Tryptophan of facultative methylotrophic bacteria Pseudomonas sp. M. II. Regulation of tryptophan biosynthesis

Regulation of tryptophan biosynthesis of facultative methylotrophic Pseudomonas sp. M was studied. Repression of the trpE, trpD and trpC genes by tryptophan was demonstrated. It was also shown that the trpE and trpDC genes are derepressed noncoordinately. No regulation of the trpF gene product could be demonstrated, indicating that its synthesis is constitutive. The trpA and trpB genes are inducible by indol-3-glycerophosphate. Anthranilate synthase and tryptophan synthase were sensitive to the feedback inhibition. The tryptophan concentrations giving 50% inhibition were estimated to be 9 microM and 1 microM, respectively. Experimental evidence for activation of the N-5-phosphoribosyl anthranilate isomerase and for inhibition of the indol-3-glycerophosphate synthase by some tryptophan intermediates was obtained.     

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.     

Isolation of Tyr- mutants of facultative methylotrophic Pseudomonas sp. M

A method for isolation of Tyr- mutants of facultative methylotrophic bacteria Pseudomonas sp. M which possess two tyrosine synthesis pathways is presented. The method is based on the two-step blocking of the tyrosine synthesis: the first step of the supplementary pathway of synthesis from phenylalanine, the second being the main pathway from 4-hydroxyphenylpyruvate.     

The tryptophan operon of the facultative methylotrophic bacteria Pseudomonas sp. M. III. Characteristics of regulatory 5-MTR mutants

Regulatory 5-DL-methyltryptophan (5-MT)-resistant mutants of facultative methylotrophic Pseudomonas sp. M. were obtained. They are able to excrete tryptophan into the growth medium (60 to 300 g/ml). 5-MTR regulatory mutants are characterized by depression of trpE, trpD and trpC genes, which causes the production of intermediates of tryptophan biosynthesis and results in trpA and trpB genes induction as well as in two-fold activation of N-5-phosphoribosyl anthranilateisomerase (trpF gene product). Besides, all mutants demonstrate reduction of synthase feed-back inhibition about 4-11-fold. Together with tryptophan excretion, 5-MTR regulatory mutants are able to excrete tyrosine and unable to utilize this amino acid as the sole carbon source, which points to multiple nature of the selective effect of 5-MT.     

NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. I. Amino acid sequence

The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.     

Repression of the synthesis and allosteric inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase in facultative methylotrophic bacterium Pseudomonas sp. M

The synthesis of 3-deoxy-D-arabinoheptulosonate-7-phosphate-synthase has been shown to be repressed by tyrosine and phenylalanine in the cells of facultative methylotrophic bacteria Pseudomonas sp. M. Activity of the enzyme is subjected to allosteric inhibition by tyrosine, tryptophane, anthranylate and phenylpyruvate.     

Regulation of citrate synthase in facultative methylotrophic bacteria

Commonly the TCA cycle fulfils an anabolic and a catabolic function in case of aerobic chemoorganoheterotrophic nutrition. In methylotrophic growth the TCA cycle is dispensable as a bioenergetic pathway. This is reflected by properties of citrate synthase in facultative methylotrophic bacteria. Two citrate synthases, a "chemoorganoheterotrophic" one, which is inhibited by NADH (or ATP in Acetobacter MB 58), and a "methylotrophic" one, which is not or less affected by energy indicators, were found in Pseudomonas oleovorans, Pseudomonas MS, Pseudomonas MA, and Acetobacter MB 58. The concentration of these citrate synthases depends on the manner of nutrition. Bacteria with ICL-negative-variant of the serine pathway and with ribulosebisphosphate pathway seem to possess only a "chemoorganoheterotrophic" citrate synthase. Possibly the anabolic function of this citrate synthase can be realized by metabolites.     

NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. III. Comparative analysis

The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.     

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     

Isolation and characterization of a novel methylhopanoid from a facultative methylotrophicCorynebacterium sp.

Several compounds such as a methylhopanoid and carotenoids have been isolated and characterized from a facultative methylotrophicCorynebacterium sp., a vitamin B¹² producer. A novel pentacyclic triterpene, 2-methyl-22-hydroxyhopane has been identified by IR,₁H-and¹³C-NMK and mass spectrometry. During the purification procedure a red pigment has been characterized as a mixture of several carotenoids by TLC and UV-VIS spectroscopy.     

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP—BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l^−l), MDW 80 (301 mg l^−l), M. komagatae (279 mg l^−l), and MSF 34 (202 mg l^−l), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.     

Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae.     

Siderochromes from Pseudomonas fluorescens. I. Isolation and characterization

Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin.     

