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Archives of Microbiology (v.190, #2)

Expression of nuclease gene nucA, a member of an operon putatively involved in uracil removal from DNA and its subsequent reuse in Prevotella bryantii by Tomaž Accetto; Gorazd Avguštin (pp. 111-117).

The genomic region of Prevotella bryantii TC1-1 that conferred an increased nucleolytic activity on Escherichia coli was characterized. It contains two divergent transcriptional units separated by an AT-rich promoter region. One unit is comprised of three genes involved in nucleotide metabolism. nucA, the first gene of this unit, whose product belongs to exonuclease/endonuclease/phosphatase Pfam family, was thought to be required for the increased nucleolytic activity and various expression strategies were employed to confirm its role. The nucA expression was only successful in cell free system where DNase and RNase activity was observed. Two genes downstream of nucA code for a putative uracil DNA glycosylase and uridine kinase which could be involved in the removal of misincorporated uracil from DNA and its reuse. Given that apurinic/apyrimidinic nuclease activity is required after uracil removal from DNA, it was somewhat surprising to find out that nucA, whose product belongs to protein family consisting mostly of apurinic/apyrimidinic nucleases, has no apurinic/apyrimidinic activity.

Keywords: Nuclease; Expression; Prevotella bryantii; Anaerobe; Rumen

Characteristics of the surface-located carbohydrate-binding protein CbpC from Streptomyces coelicolor A3(2) by Stefan Walter; Hildgund Schrempf (pp. 119-127).

Streptomyces coelicolor A3(2) produces a 35.6-kDa carbohydrate-binding protein (named CbpC) in the presence of cellobiose, cellulose or chitin as sole carbon source. The protein was found secreted (a typical signal sequence was present at the N-terminus) and linked to the peptidoglycan layer of the mycelia. At its C-terminal end a putative cell-wall sorting signal was identified, consisting of (1) Streptomyces specific recognition site for a transpeptidase (LAETG instead of generic LPXTP consensus), (2) a hydrophobic region and (3) a tail of positively charged residues. The deletion of this sorting signal abolished the cell-wall attachment because the resulting CbpC-form was found extracellular. After purification this protein was shown to interact strongly with crystalline cellulose; different crystalline chitin-forms were recognised moderately and chitosan not. As demonstrated by analysing further truncated CbpC-forms a glycine-aspartate/serine rich region, which separates the carbohydrate-binding module from the sorting signal, plays an important role in protein stability.

Keywords: Carbohydrate-binding protein; CbpC; Streptomyces coelicolor A3(2); Cell-wall attachment; LAETG-motif; Glycine-aspartate/serine repeats; Sortase

Diversity of soil fungal communities of Cerrado and its closely surrounding agriculture fields by Alinne Pereira de Castro; Betania Ferraz Quirino; Georgios Pappas Jr; Adriane Silva Kurokawa; Eduardo Leonardecz Neto; Ricardo Henrique Krüger (pp. 129-139).

Cerrado is a savanna-like region that covers a large area of Brazil. Despite its biological importance, the Cerrado has been the focus of few microbial diversity studies. A molecular approach was chosen to characterize the soil fungal communities in four areas of the Cerrado biome: a native Cerrado, a riverbank forest, an area converted to a soybean plantation, and an area converted to pasture. Global diversity of fungal communities in each area was assessed through Ribosomal intergenic spacer analysis which revealed remarkable differences among the areas studied. Sequencing of approximately 200 clones containing 18S rDNA sequences from each library was performed and, according to the genetic distance between sequences, these were assigned to operational taxonomic units (OTUs). A total of 75, 85, 85, and 70 OTUs were identified for the native Cerrado, riverbank forest, pasture, and soybean plantation, respectively. Analysis of sequences using a similarity cutoff value of 1% showed that the number of OTUs for the native Cerrado area was reduced by 35%; for the soybean plantation, a reduction by more than 50% was observed, indicating a reduction in fungal biodiversity associated with anthropogenic activity. This is the first study demonstrating the anthropogenic impact on Cerrado soil fungal diversity.

