- Volume 152, Issue 4, 2006

MAP (mitogen-activated protein) kinase-mediated pathways are key elements in sensing and transmitting the response of cells to environmental conditions by the sequential action of phosphorylation events. In the fungal pathogen Candida albicans, different routes have been identified by genetic analysis, and especially by the phenotypic characterization of mutants altered in the Mkc1, Cek1/2 and Hog1 MAP kinases. The cell integrity (or MKC1-mediated) pathway is primarily involved in the biogenesis of the cell wall. The HOG pathway participates in the response to osmotic stress while the Cek1 pathway mediates mating and filamentation. Their actual functions are, however, much broader and Mkc1 senses several types of stress, while Hog1 is also responsive to other stress conditions and participates in two morphogenetic programmes: filamentation and chlamydospore formation. Furthermore, it has been recently shown that Cek1 participates in a putative pathway involved in the construction of the cell wall and which seems to be operative under basal conditions. As these stimuli are frequently encountered in the human host, they provide a reasonable explanation for the significant reduction in pathogenicity that several signal transduction mutants show in certain animal models of virulence. MAPK pathways therefore represent an attractive multienzymic system for which novel antifungal therapy could be designed.

In the yeast Saccharomyces cerevisiae, the Cdc25/Ras/cAMP/protein kinase A (PKA) pathway plays a major role in the control of metabolism, stress resistance and proliferation, in relation to the available nutrients and conditions. The budding yeast RasGEF Cdc25 was the first RasGEF to be identified in any organism, but very little is known about its activity regulation. Recently, it was suggested that the dispensable N-terminal domain of Cdc25 could negatively control the catalytic activity of the protein. In order to investigate the role of this domain, strains were constructed that produced two different versions of the C-terminal domain of Cdc25 (aa 907–1589 and 1147–1589). The carbon-source-dependent cell size control mechanism present in the wild type was found in the first of these mutants, but was lost in the second mutant, for which the cell size, determined as protein content, was the same during exponential growth in both ethanol- and glucose-containing media. A biparametric analysis demonstrated that this effect was essentially due to the inability of the mutant producing the shorter sequence to modify its protein content at budding. A similar phenotype was observed in strains that lacked CDC25, but which possessed a mammalian GEF catalytic domain. Taken together, these results suggest that Cdc25 is involved in the regulation of cell size in the presence of different carbon sources. Moreover, production of the aa 876–1100 fragment increased heat-stress resistance in the wild-type strain, and rescued heat-shock sensitivity in the ira1Δ background. Further work will aim to clarify the role of this region in Cdc25 activity and Ras/cAMP pathway regulation.

The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids.

The widely conserved phage-shock-protein A (pspA) operon encodes an extracytoplasmic stress response system that is essential for virulence in Yersinia enterocolitica, and has been linked to other important phenotypes in Escherichia coli, Salmonella enterica and Shigella flexneri. Regulation of pspA operon expression is mediated through a promoter upstream of pspA that depends on sigma factor RpoN (σ 54) and the enhancer binding protein PspF. PspA, PspB and PspC, encoded within the pspA operon, also regulate expression by participating in a putative signal transduction pathway that probably serves to modulate PspF activity. All of this suggests that appropriate expression of the pspA operon is critical. Previous genetic analysis of the Y. enterocolitica pspA operon suggested that an additional level of complexity might be mediated by PspF/RpoN-independent expression of some psp genes. Here, an rpoN null mutation and interposon analysis were used to confirm that PspF/RpoN-independent gene expression does originate within the psp locus. Molecular genetic approaches were used to systematically analyse the two large non-coding regions within the psp locus. Primer extension, control region deletion and site-directed mutagenesis experiments led to the identification of RpoN-independent promoters both upstream and downstream of pspA. The precise location of the PspF/RpoN-dependent promoter upstream of pspA was also determined. The discovery of these RpoN-independent promoters reveals yet another level of transcriptional complexity for the Y. enterocolitica pspA operon that may function to allow low-level constitutive expression of psp genes and/or additional regulation under some conditions.

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3–4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive β-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10–15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

5-Methyl cytosine (m5C) was detected in genomic DNA of the enteric pathogen Vibrio cholerae by HPLC analysis and immunoblotting with m5C-specific antibody. Although cleavage with the restriction endonuclease EcoRII revealed the absence of a Dcm homologue in V. cholerae, analysis of the genome sequence indicated the presence of a gene, designated in this study as vchM, which encodes a DNA (cytosine-5-)-methyltransferase (m5C-MTase) designated M.Vch. M.Vch is not associated with a restriction endonuclease or a mismatch very short patch repair (Vsr)-like endonuclease and is hence an ‘orphan’ or solitary MTase, although analysis of a phylogenetic tree indicated that related cytosine MTases are all components of restriction-modification systems. M.Vch recognizes and methylates the first 5′ C in the degenerate sequence 5′-RCCGGY-3′. RT-PCR analysis suggested that vchM gene expression is increased during the stationary phase of growth. During stationary phase, the spontaneous mutation frequency in the V. cholerae wild-type strain was significantly higher than in the corresponding vchM mutant strain, suggesting that the presence of M.Vch and the absence of a very short patch (VSP) repair-like system imposes upon V. cholerae a mutator phenotype.