Isolation and characterization of Pseudomonas sp. strain capable of degrading diethylstilbestrol

Since diethylstilbestrol (DES) interrupts endocrine systems and generates reproductive abnormalities in both wildlife and human beings, methods to remove DES from the environments are urgently recommended. In this study, bacterial strain J51 was isolated and tested to effectively degrade DES. J51 was identified as Pseudomonas sp. based on its nucleotide sequence of 16S rRNA. The quinoprotein alcohol dehydrogenase and isocitrate lyase were identified to be involved in DES degradation by MALDI–TOF–TOF MS/MS analysis. In the presence of 40 mg/l DES, increase of the genes encoding quinoprotein alcohol dehydrogenase and isocitrate lyase in both RNA and protein levels was determined. The HPLC/MS analysis showed that DES was hydrolyzed to a major degrading metabolite DES-4-semiquinone. It was the first time to demonstrate the characteristics of DES degradation by specific bacterial strain and the higher degradation efficiency indicated the potential application of Pseudomonas sp. strain J51 in the treatment of DES-contaminated freshwater and seawater environments.     

NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. II. Enzyme conformation at 3.0 A resolution

Three heavy atom isomorphous derivatives were used for the X-ray analysis of the holo form of NAD-dependent bacterial formate dehydrogenase (ternary complex enzyme-NAD-azide) at 3.0 A resolution. The enzyme subunit contains a catalytic and a coenzyme binding domain, with the active centre and the coenzyme binding site in the cleft between the domains. The polypeptide chain's fold and the position of 393 C alpha-atoms were determined. The secondary structure of the formate dehydrogenase was resolved. The structure of the NAD-binding domain is shown to be similar to that of other NAD-dependent enzymes.     

假单胞菌株M18中pqsA突变株的构建及其对plt的调控

【目的】根际铜绿假单胞菌M18能产生藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)两种主要的抗生素。其PqsR/PQS群体感应系统由应答调控蛋白PqsR与信号分子PQS组成。前期研究已经表明pqsR负调控Plt生物合成及基因簇表达。本论文旨在研究PQS分子对Plt合成及基因表达的调控作用。【方法】从M18基因组中扩增PQS合成基因pqsA,通过同源重组技术构建假单胞菌M18的pqsA突变菌株M18pqsA。利用lacZ报告基因分析、信号分子添加实验等,研究PQS对Plt合成及基因表达的调控作用。【结果】在KMB培养基中,分别比较野生型菌株M18和突变菌株M18pqsA的Plt产量,突变菌株的Plt产量存在较小幅度的升高,约为野生型菌株的1.53倍。添加PQS对plt表达存在一定程度但不是很显著的负调控作用。【结论】PQS分子对Plt生物合成及基因表达存在部分负调控作用。     

Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp.

The enzyme 1-aminocyclopropane-1-carboxylate deaminase catalyzes the degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The enzyme has been detected in a limited number of bacteria and plays a significant role in sustaining plant growth and development under biotic and abiotic stress conditions by reducing stress-induced ethylene production in plants. We have screened 32 fluorescent Pseudomonas sp. isolated from rhizosphere and non-rhizosphere soils of different crop production systems for drought tolerance using polyethylene glycol 6000 (PEG 6000). Nine of these isolates were tolerant to a substrate metric potential of ?0.30 MPa (15 % PEG 6000) and therefore considered to be drought-tolerant. All of these drought-tolerant isolates were screened for ACC deaminase activity using ACC as the sole nitrogen source, and one (SorgP4) was found to be positive for ACC, producing 3.71?±?0.025 and 1.42?±?0.039 μM/mg protein/h of α-ketobutyrate under the non-stress and drought stress condition, respectively. The isolate SorgP4 also showed other plant growth-promoting traits, such as indole acetic acid production, phosphate solubilization, siderophore and hydrogen cyanide production. The ACC deaminase gene (acdS) from the isolate SorgP4 was amplified, and the nucleotide sequence alignment of the acdS gene showed significant homology with acdS genes of NCBI Genbank. The 16S rRNA gene sequencing analysis identified the isolate as Pseudomonas fluorescens. Both sequences have been submitted to the NCBI GenBank under the accession numbers JX885767 and KC192771 respectively.     

Isolation and characterization of sulfonamide-degrading bacteria Escherichia sp. HS21 and Acinetobacter sp. HS51

With the intensive application of sulfonamides in aquaculture and animal husbandry and the increase of sulfonamides discharged into the environments, there is an increasing need to find a way to remediate sulfonamide-contaminated environments. Two bacterial strains capable of degrading sulfonamides, HS21 and HS51, were isolated from marine environments. HS21 and HS51 were identified as members of Escherichia sp. and Acinetobacter sp., respectively, based on 16S rRNA gene sequencing. Degradation of each sulfonamide by Escherichia sp. HS21 and Acinetobacter sp. HS51 was characterized using capillary electrophoresis. About 66 or 72% of sulfapyridine and 45 or 67% of sulfathiazole contained in the media was degraded by Escherichia sp. HS21 or Acinetobacter sp. HS51, respectively, after incubation for 2 days. The supernatant from culture of Escherichia sp. HS21 or Acinetobacter sp. HS51 grown in sulfapyridine or sulfathiazole contained media had much attenuated cytotoxicity against HeLa cells. These results suggest that Escherichia sp. HS21 and Acinetobacter sp. HS51 are new bacterial resources for biodegrading sulfonamides and indicate the potential of isolated strains for the bioremediation of sulfonamide-polluted environments.     

Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol

Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.