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Archives of Microbiology (v.191, #6)

Genetic and biochemical analyses of chlorobenzene degradation gene clusters in Pandoraea sp. strain MCB032 by Xi-Wen Jiang; Hong Liu; Ying Xu; Shu-Jun Wang; David J. Leak; Ning-Yi Zhou (pp. 485-492).

Pandoraea sp. strain MCB032 was isolated as an emerging chlorobenzene degrader from a functionally stable bioreactor where species succession had occurred. In this study, two gene clusters encoding chlorobenzene metabolic functions have been cloned. Within the cbs gene cluster, CbsA and CbsB are similar to the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase in Ralstonia sp. JS705 and shown to transform chlorobenzene to 3-chlorocatechol. The clc gene cluster shows strong similarity to the clc genes of Ralstonia sp. JS705 and encodes chlorocatechol 1,2-dioxygenase (ClcA) and other enzymes, which catalyze the conversion of chlorocatechol to 3-oxoadipate. The Michaelis constants (K _m) values of ClcA for catechol, 3-methylcatechol and 3-chlorocatechol were determined as 10.0, 8.9 and 3.4 μM, respectively. CbsX, a putative transport protein present in the cbs cluster of strain MCB032 but not in those of other chlorobenzene degraders, shows 76 and 53% identities to two previously identified transport proteins involved in toluene degradation, TbuX from Ralstonia pickettii PKO1 and TodX from Pseudomonas putida F1. The presence of the transport protein in strain MCB032 likely provides a mechanistic explanation for its higher chlorobenzene affinity and may well be the basis for the competitive advantage of this strain in the bioreactor.

Keywords: Chlorobenzene; Chlorocatechol; Degradation; Pandoraea

Enzymatic, outer membrane proteins and plasmid alterations of starved Vibrio parahaemolyticus and Vibrio alginolyticus cells in seawater by Fethi Ben Abdallah; Héla Kallel; Amina Bakhrouf (pp. 493-500).

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 μm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.

Keywords: Vibrio ; Seawater; Starvation; Enzymatic; Morphological; Outer membrane proteins; Plasmids; Alterations

Characterization of chsA, a new gene controlling the chemotactic response in Azospirillum brasilense Sp7 by Ricardo Carreño-López; Araceli Sánchez; Nohemí Camargo; Claudine Elmerich; Beatriz Eugenia Baca (pp. 501-507).

We report, here, the characterization of a mutant strain of Azospirillum brasilense Sp7 impaired in surface motility and chemotactic response. Presence of flagella in the mutant strain was confirmed by western blot analysis, using antisera raised against the polar and lateral flagellins, and by electron microscopy. Genetic complementation and nucleotide sequencing led to the identification of a new gene, named chsA. The deduced translation product, ChsA protein, contained a PAS sensory domain and an EAL domain. As ChsA displayed characteristic signaling protein architecture, it is thought that this protein is a component of the signaling pathway controlling chemotaxis in Azospirillum.

Keywords: Azospirillum brasilense ; ChsA signaling protein; Chemotactic response

Rapid oxidation of ring methyl groups is the primary mechanism of biotransformation of gemfibrozil by the fungus Cunninghamella elegans by Su-Il Kang; Seo-Young Kang; Robert A. Kanaly; Eunjung Lee; Yoongho Lim; Hor-Gil Hur (pp. 509-517).

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1–FM9 and FM6′) from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2′-hydroxymethyl GEM. FM6 was considered to be 5′-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2′,5′-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2′,5′-dihydroxymethyl GEM from 2′-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.

Keywords: Cunninghamella elegans ; Fungal metabolism; Gemfibrozil; Hydroxylation; Hypolipidemic

A genetic analysis of in vivo selenate reduction by Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 by David Guymer; Julien Maillard; Frank Sargent (pp. 519-528).

The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Salmonella enterica serovar Typhimurium and Escherichia coli many Tat substrates are known or predicted to bind a molybdenum cofactor in the cytoplasm prior to export. In the case of N- and S-oxide reductases, co-ordination of molybdenum cofactor insertion with protein export involves a ‘Tat proofreading’ process where chaperones of the TorD family bind the signal peptides, thus preventing premature export. Here, a genetic approach was taken to determine factors required for selenate reductase activity in Salmonella and E. coli. It is reported for both biological systems that an active Tat translocase and a TorD-like chaperone (DmsD) are required for complete in vivo reduction of selenate to elemental red selenium. Further mutagenesis and in vitro biophysical experiments implicate the Salmonella ynfE gene product, and the E. coli YnfE and YnfF proteins, as putative Tat-targeted selenate reductases.

