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Archives of Microbiology (v.168, #6)

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis by Dorothee Steinmann; R. Köplin; A. Pühler; Karsten Niehaus (pp. 441-447).

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged.

Keywords: Key words Xanthomonas campestris pv. campestris; lpsI; lpsJ; 3-Oxoacid CoA-transferase; Xanthan; Lipopolysaccharide

Purine metabolism and the microaerophily of Helicobacter pylori by G. L. Mendz; Andrew J. Shepley; Stuart L. Hazell; Mark A. Smith (pp. 448-456).

The requirements for purine nucleotide synthesis, the effects of purine analogues, and the metabolism of adenine in the bacterium Helicobacter pylori were investigated employing cell culture techniques and one-dimensional NMR spectroscopy. Bacterial cells grew and proliferated in media lacking preformed purines, indicating that H. pylori can synthesize purine nucleotides de novo to meet its requirements. Blocking of this pathway in the absence of sufficient preformed purines for salvage nucleotide synthesis led to cell death. Analogues of purine nucleobases and nucleosides taken up by the cells were cytotoxic, suggesting that salvage routes could be exploited for therapy. Adenine or hypoxanthine were able to substitute for catalase in supporting cell growth and proliferation, suggesting a role for these bases in maintaining the microaerophilic conditions essentially required by the bacterium.

Keywords: Key words Purine biosynthesis; Adenine; Purine; analogues; Adenine deaminase; 1H-NMR spectroscopy; Helicobacter pylori; Microaerophily

3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae by Yasuo Kato; Y. Asano (pp. 457-463).

The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4.3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions. The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity. The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration. The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme. Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions. Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

Keywords: Key words 3-Methylaspartase; Glutamate mutase; (S)-citramalate hydrolyase; Enterobacteriaceae; Citrobacter; Morganella; (S)-glutamate fermentation

Regulation of N-acetylglucosaminidase production in Candida albicans by Kyoko Niimi; Masakazu Niimi; Maxwell G. Shepherd; R. D. Cannon (pp. 464-472).

The N-acetylglucosaminidase of Candida albicans is a secreted hydrolytic enzyme that contributes to the yeast’s virulence. There was a significant increase in the N-acetylglucosaminidase activity of C. albicans cells released from carbon starvation in medium containing N-acetylglucosamine. The increased enzyme activity in N-acetylglucosamine-grown cells correlated with increased transcription of the HEX1 gene, which encodes C. albicans N-acetylglucosaminidase. In contrast, glucose repressed HEX1 transcription, and glucose-grown cells had on average 94-fold lower N-acetylglucosaminidase activities than did N-acetylglucosamine-grown cells. N-acetylglucosaminidase induction in cells grown on N-acetylglucosamine was also repressed by fructose, mannose or galactose, although to a lesser extent than by glucose, and sucrose repressed enzyme production by only 10%. Eighty-eight percent of the enzyme in N-acetylglucosamine-grown cells was localised in the periplasm, and after incubation for 5 h, 30 or 70% of the total enzyme activity was secreted into the medium by yeast or mycelial cells, respectively. The cellular location of the enzyme and the regulation of production by the carbon source indicate a scavenging role for C. albicans N-acetylglucosaminidase.

Keywords: Key wordsCandida albicans; N-acetylglucosaminidase; HEX1; Glucose repression

Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology by Leonilde M. Moreira; I. Sá-Correia (pp. 473-479).

Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates). The estimated size of the large plasmids purified from T. oshimai SPS-18 from S. Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively. No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis. Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels. Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring. Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined. The 16-kb HindIII–HindIII fragment from isolate SPS-18 megaplasmid showed DNA–DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas. This suggests a high degree of sequence conservation in T. oshimai megaplasmids.

Keywords: Key words Thermus oshimai; Megaplasmids; Pulsed-field gel electrophoresis; RFLP; Southern; hybridization

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes by G. Zellner; Anke Jargon (pp. 480-485).

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity.

Keywords: Key words Aldehyde; Aldehyde dehydrogenase; FMN; FAD; Tungstate; Sulfate-reducing bacteria; Desulfovibrio

Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase by Robin A. Henderson; C. W. Jones (pp. 486-492).

Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, dl-lactate or l-lactate. Unlike growing cultures, washed cells excreted significant amounts of pyruvate. The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production. The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA). Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP. Citrate synthase was purified and shown to be a “large” form of the enzyme (M _r 227,000), comprising a single type of subunit (M _r 47,000) as found in several other gram-negative aerobes. The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed.

Keywords: Key words Poly-3-hydroxybutyrate; Washed cells; Alcaligenes eutrophus; Citrate synthase

Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov. by Levente Bodrossy; E. M. Holmes; A. J. Holmes; Kornél L. Kovács; J. C. Murrell (pp. 493-503).

Two methanotrophic bacteria with optimum growth temperatures above 40° C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14L^T and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile.

Keywords: Key words Methylocaldum szegediense; Methylocaldum tepidum; Methylocaldum gracile; Thermophilic methanotrophs; 16S rRNA phylogeny; Particulate methane monooxygenase gene

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method by J. Ramón De Lucas; Susana Valenciano; A. I. Domínguez; Geoffrey Turner; F. Laborda (pp. 504-512).

