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Archives of Microbiology (v.189, #3)

Identification and functional characterisation of cellobiose and lactose transport systems in Lactococcus lactis IL1403 by Magdalena Kowalczyk; Muriel Cocaign-Bousquet; Pascal Loubiere; Jacek Bardowski (pp. 187-196).

Physiological, biochemical and macroarray analyses of Lactococcus lactis IL1403 and its ccpA and bglR single and double mutants engaged in lactose and β-glucosides catabolism were performed. The kinetic analysis indicated the presence of different transport systems for salicin and cellobiose. The control of salicin catabolism was found to be mediated by the transcriptional regulator BglR and the CcpA protein. The transcriptional analysis by macroarray technology of genes from the PEP:PTS regions showed that several genes, like ybhE, celB, ptcB and ptcA, were expressed at higher levels both in wild type cells exposed to cellobiose and in the ccpA mutant. We also demonstrated that in L. lactis IL1403 cultured on medium with cellobiose and lactose as carbon sources, after the first phase of cellobiose consumption and then co-metabolism of the two sugars, when cellobiose is exhausted the strain uses lactose as the only carbon source. These data could indicate that lactose and cellobiose are transported by a unique system—a PTS carrier induced by the presence of cellobiose, and negatively controlled by the CcpA regulator.

Keywords: CcpA; Sugar catabolism; Cellobiose; Lactose; PTS

Porphyromonas gingivalis HmuY and HmuR: further characterization of a novel mechanism of heme utilization by Teresa Olczak; Aneta Sroka; Jan Potempa; Mariusz Olczak (pp. 197-210).

Porphyromonas gingivalis HmuY is a putative heme-binding lipoprotein associated with the outer membrane. It is part of an operon together with a gene encoding an outer-membrane hemin utilization receptor (HmuR) and four uncharacterized genes. A similar operon organization was found in Bacteroides fragilis and B. thetaiotaomicron, with the former containing an additional HmuY homologue encoded upstream of the hmuR-like gene. In P. gingivalis cultured under heme-limited conditions, a ∼1-kb hmuY transcript was produced at high levels along with some ∼3.5 and ∼9-kb transcripts. Compared with the parental strain, mutants deficient in hmuY or hmuR or hmuY–hmuR gene function grew more slowly and bound lower amounts of hemin and hemoglobin. Significantly, they grew more slowly or were unable to grow when human serum was used as the sole iron/heme source. Analysis of the hmu promoter showed that it is regulated by iron. The HmuY protein normally occurs as a homodimer, but in the presence of hemin it may form tetramers. These results show that HmuY may be the first reported member of a new class of proteins in Porphyromonas and Bacteroides species involved in heme utilization, a function being exerted in conjunction with HmuR, an outer-membrane heme transporter.

Keywords: Porphyromonas gingivalis ; HmuR; Heme outer-membrane receptor; HmuY; Heme-binding protein; Heme uptake; Lipoprotein; His-tag

The omlA gene is involved in multidrug resistance and its expression is inhibited by coumarins in Xanthomonas campestris pv. phaseoli by Mayuree Fuangthong; Ratiboot Sallabhan; Sopapan Atichartpongkul; Nuchanart Rangkadilok; Ruchadaporn Sriprang; Jutamaad Satayavivad; Skorn Mongkolsuk (pp. 211-218).

A gene encoding the outer membrane lipoprotein, OmlA, from the bacterial phytopathogen Xanthomonas campestris pv. phaseoli was isolated and characterized. An omlA insertion mutant showed an increased susceptibility to novobiocin and coumermycin, antibiotics with gyrase inhibitor activity. The omlA mutant accumulated novobiocin. Additionally, the omlA mutant was more sensitive than the wild type to chloramphenicol, a protein synthesis inhibitor; SDS, a detergent; and menadione, a superoxide generator. The susceptibility of the mutant to unrelated chemicals indicated a general role for OmlA in maintaining membrane integrity. Transcription of omlA was downregulated in the presence of both gyrase inhibitors, suggesting that DNA supercoiling might regulate the synthesis of OmlA. The omlA gene was divergently transcribed from the gene encoding the ferric uptake regulator Fur. Although the promoters of omlA and fur overlapped, Fur did not play any regulatory role in the expression of omlA due to the fact that inactivation of Fur did not affect the expression of omlA either in the presence or absence of iron.

Keywords: SmpA/OmlA family; Xanthomonas ; Gyrase

Induction of the yjbEFGH operon is regulated by growth rate and oxygen concentration by Michael Ionescu; Alessandro Franchini; Thomas Egli; Shimshon Belkin (pp. 219-226).

