Bergey's Manual of Determinative Bacteriology (8th ed.) as a species of the family Azotobacteraceae. Although taxonomically close to Beijerinckia and Derxia, the nitrogen-fixing, hydrogen-oxidizing bacteria are regarded as representing a new genus, for which we propose the name Xanthobacter. C. autotrophicum Baumgarten, Reh, and Schlegel is transferred to the new genus as Xanthobacter autotrophicus (Baumgarten et al.) comb. nov.

N2-fixation was investigated in the chemoautotrophic hydrogen bacterium Xanthobacter H4-14. N2-fixing batch cultures of this organism could only be grown at pO2 values of around 0.02 bar, and in continuous culture dissolved oxygen tensions above 16 μM were found to inhibit N2-fixation. Xanthobacter H4-14 utilized a variety of amino acids, nitrate and ammonia as nitrogen sources. Cell-free extracts from steady-state continuous cultures of ammonia grown, nitrate grown and N2-fixing Xanthobacter were assayed for the presence of ammonia assimilation enzymes. No alanine dehydrogenase or glutamate dehydrogenase activity was detected. Ammonia was assimilated exclusively via the glutamine synthetase/glutamate synthase pathway, irrespective of the extracellular concentration of ammonia.

Newly isolated Xanthobacter spp. were able to grow on the gaseous alkenes like ethene, propene, 1-butene and 1,3-butadiene. Resting-cell suspensions of propene-, 1-butene- or 1,3-butadiene-grown Xanthobacter Py10 accumulated 1,2-epoxyethane from ethene. Ethene-grown Xanthobacter Py10 did not produce any 1,2-epoxyalkane from the alkenes tested. Furthermore, propenegrown Xanthobacter Py2 accumulated 2,3-epoxybutane from trans-butene and cis-butene but did not form epoxides from other substrates tested.

Xanthobacter 124X when grom on 4-hydroxyphenylacetate was able to hydroxylate this compound yielding homogenisate. Ring fission of this latter compound gave maleylacetoacetate which was isomerized to fumarylacetoacetate. The isomerase involved resembled maleylacetoacetate isomerases in Gram-negative bacteria in that glutathione was required for activity. Fumarate and acetoacetate were both detected as products of the hydrolysis of fumarylacetoacetate.

A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, Xanthobacter sp. CP, was isolated after enrichment in aerated soil columns. A limited number of chlorinated phenols and chlorinated phenoxyalkanoic acids with an even number of carbon atoms in the side chain served as substrates for growth, although whole cells exhibited oxygen uptake with a wide range of those compounds. The maximal growth rate with 2,4-D was 0.13·h-1 at a growth yield of 0.1 g biomass/g 2,4-D. Chloride ions were released quantitatively from 2,4-D and related chlorinated aromatic compounds which served as growth substrates. No by-products of 2,4-D metabolism were detected in oxygen-sufficient cultures of Xanthobacter sp. CP and catechols were cleaved exclusively by catechol 1,2-dioxygenase.

With propene-grown cells of Xanthobacter Py2 it was possible to resolve racemic mixtures of 2,3-epoxyalkanes. Only 2S-forms were metabolized by this organism, resulting in pure 2R-2,3-epoxyalkanes. Chiral resolution was obtained with trans-2,3-epoxybutane, trans-2,3-epoxypentane and cis-2,3-epoxypentane. Xanthobacter Py2 was however not able to discriminate between the enantiomeric forms of 1,2-epoxyalkanes, resulting in the complete degradation of both chiral forms of 1,2-epoxyalkanes.

Immobilization of Mycobacterium PY1 and Xanthobacter PY2 in alginate and in or on hydroculture has a minor influence on the maximum rate of oxidation of propene and ethane. The apparent K(m) values of the immobilized cells are slightly higher than those of the free cells, indicating the presence of diffusion limitation in the immobilized systems. Both bacterial strains rapidly lose their alkene-oxidizing activity when the water activity is decreased. This decrease in activity is so rapid that most of the activity is lost already when the pores of the pertinent supports are still filled with water. Therefore, it is not possible with this system to study the transition of mass transfer of substrates entirely in the water phase to mass transfer to substrates entirely through the gas phase.

The aerobic nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus strain GZ29 [1] can grow autotrophically with hydrogen, oxygen, carbon dioxide, and molecular nitrogen as sole sources of electrons, energy, carbon, and nitrogen. Carbon dioxide is fixed via the Calvin cycle, and for activation of hydrogen only a membrane-bound hydrogenase activity was detected which does not reduce NAD [2]. This is true also for many other aerobic hydrogen bacteria [2,3], and it is assumed that in these organisms the membrane-bound hydrogenase has to provide electrons for NAD reduction in a reversed electron transport as well as for energy conservation. In X. autotrophicus the hydrogenase beyond it has to supply electrons for reduction of nitrogen by nitrogenase at the redox potential of ferredoxin (around - 4 2 0 mV). For this reason the hydrogenase of X. autotrophicus should differ from the type of membrane-bound hydrogenase found in Alcaligenes eutrophus which reduces electron acceptors only at the redox potential of quinones [4]. It was also considered that even two different hydrogenases might be present, one reducing ferredoxin and one providing electrons for the respiratory chain. The involvement of ferredoxin in the nitrogen fixation process of X. autotrophicus GZ29 has recently been shown by electron transfer and regulation studies [5]. In the present study the membrane-bound hydrogenase system of S. autotrophicus is shown to be associated with a cytochrome b complex, and catalytic as well as molecular properties are described.

SUMMARY: A facultatively methylotrophic Mycobacterium was isolated from Cleveland Harbor, Ohio, USA. The isolate, designated ID-Y, used a wide range of carbon and energy sources including methane and several other hydrocarbons. It displayed a growth cycle from rod-shaped exponential-phase cells, with many cell pairs exhibiting V-formation, to cocco-bacillary stationary-phase cells. A fixation technique involving glutaraldehyde/alcian blue resulted in the observation of a three-layered cell wall. Isolate ID-Y has an ultrastructure similar to that of other mycobacteria, particularly Mycobacterium phlei and Mycobacterium flavum, which is presently classified as a Xanthobacter species.

Summary Unusually high contents of glutamic acid in cell wall preparations of various bacteria were found to originate from glutamyl polymers associated with cell walls. Determination of the configuration of glutamyl residues, isolation and identification of a dipeptide from a partial acid hydrolysate and the result of hydrazinolysis showed that strains of Xanthobacter and Flexithrix contain an α -L-glutaminyl polymer, whereas strains of Sporosarcina halophila and Planococcus halophilus form a γ -D-glutamyl polymer.

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.

Intensive screening, of soil microbial cultures for agglutinating activity ofAgrobacterium tumefaciens resulted in the discovery of a bacterium,Xanthobacter sp. KB-3001, which produced an agglutinin ofA. tumefaciens. This compound is an acidic polysaccharide consisting of glucose and galacturonic acid in the ratio 4∶1. This compound assists the attachment ofA. tumefaciens to plant cells and promotes crown gall formation, owing to its affinity to bothA. tumefaciens and plant cells.