Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT.

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.     

Expression of polyhydroxyalkanoic-acid-biosynthesis genes in methylotrophic bacteria relying on the ribulose monophosphate pathway

Four representatives of methylotrophic bacteria relying on the ribulose monophosphate (RMP) pathway were investigated for their capability to synthesize polyhydroxyalkanoic acids (PHA). In Methylophilus methylotrophus B115, Methylobacillus glycogenes strains B121 and B53 and Acetobacter methanolicus B58 no \-ketothiolase, acetoacetyl-coenzyme A (CoA) reductase or PHA synthase could be detected, and hybridization experiments using heterologous DNA probes derived from PHA-biosynthesis genes of Methylobacterium extorquens or Alcaligenes eutrophus gave no evidence for the presence of the corresponding genes in these PHA-negative methylotrophic bacteria. Fragments harbouring a cluster of PHA-biosynthesis genes of A. eutrophus or Chromatium vinosum or isolated PHA synthase structural genes of M. extorquens, Rhodospirillum rubrum or Rhodobacter sphaeroides were mobilized into the RMP pathway bacteria mentioned above. Only transconjugants, which harboured the PHA-biosynthesis genes of A. eutrophus or C. vinosum, expressed active \-ketothiolase, acetoacetyl-CoA reductase and PHA synthase and accumulated poly(3-hydroxybutyric acid) (PHB). Highest amounts of PHB (up to 15% of the cellular dry weight) were accumulated in transconjugants of Methylophilus methylotrophus B115 or of Methylobacillus glycogenes strains B121 and B53 harbouring the PHA-biosynthesis genes of C. vinosum. Correspondence to: A. Steinbüchel     

Conjugative R-plasmid of facultative methylotrophic bacteria Pseudomonas sp. M

50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.     

l-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

Abstract The accumulation of l -threonine by the methylotrophic bacterium Methylobacillus flagellatum KT occurs via a specific system that is capable of transporting l -threonine against a 100-fold concentration gradient. This transport system demonstrates the following kinetic parameters: K _m= 0.2 mM and V _max= 2.5 nmol/min/mg of cells (dry weight). The activity of the system is inhibited by oxidative phosphorylation uncouplers and valinomycin. Cytoplasmic l -threonine does not leak from the cell, but bacteria are capable of exchanging exogenous l -threonine for its intracellular counterpart.     

Methylovorus mays--novel species of aerobic, obligatory methylotrophic bacteria associated with plants

A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere. Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission. The strain does not require vitamins or supplementary growth factors. It is obligately aerobic and urease-, oxidase-, and catalase-positive. The optimum growth temperature is 35-40 degrees C; the optimum pH is 7.0-7.5. The doubling time is 2 h. The bacterium implements the ribulose monophosphate pathway and possesses NAD(+)-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle. alpha-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent. Fatty acids are dominated by palmitic (C16:0) and palmitoleic (C16:1) acids. The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. Cardiolipin is present in minor amounts. The dominant ubiquinone is Q8. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The G + C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature (Tm). The bacterium shows low DNA homology (< 10%) with restricted facultative methylotrophic bacteria of the genus Methylophilus (M. methylotrophus NCIMB 10515T and M. leisingerii VKM B-2013T) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475T). DNA homology with the type representative of the genus Methylovorus, M. glucosetrophus VKM B-1745T, is high (58%). The new isolate was classified as a new species, Methylovorus mays sp. now.     

Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans.

Nine acetotrophic and three methylotrophic strains of methane-producing bacteria were screened for the presence of plasmid DNA. Plasmids were detected in three marine isolates, including Methanosarcina acetivorans. All three plasmids appeared to be similar based on size and restriction site analyses. The plasmid from M. acetivorans, designated pC2A, was approximately 5.1 kilobase pairs in size and was estimated to be present in a low copy number of six plasmids per genome. Multimers were also observed. A restriction map was constructed. The function of this plasmid is cryptic.     

Use of methylotrophic bacteria Methylobacillus flagellatum KT for isolation of deuterated exogenous carbohydrates

The cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, while deuterated exogenous polysaccharides included 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.     

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.     

Use of Methylotrophic Bacteria Methylobacillus flagellatumKT for Isolation of Deuterated Exogenous Carbohydrates

Cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum KT were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD₃OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by HPLC and NMR spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, whereas deuterated exogenous polysaccharides contained 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.     