Keywords: Fungal diversity; 18S rDNA; Diversity indices; Anthropogenic modified soils

Physiological states and energetic adaptation during growth of Pseudomonas putida mt-2 on glucose by Leith Latrach Tlemçani; David Corroler; Daniel Barillier; Ridha Mosrati (pp. 141-150).

Kinetic study of growth of Pseudomonas putida mt-2 was investigated in batch culture under aerobic conditions, on glucose as initial carbon and energy source. Cell growth was continuous and three phases were found regarding accumulation of intermediates: (1) glucose was largely converted to gluconate and 2-ketogluconate, (2) then gluconate was converted to 2-ketogluconate and (3) the latter was consumed after gluconate depletion. Examination of growth kinetics and yields showed that glucose flux was mainly oriented to oxidation reduction in the periplasm and less towards biosynthesis. Values of respiratory quotient and of CO₂/biomass and O₂/biomass yields were characteristic of each phase. Main enzymatic activities involved in the use of these substrates were always detected meaning that concomitant assimilation is possible. However the levels of these activities varied during growth. Membrane conversions seem to have a significant energetic contribution explaining the higher specific growth rate obtained in glucose phase compared to gluconate and 2-ketogluconate ones. This is also noticeable through the evolution of the yields $$ Y_{{ ext{O}}_2/{ ext{X}}} $$ and $$ Y_{{ ext{CO}}_2 /{ ext{X}}} $$ . Although the three convergent pathways are operational and can be genetically controlled, the progression of the culture in successive phases highlights an overall level of regulation in response to the energetic needs.

Keywords: Pseudomonas putida mt-2; Growth phases; Glucose; On-line respiratory quotient; Physiology; Cell-energetics

Function and transcriptional regulation of the isocitrate lyase in Pseudomonas aeruginosa by Utta Kretzschmar; Viola Khodaverdi; Jae-Hun Jeoung; Helmut Görisch (pp. 151-158).

Pseudomonas aeruginosa ATCC 17933 is capable of growing aerobically on ethanol as sole source of carbon and energy. This requires the glyoxylate cycle for replenishing C₄-compounds to the TCA cycle. The enzyme isocitrate lyase (ICL) catalyzes the first step of this glyoxylate shunt. Its activity was induced more than 10-fold in response to the carbon sources ethanol or acetate instead of glucose or succinate. We could prove that in P. aeruginosa ICL is essential for aerobic as well as anaerobic utilization of C₂-sources. Transcriptional regulation of icl gene (aceA) expression was monitored on different carbon sources by using an aceA–lacZ gene fusion. A strong correlation between promoter and ICL activity indicated regulation at the transcriptional level. But ICL was not simply induced by the mere presence of ethanol in the growth medium as was demonstrated by cultivation on mixed substrates. P. aeruginosa showed diauxic growth on media containing ethanol–succinate or ethanol–glucose mixtures and did not transcribe the aceA gene to metabolize ethanol until succinate or glucose, respectively, were exhausted. Inactivation of the chromosomal aceA gene in P. aeruginosa led to an inability to grow on ethanol and acetate. Promoter activity studies showed that all genes necessary to oxidize ethanol were downregulated in the ICL-negative mutant. But on mixed substrates like ethanol–succinate or ethanol–glucose the mutant exhibited growth and utilized ethanol as well, probably as energy source only.

Keywords: Aerobic and anaerobic growth; Diauxie; Ethanol oxidation; Isocitrate lyase; Pseudomonas aeruginosa ; Transcriptional regulation

Sulfoacetate released during the assimilation of taurine-nitrogen by Neptuniibacter caesariensis: purification of sulfoacetaldehyde dehydrogenase by Zdenĕk Krejčík; Karin Denger; Sonja Weinitschke; Klaus Hollemeyer; Václav Pačes; Alasdair M. Cook; Theo H. M. Smits (pp. 159-168).