Keywords: Enteric bacteria; Bacterial respiration; Twin-arginine translocation pathway; Molybdo-enzymes; Selenate reductase; Molecular chaperone; Mutagenesis; Isothermal titration calorimetry

Characterization of multiple promoters and transcript stability in the sacB–sacC gene cluster in Zymomonas mobilis by V. Senthilkumar; J. Rajendhran; S. J. W. Busby; P. Gunasekaran (pp. 529-541).

In Zymomonas mobilis, the extracellular levansucrase (SacB) and extracellular sucrase (SacC) are involved in sucrose hydrolysis. Genes coding for these two enzymes (sacB and sacC) are arranged in a cluster in the genome and separated by a short intervening sequence. The level of sacC transcript was 12-fold higher than that of sacB transcript. On the other hand, transcript stability analysis in sucrose grown cultures revealed that the half-life of the sacB transcripts (153 s) was more than twofold higher than that of sacC transcript (66 s). The decay curves of sacB and sacC transcripts analyzed by the semi-quantitative RT-PCR correlated well with the decay curves of the respective enzyme activities. In the sacB promoter disruption mutant, Z. moblis BT2, the extracellular sucrase activity decreased from 2.6 to 2.0 U mg⁻¹ in sucrose medium due to the loss of SacB expression. The expression of sacC in the absence of the sacB promoter suggested that these two genes could be transcribed as different mRNAs. The promoter-lacZ fusion studies in Escherichia coli proved that the short intervening region acts as a strong promoter for the sacC gene.

Keywords: Zymomonas mobilis ; Levansucrase; Sucrase; Promoter disruption; Transcript stability; lacZ fusion

Conjugative plasmid pLD-TEX-KL promotes growth of host bacterium Legionella dumoffii at low temperatures by Tian Qin; Ken-ichiro Iida; Hideki Hirakawa; Susumu Shiota; Hiroaki Nakayama; Shin-ichi Yoshida (pp. 543-551).

Legionella (Fluoribacter) dumoffii is a resident of various aquatic environments and occasionally causes pneumonia in humans. We found that L. dumoffii strain TEX-KL carries a 66-kb circular plasmid. As predicted by the presence of tra genes similar to those of other transferable plasmids, we showed that pLD-TEX-KL was actually capable of transferring itself to a plasmid-cured derivative of the original strain. Unexpectedly, this plasmid-free derivative turned out to be partially defective in terms of growth at temperatures 30°C or lower. Subsequent works revealed that the growth defect was attributable to the loss of the plasmid gene traA(Ti) homologous to the traA gene of Ti plasmid from Agrobacterium tumefaciens, and that the growth was restored by the introduction of the mobA/repB gene of plasmid pMMB207. Since the existence of a DNA nickase domain is the only feature common to the traA(Ti) and mobA/repB gene products, we hypothesized that this growth defect at low temperature is related to insufficient DNA transactions, which can somehow be alleviated by the nickase activity of those plasmid-encoded proteins. It was also noted that the above features of growth defect at low temperatures were seen in L. dumoffii cells parasitizing the amebic host Acanthamoeba culbertsoni.

Keywords: Legionella dumoffii ; pLD-TEX-KL; Conjugative transfer; Low temperature; TraA

The hybrid histidine kinase Slr1759 of the cyanobacterium Synechocystis sp. PCC 6803 contains FAD at its PAS domain by Klaus-Peter Michel; Ann-Kristin Schröder; Maike Zimmermann; Sonja Brandt; Elfriede K. Pistorius; Nicole Frankenberg-Dinkel; Dorothee Staiger (pp. 553-559).

The cyanobacterium Synechocystis sp. PCC 6803 harbours 47 histidine kinases (Hiks). Among these are hybrid histidine kinases with one or two response regulator domains as well as numerous Hiks with several sensory domains. One example is the hybrid histidine kinase Slr1759 (Hik14) that has two PAS domains arranged in tandem linked to a predicted GAF domain. Here, we show that a Slr1759 derivative recombinantly expressed in Escherichia coli has a flavin cofactor. Using truncated Slr1759 variants, it is shown that the flavin associates with the first PAS domain. The cofactor reconstitutes the activity of d-amino acid oxidase apoprotein from pig kidney, indicating that the flavin derivative is FAD. Furthermore, the Slr1759 histidine kinase domain indeed undergoes autophosphorylation in vitro. The phosphorylated product of a recombinant Slr1759 derivative is sensitive to acids, pointing to a histidine residue as the phosphate-accepting group.

Keywords: Hybrid histidine kinase; FAD; PAS domain; Synechocystis

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Archives of Microbiology (v.191, #6)

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Archives of Microbiology (v.191, #6)