Characterization of a methionine sulfoximine resistant strain of Rhodobacter capsulatus E1F1

The transposon-loaded plasmid pAS8-121, incapable of autonomous replication in Gram-negative bacteria of non-enteric group, was transferred to Methylobacillus flagellatum KT wild type strain MFK1. The transconjugants arose at a frequency of 10(-7) per donor cell. The majority of the transconjugants tested exhibited the transfer of all selected chromosomal markers at rather high (10(-4)-10(-6) per donor cell) but similar frequencies. Only one of the obtained donors, designated MFK 64, was capable of mobilizing M. flagellatum KT chromosome in a polarized manner. The integrated nature of the plasmid in this and other MFK1 (pAS8-121) derivatives was supported by the results of DNA-DNA hybridization.     

Application of pM3, a Broad Host Range IncP-9 Plasmid, for Genetic Analysis in Enterobacteriaceae

The possibility of using a transposon-carrying variant of broad host range plasmid pM3 (IncP-9) as a universal vector for transposon mutagenesis and as a chromosome-mobilizing factor was demonstrated in bacteria of the family Enterobacteriaceae.     

Growth of the obligate methylotroph Methylobacillus flagellatum under stationary and nonstationary conditions during continuous cultivation

The growth characteristics of a chemostat culture of the obligate methylotrophic bacterium Methylobacillus flagellatum have been determined. Steady-state cultures growing at a rate of 0.73-0.74 h(-1), equal to the maximal growth rate, were obtained under oxyturbidostat cultivation conditions. The response of a chemostat culture to a pulse increase of methanol concentration was studied. It was shown that slow and rapidly growing cultures of M. flagellatum responded differently to pulse methanol addition. The growth characteristics of slow-growing cultures decreased after methanol addition compared to those of stationary chemostat cultures. The growth characteristics of rapidly growing cultures were practically unchanged with and without pulse methanol addition.     

Application of pM3, a broad host range IncP-9 plasmid, for genetic analysis in Enterobacteriaceae

The possibility of using a transposon-carrying variant of broad host range plasmid pM3 (IncP-9) as a universal vector for transposon mutagenesis and as a chromosome-mobilizing factor was demonstrated in bacteria of the family Enterobacteriaceae.     

Cloning of the REP-region of plasmids from a broad circle of pM3 hosts (INC P-9)]

The rep-region of a broad host range plasmid pM3 (IncP-9) has been cloned by connecting the 7.2 kb PstI fragment of the plasmid with the 1.4 kb PstI fragment of the pUC4K vector plasmid. The latter includes the gene for kanamycin resistance and is unable to replicate by itself. The obtained minireplicon designated pMT2 contains rep-genes permitting its inheritance in the different gram-negative cells and a mob-site coding for incompatibility and temperature instability characteristic of the parent plasmid pM3.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

吡咯喹啉醌产生菌筛选方法建立及菌种筛选

吡咯喹啉醌(PQQ)是一种氧化还原酶的辅酶,具有多种生理功能。扩增得到大肠杆菌葡萄糖脱氢酶(GDH)基因,并利用表达载体pET28a在E.coli BL21(DE3)中进行了表达。纯化了可溶性表达产物,并建立了基于GDH的重组酶法分析PQQ的方法。确定了甲基营养菌筛选模型,从2000余份土样中分离得到一株PQQ高产生菌MP606,在未经培养条件优化及诱变选育的条件下PQQ产量达113mg/L。从该菌培养液中制备得到了产物的结晶,HPLC分析、特征光谱分析以及酶法分析均证实该产物为PQQ。扩增并分析了MP606的16S rDNA序列,结果显示该菌16S rDNA序列与12种甲基营养菌都具有95%以上同源性,其中与食甲基菌属两菌株的16S rDNA序列同源性达99%。     

Intergeneric conjugal transfer ofEscherichia coli/Methylobacterium sp. shuttle vector

To develop a host-vector system forMethylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, theE. coli —Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Ap^r, Tc^r) was constructed by joining theE. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium,Methylobacterium sp. strain M17.Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidlessMethylobacterium sp. strain R2b. The stability of pWUBR in Tc^r methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90 % in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.     

Regulation of phenylalanine biosynthesis in the obligate methylotroph Methylobacillus M75

Regulation of phenylalanine biosynthesis has been studied in the bacterium Methylobacillus M75 on the level of enzymes 3-deoxy-D-arabinoheptulose-7-phosphate-synthase, chorismatmutase, prephenatdehyrataze. The DAHP-synthase is shown to be synthesized constitutively and its activity is inhibited by all aromatic aminoacids and antranilate. The synthesis of chorismatmutase and prephenatdehydratase is repressed by tyrosine, the activity of the latter enzyme, besides that, is inhibited by phenylalanine, the effect of which is decreased in the presence of tyrosine.     

Alcohol dehydrogenase from Methylobacterium organophilum.

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 x 10(-5) M for methanol and 8.2 x 10(-5) M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.