Taurine (2-aminoethanesulfonate) is a widespread natural product whose nitrogen moiety was recently shown to be assimilated by bacteria, usually with excretion of an organosulfonate via undefined novel pathways; other data involve transcriptional regulator TauR in taurine metabolism. A screen of genome sequences for TauR with the BLAST algorithm allowed the hypothesis that the marine gammaproteobacterium Neptuniibacter caesariensis MED92 would inducibly assimilate taurine-nitrogen and excrete sulfoacetate. The pathway involved an ABC transporter (TauABC), taurine:pyruvate aminotransferase (Tpa), a novel sulfoacetaldehyde dehydrogenase (SafD) and exporter(s) of sulfoacetate (SafE) (DUF81). Ten candidate genes in two clusters involved three sets of paralogues (for TauR, Tpa and SafE). Inducible Tpa and SafD were detected in cell extracts. SafD was purified 600-fold to homogeneity in two steps. The monomer had a molecular mass of 50 kDa (SDS-PAGE); data from gel filtration chromatography indicated a tetrameric native protein. SafD was specific for sulfoacetaldehyde with a K _m-value of 0.12 mM. The N-terminal amino acid sequence of SafD confirmed the identity of the safD gene. The eight pathway genes were transcribed inducibly, which indicated expression of the whole hypothetical pathway. We presume that this pathway is one source of sulfoacetate in nature, where this compound is dissimilated by many bacteria.

Keywords: Assimilation of taurine-nitrogen; DUF81; Sulfoacetaldehyde dehydrogenase; Sulfoacetate exporter; Taurine; Taurine:pyruvate aminotransferase

Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture by Jeffrey L. Bose; Charles S. Rosenberg; Eric V. Stabb (pp. 169-183).

Production of bioluminescence theoretically represents a cost, energetic or otherwise, that could slow Vibrio fischeri growth; however, bioluminescence is also thought to enable full symbiotic colonization of the Euprymna scolopes light organ by V. fischeri. Previous tests of these models have proven inconclusive, partly because they compared nonisogenic strains, or undefined and/or pleiotropic mutants. To test the influence of the bioluminescence-producing lux operon on growth and symbiotic competence, we generated dark ∆luxCDABEG mutants in strains MJ1 and ES114 without disrupting the luxR-luxI regulatory circuit. The MJ1 ∆luxCDABEG mutant out-competed its visibly luminescent parent ~26% per generation in a carbon-limited chemostat. Similarly, induction of luminescence in the otherwise dim ES114 strain slowed growth relative to ΔluxCDABEG mutants. Some culture conditions yielded no detectable effect of luminescence on growth, indicating that luminescence is not always growth limiting; however, luminescence was never found to confer an advantage in culture. In contrast to this conditional disadvantage of lux expression, ES114 achieved ~fourfold higher populations than its ∆luxCDABEG mutant in the light organ of E. scolopes. These results demonstrate that induction of luxCDABEG can slow V. fischeri growth under certain culture conditions and is a positive symbiotic colonization factor.

Keywords: Photobacterium ; Aliivibrio ; Autoinduction; Competition; Evolution

Genome analysis of the coral bleaching pathogen Vibrio shiloi by Leah Reshef; Eliora Ron; Eugene Rosenberg (pp. 185-194).

The past few decades have seen a world-wide increase in coral diseases, yet little is known about coral pathogens. In this study, techniques commonly used in pathogenomic research were applied to the coral pathogen Vibrio shiloi in order to identify genetic elements involved in its virulence. Suppressive subtractive hybridization was used to compare the gene content of V. shiloi to that of a closely related but non-pathogenic bacterium, Vibrio mediterranei, resulting in identification of several putative virulence factors and of three novel genomic islands. The entire genome of V. shiloi was further screened for genes related to previously characterized steps in infection: adhesion, superoxide dismutase production and toxin production. Exposure of pure cultures of V. shiloi to crushed coral tissues strongly affected the expression of seven genes encoding pili, zona occludins toxin (Zot) and a superoxide dismutase. Analysis of eight V. shiloi strains isolated in the last decade shows a shift of the natural population from strains carrying all three genomic islands to strains carrying none of them. This shift occurred following appearance of resistance in the coral Oculina patagonica to infection by V. shiloi. The relevance of these findings to the bleaching disease caused by V. shiloi is discussed.

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Archives of Microbiology (v.190, #2)