Deoxyribonucleic Acid Homologies of Some So-Called “Hydrogenomonas” Species

Evidence based on deoxyribonucleic acid homology supports the abandonment of the genus Hydrogenomonas. Pseudomonas facilis (formerly Hydrogenomonas facilis) is closely related to the nonautotrophic species P. delafieldii. P. facilis and Alcaligenes eutrophus (often called H. eutropha) are not related to each other or to other hydrogen bacteria and pseudomonads studied.     

Isolation and characterization of Hydrogenomonas facilis bacteriophages under heterotrophic growth conditions

Pootjes, Christine F. (The Pennsylvania State University, University Park), R. B. Mayhew, and B. D. Korant. Isolation and characterization of Hydrogenomonas facilis bacteriophages under heterotrophic growth conditions. J. Bacteriol. 92:1787-1791. 1966.-We have isolated five strains of bacteriophage specific for Hydrogenomonas facilis. The host range of the phage is limited to H. facilis. Morphologically, the phage particles consist of a head 580 A in diameter and a short tail 200 A in length. The particles share a common surface antigen, and all contain deoxyribonucleic acid. The five strains differ from each other in growth characteristics, heat stability, and neutralizing antigens.     

Transformation of the Acidophilic Heterotroph Acidiphilium facilis by Electroporation

We constructed a cloning vector for use in the acidophilic heterotroph Acidiphilium facilis. The vector pAH1O1 (8.8kb) was constructed from a 6.1 kb restriction fragment of the Acidiphilium plasmid pAU1 and a pUC19 carrying a β-lactamase gene. The antibiotic resistance gene was efficiently expressed in A. facilis. Several factors which influenced the transformation efficiency were optimized, resulting in a transformation efficiency of up to 3 X 10³ transformants per μg of plasmid DNA at a field strength of 10kV/cm with a 7.0ms pulse.     

An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation.

Pleiotropic mutants of Alcaligenes eutrophus with the phenotype Hno- have been characterized previously. They are deficient in several diverse metabolic activities, including hydrogen oxidation, nitrate and urea assimilation, denitrification, and various substrate transport systems. Phenotypically similar mutants were identified among hydrogenase-deficient strains of Pseudomonas facilis. The Tn5-labeled hno gene was cloned from a genomic DNA library of A. eutrophus and used to identify the corresponding unimpaired wild-type DNA sequence. The recombinant plasmid pCH148 contained an insert of 12.3 kilobase pairs and was shown to restore the Hno+ phenotype to mutants of A. eutrophus and P. facilis. A cosmid isolated from a DNA library of P. facilis also exhibited intergeneric Hno-complementing activity. The cloned hno loci from both organisms showed DNA homology by Southern blot hybridization. A subclone of pCH148 which contained a 6.5-kilobase-pair insert was constructed. The resulting hybrid, pCH170, not only was able to complement Hno- mutants but also relieved glutamine auxotrophy in NtrA- mutants of enteric bacteria. This suggests that the hno gene product from A. eutrophus is functionally similar to the NtrA protein, which has been identified as a novel sigma factor (sigma 54) of RNA polymerase.     

Cell surface protein of Pseudomonas (Hydrogenomonas) facilis

Intact cells of Pseudomonas facilis contain one major molecular weight class of protein that is exposed at the cell surface as revealed by lactoperoxidase-catalyzed iodination with (125)I. All molecular weight classes of protein in derived cell envelope preparations are apparently saturated by iodination by lactoperoxidase after prolonged sonic treatment. The molecular weight of the predominantly exposed protein in intact cells is approximately 16,000, which is the minimal molecular weight of a cell envelope protein that precipitates as a complex with phospholipid from extracts of P. facilis. The isolation of labeled phospholipoprotein (PLP) after labeling intact cells with (125)I corroborates previous experiments which suggested a surface location for the protein portion of the phospholipoprotein (P(PLP)). Solvent extraction of cells and immunological evidence, including studies with ferritin-coupled antibodies, indicate that P(PLP) is located at the cell surface and may also be within the cell envelope. These experiments suggest that P(PLP) is the major cell surface protein in P. facilis.     

Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522.

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.     

Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov

Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).     

