NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.     

Genetic characterization of ompH mutants in the deep-sea bacterium Photobacterium sp. strain SS9

OmpH is an outer membrane protein produced by the deep-sea bacterium Photobacterium species strain SS9 in response to elevated hydrostatic pressure. In order to facilitate studies of the function of this protein, a series of OmpH⁺ and OmpH^- strains were obtained from SS9 by Tn5 gene replacement mutagenesis. A previously isolated ompH::lacZ strain and a derivative of this strain harboring a plasmid expressing the wild-type ompH gene were also utilized. The acridine mutagen ICR 191 preferentially inhibited the growth of OmpH⁺ over OmpH^- cells. Indeed, OmpH⁺ cultures treated with the mutagen rapidly accumulated mutants producing reduced levels of OmpH. In addition. OmpH⁺ cells took up the peptide Met-Leu-Phe approximately 15 times more rapidly than OmpH^- cells. The results are consistent with the hypothesis that OmpH functions as a relatively large, nonspecific diffusion channel.Abbreviations OMP Outer membrane protein     

Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium.

A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides. This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium. The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism. The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol. Strain ER2 contained two plasmids (120 and 130 kb). A probe cloned from the pDL11 plasmid of Achromobacter sp. strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P. H. Tomasek and J. S. Karns, J. Bacteriol. 171:4038-4044, 1989), hybridized to the 120-kb plasmid. Restriction fragment profiles of pDL11 and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11. On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogenetically unique type II methylotroph. The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose. Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation. These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment.     

Isolation and characterization of a plasmid in a methylotrophic bacterium

A relatively small plasmin (9 kilobase pairs, pUK1) was isolated from a facultative methylotroph and its restriction map was constructed. pUK1 was joined with the Escherichia coli plasmid pACYC177 to produce pUK11. The presence of proteins expressed by pUK11 was examined by in vivo protein synthesis. The results of fluorography indicated that at least one protein seemed to be coded by pUK1.     

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

Aims:  To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field.
Methods and Results:  Production water collected from 'Sercina' petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l⁻¹ NaCl and at 37°C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC–MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C₁₆). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100.
Conclusions:  A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation.
Significance and Impact of the Study:  The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

Complete genome sequence of the bacterium Methylovorus sp. strain MP688, a high-level producer of pyrroloquinolone quinone

Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Tryptophan operon of methylotrophic facultative Pseudomonas sp. M bacteria. I. Isolation and characterization of auxotrophic Trp-mutants

Eighteen auxotropic trp- mutants of the facultative methylotrophic bacteria Pseudomonas sp. M. induced by nitrosoguanidine were characterized. Trp- mutants were tested for a number of biochemical properties: the capacity to grow on tryptophan intermediates, their accumulation in growth medium and activities of key enzymes. The trpE, trpD, trpC, trpF, trpB and trpA mutants were identified. The trpDC121 mutant with a one-point mutation has been obtained. This mutation caused inactivation of two enzymes--anthranilate-5-phosphoribosyl transferase and indole-3-glycerophosphate synthase. Unusual trpA and trpB auxotrophs with TrpAB- phenotype were described. It may be concluded that this type of mutations cause loss of catalytic activity of a subunit of tryptophan synthase as well as its structural modification. As a result, no active tryptophan synthase complex is formed and hence, the activity of the opposite intact subunit is inhibited.     

Cloning and characterization of extracellular metal protease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.     

Cloning and characterization of monofunctional catalase from photosynthetic bacterium Rhodospirillum rubrum S1

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from 20 degrees C to 60 degrees C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's K(m) value and V(max) of the catalase for H2O2 were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of A406 to A280 for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.     

Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8.

Dibenzothiophene (DBT), a model compound for sulfur-containing organic molecules found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl (2-HBP) by Rhodococcus sp. strain IGTS8. Complementation of a desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfurization. A 6.7-kb TaqI fragment cloned in Escherichia coli-Rhodococcus shuttle vector pRR-6 was found to both complement this mutation and confer desulfurization to Rhodococcus fascians, which normally is not able to desulfurize DBT. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. A molecular analysis of the cloned fragment revealed a single operon containing three open reading frames involved in the conversion of DBT to 2-HBP. The three genes were designated dszA, dszB, and dszC. Neither the nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes. Subclone analyses revealed that the gene product of dszC converts DBT directly to DBT-sulfone and that the gene products of dszA and dszB act in concert to convert DBT-sulfone to 2-HBP.     

Complete genome sequence of the nitrogen-fixing and rhizosphere-associated bacterium Pseudomonas stutzeri strain DSM4166

We present here the analysis of the whole-genome sequence of Pseudomonas stutzeri strain DSM4166, a diazotrophic isolate from the rhizosphere of a Sorghum nutans cultivar. To our knowledge, this is the second genome to be sequenced for P. stutzeri. The availability and analysis of the genome provide insight into the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in interactions with host plants.     

Cloning and molecular characterization of a strawberry fruit ripening-related cDNA corresponding a mRNA for a low-molecular-weight heat-shock protein

We have isolated and characterized a cDNA from a strawberry fruit subtractive library that shows homology to class-I low-molecular-weight (LMW) heat-shock protein genes from other higher plants. The strawberry cDNA (clone njjs4) was a 779 bp full-length cDNA with a single open reading frame of 468 bp that is expected to encode a protein of ca. 17.4 kDa with a pI of 6.57. Southern analysis with genomic DNA showed several high-molecular-weight hybridization bands, indicating that the corresponding njjs4 gene is not present as a single copy in the genome. This strawberry gene was not expressed in roots, leaves, flowers and stolons but in fruits at specific stages of elongation and ripening. However, a differential pattern of mRNA expression was detected in the fruit tissues achenes and receptacle. The njjs4 gene expression increased in achenes accompanying the process of seed maturation whereas in the receptacle, a high mRNA expression was detected in the W2 stage, during which most of the metabolic changes leading to the fruit ripening are occurring. Our results clearly show a specific relationship of this njjs4 strawberry gene with the processes of seed maturation and fruit ripening, and strongly support that at least some of the class-I LMW heat-shock protein-like genes have a heat-stress-independent role in plant development, including fruit ripening.