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 × 10⁷ viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole^–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole^– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim^– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 ×g supernatant, but produced wild-type levels of penicillin.

Keywords: Key words Aspergillus nidulans; Peroxisomes; Oleate mutants; per mutants; pas mutants; Pim; mutants; acu mutants; Peroxisome biogenesis; Penicillin

Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain PCE-S by Evelyn Miller; Gert Wohlfarth; G. Diekert (pp. 513-519).

Tetrachloroethene reductive dechlorination was studied with cell extracts of a newly isolated, tetrachloroethene-utilizing bacterium, Desulfitobacterium sp. strain PCE-S. Tetrachloroethene dehalogenase mediated the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with artificial electron donors such as methyl viologen. The chlorinated aromatic compounds tested so far were not reduced. A low-potential electron donor (E ₀′ < –0.4 V) was required for tetrachloroethene reduction. The enzyme in its reduced state was inactivated by propyl iodide and reactivated by light, indicating the involvement of a corrinoid in reductive tetrachloroethene dechlorination.

Keywords: Key words Corrinoids; Dehalospirillum multivorans; Desulfitobacterium; Tetrachloroethene dehalogenase; Propyl iodide; Reductive dechlorination; Strain PCE-S; Tetrachloroethene; Trichloroethene

Purification, characterization and gene sequence analysis of a novel cytochrome c co-induced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1 by Tai Man Louie; Shuisong Ni; Luying Xun; William W. Mohn (pp. 520-527).

The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate via halorespiration. To understand this respiratory process better, we examined electron carriers from different cellular compartments of D. tiedjei. A 50-kDa cytochrome from the membrane fraction was found to be co-induced with dechlorination activity. This inducible cytochrome was extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoretic homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatography. The purified cytochrome had a high-spin absorption spectrum. In a pH titration experiment, the absorption spectrum of the inducible cytochrome shifted to low spin at pH 13.2. The midpoint potential of the inducible cytochrome at pH 7.0 was –342 mV. The NH₂-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse PCR products containing the sequence of the gene encoding the inducible cytochrome. The ORF was 1398 bp and coded for a protein of 52.6 kDa. Two c-type heme-binding domains were identified in the COOH-terminal half of the protein. A putative signal peptide of 26 residues was found at the NH₂-terminal end. The protein sequence was not found to have substantial sequence similarity to any other sequence in GenBank. We conclude that this is a c-type cytochrome substantially different from previously characterized c-type cytochromes.

Keywords: Key words Desulfomonile tiedjei DCB-1; Halorespiration; Reductive dechlorination; Electron; transport; Cytochrome c

pp1-cyano2, a protein serine/threonine phosphatase 1 gene from the cyanobacterium Microcystis aeruginosa UTEX 2063 by L. Shi; W. W. Carmichael (pp. 528-531).

Polymerase chain reaction (PCR) products similar to protein serine/threonine family I phosphatase genes were identified in five strains of cyanobacteria from three species. The gene for one of these protein phosphatase PCR products, pp1-cyano2 from Microcystis aeruginosa UTEX 2063, was cloned and sequenced. The deduced protein sequence PP1-cyano2 contains 264 amino acid residues (∼30.3 kDa). In its N-terminal region, PP1-cyano2 had a GDXXHG(X)_nGDXXDRG(X)_nGNHE (nP23) sequence that is well-conserved in all protein serine/threonine family I phosphatases. Of 19 amino acid residues important for either metal binding, structure of the active site, or catalysis in eukaryotic PP1, 18 were present in PP1-cyano2. Reverse-transcription-PCR results showed that pp1-cyano2 was expressed under laboratory culture conditions.

Keywords: Key words Protein serine/threonine phosphatase 1; Cyanobacterium; Microcystis aeruginosa; pp1-cyano2; PP1-cyano2

Detection and preliminary characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus occultus by C. A. Studdert; Rosana E. De Castro; Karina Herrera Seitz; Jorge J. Sánchez (pp. 532-535).

Extracellular proteolytic activity was detected in the haloalkaliphilic archaeon Natronococcus occultus as the culture reached the stationary growth phase. Proteolytic activity was precipitated with ethanol and subjected to a preliminary characterization. Optimal conditions for activity were attained at 60° C and 1–2 M NaCl or KCl. Gelatin zymography in the presence of 4 M betaine revealed a complex pattern of active species with apparent molecular masses ranging from 50 to 120 kDa. Experiments performed with inhibitors of the various groups of proteases indicated that the extracellular proteolytic enzymes of N. occultus are of the serine type. Individual protein species showed some differences in salt and thermal stability.

Keywords: Key words Archaea; Haloalkaliphiles; Natronococcus; occultus; Proteolytic activity; Gelatin zymography; Betaine

Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus by Nina Aalén; Ida Helene Steen; Nils-Kåre Birkeland; Torleiv Lien (pp. 536-539).

NADP⁺-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95° C. The enzyme was highly thermostable, having a half-life of 140 min at 100° C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp.

Keywords: Key words Archaeoglobus fulgidus; Archaea; Glutamate dehydrogenase; Thermostable enzyme; Amino acid dehydrogenase

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Archives of Microbiology (v.168, #6)