Replication of Bacteriophage SH-133 in the Facultative Autotroph Hydrogenomonas facilis I. Bacteriophage Synthesis Under Heterotrophic, Autotrophic, and Mixotrophic Growth Conditions

The ability of bacteriophage SH-133 to replicate in heterotrophically (H-) and autotrophically (A-) grown Hydrogenomonas facilis was examined. Both the synthesis of infectious phage particles and the efficiency of plating (EOP) were reduced by 90% in A-grown cells. Adsorption of phage and lethal effects on H. facilis were identical in both systems. One-step growth experiments showed that cell lysis preceded the appearance of infectious particles in A-grown cells. Burst size studies with mixotrophically grown cells did not indicate the presence of an inhibitor of phage synthesis indigenous to autotrophic metabolism. DNA synthesis was identical in H- and A-grown infected cells; however, protein synthesis was significantly reduced in A-grown infected cells when compared with protein synthesis in H-grown infected cells. The data suggest that the reduction in EOP and phage synthesis in A-grown cells is caused by a defect in viral protein synthesis which results in the limited production of an essential viral protein at the time of cell lysis.     

Purification,cloning, sequencing and over-expression in<Emphasis Type="Italic"> Escherichia coli</Emphasis> of a regioselective aliphatic nitrilase from<Emphasis Type="Italic"> Acidovorax facilis</Emphasis> 72W

A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.     

Immunologically Cross-Reacting Proteins in Cell Walls of Many Bacteria

Antibodies to a protein present in the cell envelope of Hydrogenomonas facilis agglutinate many gram-negative bacteria and a few gram-positive bacteria. Immunodiffusion studies of extracts from the various organisms tested indicate the presence of components sharing antigenic determinants with the cell envelope protein in all bacteria which can be agglutinated and a few that cannot. Cross-reacting components were not detected in extracts of Mycoplasma laidlawii, Anacystis nidulans, and several eukaryotic organisms.     

Kinetics of chlorobenzene biodegradation under reduced oxygen levels

Focussing on the role of chlorocatechol 1,2-dioxygenase (CC12O), an oxygen-dependent key enzyme in the aerobic catabolism of chlorobenzene (CB), Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria were amended with CB degradation under either oxic or hypoxic conditions. All cultures readily degraded CB at high oxygen availability, but had differing abilities to completely degrade CB when exposed to oxygen limitation. For the three cultures very distinct oxygen half-saturation constants (0.3-11.7 muM) for the respective CC12Os were obtained and protein analysis showed that high affinity-type A. facilis and low affinity-type P. veronii express CC12Os, which belong to different structural clusters. From this a functional relation between CC12O type and the ability to cope with efficient ring fission under oxygen limitation is anticipated. Extremely high oxygen affinities for CC12Os support the assumption that truly oxic environments are not an essential requirement to degrade chloro(aromatic) compounds. Tiny quantities of oxygen permanently re-supplied will sufficiently maintain the growth of microaerophilic specialists with the ability to transform chloro(aromatics) via catechol intermediates.     

Metal resistance in Acidocella strains and plasmid-mediated transfer of this characteristic to Acidiphilium multivorum and Escherichia coli.

Acidophilic heterotrophic strain GS19h of the genus Acidocella exhibited extremely high resistance to CdSO4 and ZnSO4, with a MIC of 1 M for each. The respective MICs for an Acidocella aminolytica strain were 400 and 600 mM. The MICs of NiSO4 for the above strains were 200 and 175 mM, respectively. These strains were also resistant to CuSO4, the MICs being 20 and 40 mM, respectively. An Acidocella facilis strain showed resistance only to ZnSO4, with a MIC of 150 mM. The metal salts, in general, extended the lag period, log period, and generation time, with decreases in growth rate and optimum growth. A. aminolytica and strain GS19h each contain more than one plasmid, while A. facilis contains none. After transformation by electroporation with the plasmid preparation from strain GS19h, an Acidiphilium multivorum strain became highly resistant to cadmium and zinc, and the plasmid profile of the transformed cells was found to differ from that of the original Acidiphilium multivorum strain. Escherichia coli HB101 and DH5 alpha also exhibited more resistance to these metals, especially zinc, after transformation with the total plasmid preparation of strain GS19h or a 24.0-MDa plasmid of the same strain, although no plasmid was detected in the transformed cells. Thus, the results derived mainly through genetic experiments demonstrate for the first time the plasmid-mediated transfer of metal resistance for an acidophilic bacterium.     

