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Archives of Microbiology (v.191, #3)

Temperature influences the expression of fimbriae and flagella in Hafnia alvei strains: an immunofluorescence study by D. Padilla; F. Acosta; J. A. García; F. Real; Jose R. Vivas (pp. 191-198).

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.

Keywords: Fimbriae; Flagella; Hafnia alvei ; Phase variation

Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B₁₂-dependent glycerol and diol dehydratases by Takamasa Tobimatsu; Tsuneo Nishiki; Masaya Morimoto; Ryou Miyata; Tetsuo Toraya (pp. 199-206).

Coenzyme B₁₂-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.

Keywords: Chimeric enzyme; Glycerol dehydratase; Diol dehydratase; Coenzyme B₁₂ ; Low solubility

New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species by Monika Janczarek; Michał Kalita; Anna Maria Skorupska (pp. 207-219).

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.

Keywords: Diversity; Identification of R. leguminosarum ; PCR detection; Pss genes

Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE by Terry C. Hazen; Romy Chakraborty; James M. Fleming; Ingrid R. Gregory; John P. Bowman; Luis Jimenez; Dai Zhang; Susan M. Pfiffner; Fred J. Brockman; Gary S. Sayler (pp. 221-232).

Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n = 367) taken from 0 m (surface)–43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study demonstrates that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.

Keywords: Trichloroethylene; Bioremediation; Methanotrophs

Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate by Ping Wei; Liping Bai; Wengang Song; Gangping Hao (pp. 233-240).

Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20°C with an optimal pH of around 8.0, and PWTSC at 40°C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30°C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters.

Keywords: Lipase; Metagenomic library; PWTSB; PWTSC

Comparative genomic study of spo0E family genes and elucidation of the role of Spo0E in Bacillus anthracis by Gyanendra P. Dubey; Azeet Narayan; Abid R. Mattoo; Gajendra P. Singh; Raj K. Kurupati; Mohd. S. Zaman; Anita Aggarwal; Renu B. Baweja; Sharmila Basu-Modak; Yogendra Singh (pp. 241-253).

The propensity of bacterium to sporulate or retain the vegetative form depends on the amount of phosphorylated Spo0A (Spo0A^-P), regulated by Spo0E multigene family of phosphatases (Spo0E, YisI and YnzD). Phylogenetic analysis revealed that Spo0E multigene family of phosphatases (SMFP) descends in two distinct clades of aerobic (Bacillus cluster) and anaerobic (Clostridia cluster) sporulating bacteria. High sequence conservation within species gives a notion that these members could have evolved through lineage and species-specific duplication event. Of the five genes in Bacillus cereus group, three are pathogen specific, and their synteny suggests that these paralogs could be involved in the regulation of amino acid metabolism and its transport. Overexpression of B. subtilis Spo0E, an ortholog of SMFP members in B. anthracis (BAS1251), resulted in sporulation deficient phenotype in B. anthracis. B. anthracis Spo0A^-P binds to a consensus DNA sequence 5′-TGNCGAA-3′ (‘0A-like box’) and loses its DNA binding ability following treatment with B. subtilis Spo0E. Thus, B. subtilis Spo0E acts on B. anthracis Spo0A^-P and, therefore could complement the function of BAS1251. Further, since ‘0A-like box’ are present in the promoter region of abrB gene, a known regulator of anthrax toxin gene expression, cross talk among SMFP members and Spo0A^-P–AbrB could regulate the expression of anthrax toxin genes.

Keywords: Spo0E; SMFP; Sporulation; Synteny; Bacillus ; Gene duplication; Ortholog

Role of NtcA, a cyanobacterial global nitrogen regulator, in the regulation of sucrose metabolism gene expression in Anabaena sp. PCC 7120 by Clarisa Marcozzi; Andrea C. Cumino; Graciela L. Salerno (pp. 255-263).

In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.

Keywords: Cyanobacteria; Sucrose metabolism; Transcriptional regulation; NtcA

Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase by Fu-Pang Lin; Hsu-Han Chuang; Yi-Hsuan Liu; Chia-Yu Hsieh; Pei-Wen Lin; Hsu-Yang Lin (pp. 265-273).

C-Terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k _cat/K _M, of AcD1ChiAK606 with the 4MU-(GlcNAc)₂ and the 4MU-(GlcNAc)₃ chitin substrates was 15–26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble α-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.

Keywords: Aeromonas caviae D1; Chitinase; C-terminal truncation

Encystment of Azotobacter nigricans grown diazotrophically on kerosene as sole carbon source by Gabriela García-Esquivel; Graciano Calva-Calva; Ronald Ferrera-Cerrato; Luis Carlos Fernández-Linares; Refugio Rodríguez Vázquez; Fernando José Esparza-García (pp. 275-281).

Encystment of Azotobacter nigricans was induced by its diazotrophic cultivation on kerosene. Its growth and nitrogenase activity were affected by kerosene in comparison to cultures grown on sucrose. Electron microscopy of vegetative cells showed that when nitrogenase activity was higher and the poly-β-hydroxybutyrate granules were not present to a significant extent, peripheral bodies were abundant. After 8 days of culture on kerosene, the presence of cysts with intracellular bunches of poly-β-hydroxybutyrate granules was observed. Germination of cysts bears germinating multicelled yet unbroken capsule cysts with up to three cells inside. This is the first report of encystment induction of Azotobacter species grown on kerosene.

Keywords: Cyst; Kerosene removal; Nitrogen fixation; Multi-celled cysts; Nitrogenase activity; Poly-β-hydroxybutyrate; PHB; NFB

Complex quorum-sensing regulatory systems regulate bacterial growth and symbiotic nodulation in Mesorhizobium tianshanense by Huijuan Cao; Menghua Yang; Huiming Zheng; Jiang Zhang; Zengtao Zhong; Jun Zhu (pp. 283-289).

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process.

Keywords: Mesorhizobium tianshanense ; Autoinducer synthase; MarR-family; Nodulation

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Archives of Microbiology (v.191, #3)

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Archives of Microbiology (v.191, #3)