The yjbEFGH operon of Escherichia coli was previously shown to be involved in exopolysaccharide production and induced during biofilm formation by osmotic stress; in this communication we investigate some of the factors taking part in its regulation. We show that induction of yjbF’::luxCDABE transcriptional fusion in response to osmotic shock is limited to the early growth phase of batch-grown cells, and is dictated by cell density in an oxygen-related manner: induction was maximal at low cell density and at high oxygen concentrations. The dependency on cell density and oxygen availability was verified in both batch and continuous culture, using either bioluminescence (luxCDABE) or β-galactosidase (lacZ) genes as the reporter system. It is further shown that yjb is also induced by low specific growth rates, even in the absence of osmotic stress. Finally, it is demonstrated that induction of the yjbEFGH operon, though clearly a stress response, is not a part of the general stress regulon under the positive control of RpoS, as it is negatively affected by this sigma factor.

Keywords: Escherichia coli ; yjbEFGH ; Batch culture; Gene regulation; Chemostat; Continuous culture; Specific growth rate; Oxygen dependency; Stress response; RpoS

Molecular characterization of Vibrio cholerae ΔrelA ΔspoT double mutants by Bhabatosh Das; Rupak K. Bhadra (pp. 227-238).

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae.

Keywords: Vibrio cholerae ; relA ; spoT ; (p)ppGpp; Stringent response

Phylogenetic diversity based on rrs, atpD, recA genes and 16S–23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru by Nery Santillana; Martha Helena Ramírez-Bahena; Paula García-Fraile; Encarna Velázquez; Doris Zúñiga (pp. 239-247).

In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132^T and Rhizobium etli CFN42^T (=USDA 9032^T), respectively. The analysis of the 16S–23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132^T and CFN42^T. These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.

Keywords: Rhizobium ; Vicia ; Pisum ; Phylogeny; Genetic diversity; Peru

Role of σ⁵⁴ in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa by José F. da Silva Neto; Tie Koide; Cecília M. Abe; Suely L. Gomes; Marilis V. Marques (pp. 249-261).

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor σ⁵⁴ (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a σ⁵⁴-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that σ⁵⁴ differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Keywords: Xylella fastidiosa ; Sigma 54; Pili; Biofilm

The Helicobacter pylori mutY homologue HP0142 is an antimutator gene that prevents specific C to A transversions by Stefan Kulick; Claudia Moccia; Christian Kraft; Sebastian Suerbaum (pp. 263-270).

Extensive genetic variability resulting from a high mutation rate and frequent recombination is a characteristic of Helicobacter pylori. Its average mutation rate is 1 × 10⁻⁶, similar to that of Escherichia coli mutator strains. Few genes involved in DNA repair have been functionally characterized in H. pylori. In E. coli, the DNA glycosylase MutY is a part of the base excision repair system. The H. pylori mutY homolog HP0142 was analyzed in this study. HP0142 was disrupted by inserting a kanamycin resistance cassette. Mutation rates were determined by measuring the frequency of point mutations in rpoB conferring resistance against rifampicin. Inactivation of mutY in H. pylori resulted in an increase of the mutation frequency by a factor of up to 34. Sequence analysis of rpoB in rifampicin-resistant clones selected from the mutY mutant showed a modest increase of G:C/T:A transversions in comparison to clones selected from wild type strains. In contrast, inactivation of mutY had a profound impact on the distribution of mutations within rpoB. This finding suggests that the efficiency with which mutY prevents transversions is strongly dependent upon the sequence context. Inactivation of mutY was associated with a stationary phase fitness deficit in competitive cultures with the wild type strain.

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12 by Catarina C. Pacheco; João F. Passos; A. Rita Castro; Pedro Moradas-Ferreira; Paolo De Marco (pp. 271-278).

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.

Keywords: Escherichia coli ; Cadmium toxicity; Oxidative stress; Glutathione; Respiratory chain

The 5′ untranslated region of fruA mRNA is required for translational enhancement of FruA synthesis during Myxococcus xanthus development by Nianhua Ding; Ying Zheng; Qian Wu; Xiaohua Mao (pp. 279-288).

The fruA gene encodes a DNA-binding response regulator protein essential for the development of Myxococcus xanthus. This gene is transcribed with an unusually long (235 nucleotides) 5′ untranslated region (UTR) that has been shown to be absolutely necessary for the induction of FruA synthesis during development. With lacZ as a reporter, it was found in this report that each regional deletion mutation within 5′ UTR caused a decrease in β-galactosidase production. Base substitution mutations that were designed to alter local stem-loop structures also decreased fruA-lacZ expression, however their compensatory mutations could not rescue fruA-lacZ expression at all. A moderate decrease in β-galactosidase activity was observed from the fruA-lacZ transcriptional fusion lacking fruA 5′ UTR; in contrast, expression of the fruA-lacZ translational fusion lacking the 5′ UTR was severely impaired. In addition, both the amount and stability of fruA-lacZ mRNA were just moderately reduced in the absence of this 5′ UTR. These results suggest that the function of the 5′ UTR of fruA mRNA requires integrity of almost the entire region and may depend on the primary sequence. More importantly, fruA 5′ UTR modulates the expression of its own gene mainly by enhancing translation efficiency of the transcript.

Keywords: Myxococcus xanthus ; fruA gene; Untranslated region; Translational control

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Archives of Microbiology (v.189, #3)

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Archives of Microbiology (v.189, #3)