Protein engineering of nitrilase for chemoenzymatic production of glycolic acid

A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate using a nitrilase derived from Acidovorax facilis 72W. Protein engineering and over-expression of this nitrilase, combined with optimized fermentation of an E. coli transformant were used to increase the enzyme-specific activity up to 15-fold and the biocatalyst-specific activity up to 125-fold. These improvements enabled achievement of the desired volumetric productivity and biocatalyst productivity for the conversion of GLN to ammonium glycolate.     

Arthrobacter nitroguajacolicus腈水解酶基因的克隆和表达

腈水解酶是一类能将腈类化合物催化生成酸的氰基水解酶。目前已有多个菌种的腈水解酶基因序列被报道,如敏捷食酸菌Acidovoraxfacilis,粪产碱菌Alcaligenesfaecalis,睾丸丛毛单胞菌Comamonastestoteroni,肺炎克雷伯菌Klebsiellapneumoniae,假单胞菌Pseudomonas属菌株,红球菌Rhodococcus属菌株,但节杆菌属菌株Arthrobacternitroguajacolicus的腈水解酶基因序列尚未见报道。经由野生型酶的分离纯化,基因文库筛选及侧翼序列扩增等步骤,克隆得到该菌株的腈水解酶基因,从而为进一步研究该酶的特性及构建用于工业生产的重组菌打下基础。     

Effect of Growth Conditions on Morphology of Hydrogenomonas facilis and on Yield of a Phospholipoprotein

Hydrogenomonas facilis grown heterotrophically on fructose with very low aeration eventually ceased to divide and produced elongated forms. Short forms were obtained from fructose-grown long forms by increasing the availability of oxygen to the organisms. A phospholipoprotein, the protein moiety of which is known to be present in the cell envelope, precipitated upon lowering the ionic strength of extracts from cells in the earlier stages of elongation (i.e., in the middle and late log phase of growth). The maximal yield of the protein moiety of the phospholipoprotein precipitate (i.e., grams of protein/grams of soluble protein x 100) was 2%. Poly-beta-hydroxybutyric acid accumulated as growth on fructose progressed, the accumulation being more marked with lower aeration.     

The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

A simplified procedure for the determination of autotropic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphre containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccus denitrificans, Pseudomonas facilis, Pseudomonas flava, Pseudomonas palleronii, Pseudomonas saccahrophilia and Rhodococcus sp. (‘Nocardia opaca’). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria.

Nineteen characterized strains and isolates of acidophilic heterotrophic bacteria were screened for their abilities to catalyse the reductive dissolution of the ferric iron mineral schwertmannite, under oxygen-limiting conditions. Acidocella facilis, Acidobacterium capsulatum, and all of the Acidiphilium, Acidocella and Acidobacterium-like isolates that grew in liquid cultures were able to reduce iron. In contrast, neither Acidisphaera rubrifaciens nor three Acidisphaera-like isolates tested were found to have the capacity for dissimilatory iron reduction. One of two iron-oxidizing Frateuria-like isolates also reduced iron under oxygen-limiting conditions. Microbial dissolution of schwertmannite was paralleled with increased concentrations of soluble ferrous iron and sulfate in microbial cultures, together with increased pH values and decreased redox potentials. While dissimilatory ferric iron reduction has been described previously for Acidiphilium spp., this is this first report of this capacity in Acidocella and the moderate acidophile Acidobacterium. The finding has significant implications for understanding of the biogeochemistry of acidic environments.     

Anaerobic mineralization of quaternary carbon atoms: isolation of denitrifying bacteria on dimethylmalonate.

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the alpha-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans(T)), and three strains isolated from freshwater ditches were affiliated with the beta-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae(T) and Acidovorax facilis(T), respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 x 10(4) dimethylmalonate-utilizing cells ml(-1), representing up to 0.4% of the total culturable nitrate-reducing population.     

Protein engineering of Acidovorax facilis 72W nitrilase for bioprocess development

Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to chemical production since the reactions are run at ambient temperature, do not require strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion. However, native enzymes often do not perform desired reactions with the efficiency required for commercial applications. Protein engineering was used to significantly increase the specific activity of nitrilase from Acidovorax facilis 72W for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. Overexpression of engineered nitrilase enzymes in Escherichia coli, combined with immobilization of whole cells in alginate beads that can be recycled many times has facilitated the development of a commercially viable bioprocess for production of 3-hydroxyvaleric acid.