The NADP(H)-dependent enzymes glucose-6-phosphate dehydrogenase (G6PDH) and ferredoxin(flavodoxin)-NADP(H) reductase (FPR), encoded by the zwf and fpr genes, respectively, are committed members of the soxRS regulatory system involved in superoxide resistance in Escherichia coli. Exposure of E. coli cells to the superoxide propagator methyl viologen (MV) led to rapid accumulation of G6PDH, while FPR was induced after a lag period of several minutes. Bacteria expressing G6PDH from a multicopy plasmid accumulated higher NADPH levels and displayed a protracted soxRS response, whereas FPR build-up had the opposite effects. Inactivation of either of the two genes resulted in enhanced sensitivity to MV killing, while further increases in the cellular content of FPR led to higher survival rates under oxidative conditions. In contrast, G6PDH accumulation over wild-type levels of expression failed to increase MV tolerance. G6PDH and FPR could act concertedly to deliver reducing equivalents from carbohydrates, via NADP+, to the FPR acceptors ferredoxin and/or flavodoxin. To evaluate whether this electron-transport system could mediate reductive repair reactions, the pathway was reconstituted in vitro from purified components; the reconstituted system was found to be functional in reactivation of oxidatively damaged iron–sulfur clusters of hydro-lyases such as aconitase and 6-phosphogluconate dehydratase. Recovery of these activities after oxidative challenge was faster and more extensive in transformed bacteria overexpressing FPR than in wild-type cells, indicating that the reductase could sustain hydro-lyase repair in vivo. However, FPR-deficient mutants were still able to fix iron–sulfur clusters at significant rates, suggesting that back-up routes for ferredoxin and/or flavodoxin reduction might be called into action to rescue inactivated enzymes when FPR is absent.

The majority of Escherichia coli strains isolated from urinary tract infections have the potential to express multiple fimbriae. Two of the most common fimbrial adhesins are type 1 fimbriae and pyelonephritis-associated pili (Pap). Previous research has shown that induced, plasmid-based expression of a Pap regulator, papB, and its close homologues can prevent inversion of the fim switch controlling the expression of type 1 fimbriae. The aim of the present study was to determine if this cross-regulation occurs when PapB is expressed from its native promoter in the chromosome of E. coli K-12 and clinical isolates. The regulation was examined in three ways: (1) mutated alleles of the pap regulatory region, including papB and papI, that maintain the pap promoter in either the off or the on phase were exchanged into the chromosome of both E. coli K-12 and the clinical isolate E. coli CFT073, and the effect on type 1 fimbrial expression was measured; (2) type 1 fimbrial expression was determined using a novel fimS : : gfp + reporter system in mutants of the clinical isolate E. coli 536 in which combinations of complete fimbrial clusters had been deleted; (3) type 1 fimbrial expression was determined in a range of clinical isolates and compared with both the number of P clusters and their expression. All three approaches demonstrated that P expression represses type 1 fimbrial expression. Using a number of novel genetic approaches, this work extends the initial finding that PapB inhibits FimB recombination to the impact of this regulation in clinical isolates.

Bacterial fructosyltransferase (FTF) enzymes synthesize fructan polymers from sucrose. FTFs catalyse two different reactions, depending on the nature of the acceptor, resulting in: (i) transglycosylation, when the growing fructan chain (polymerization), or mono- and oligosaccharides (oligosaccharide synthesis), are used as the acceptor substrate; (ii) hydrolysis, when water is used as the acceptor. Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes are closely related at the amino acid sequence level (86 % similarity). Also, the eight amino acid residues known to be involved in catalysis and/or sucrose binding are completely conserved. Nevertheless, these enzymes differ markedly in their reaction and product specificities, i.e. in β(2→6)- versus β(2→1)-glycosidic-bond specificity (resulting in levan and inulin synthesis, respectively), and in the ratio of hydrolysis versus transglycosylation activities [resulting in glucose and fructooligosaccharides (FOSs)/polymer synthesis, respectively]. The authors report a detailed characterization of the transglycosylation reaction products synthesized by the Lb. reuteri 121 Lev and Inu enzymes from sucrose and related oligosaccharide substrates. Lev mainly converted sucrose into a large levan polymer (processive reaction), whereas Inu synthesized mainly a broad range of FOSs of the inulin type (non-processive reaction). Interestingly, the two FTF enzymes were also able to utilize various inulin-type FOSs (1-kestose, 1,1-nystose and 1,1,1-kestopentaose) as substrates, catalysing a disproportionation reaction; to the best of our knowledge, this has not been reported for bacterial FTF enzymes. Based on these data, a model is proposed for the organization of the sugar-binding subsites in the two Lb. reuteri 121 FTF enzymes. This model also explains the catalytic mechanism of the enzymes, and differences in their product specificities.

Erwinia carotovora subsp. carotovora strain ATTn10 produces the β-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid (carbapenem) by expressing the carABCDEFGH operon. Mutants exhibiting increased carbapenem gene transcription were positively selected using an engineered strain with a functional β-lactamase translational fusion in carH, the last gene of the operon. However, spontaneous ampicillin-resistant mutants were isolated even when transcription of carH : : blaM was blocked by a strongly polar mutation in carE. The mechanism of resistance was shown to be due to cryptic IS10 elements transposing upstream of carH : : blaM, thereby providing new promoters enabling carH : : blaM transcription. Southern blots showed that IS10 was present in multicopy in ATTn10. In addition, a Tn10 genetic remnant was discovered. The results offer insights into the genetic archaeology of strain ATTn10 and highlight the powerful impacts of cryptic IS elements in bacterial adaptive evolution.

The genetic relationship and population structure of Salmonella enterica subspecies I strains were analysed using nucleotide sequences of four genes (mglA, proV, torC and speC). Fifteen strains from the Salmonella reference collection B (SARB), belonging to 13 serovars, were analysed. Sequence data of two housekeeping genes, mdh and mutS, of the same 15 strains reported by Brown et al. (2003) (Proc Natl Acad Sci U S A 100, 15676–15681) were also included in the analyses. Phylogenetic analysis revealed that there was a lack of congruence among the six gene trees. Split decomposition analysis resolved only five strains with a network structure, while others showed a star phylogeny. Compatibility values for the SARB strains were the lowest in comparison to those for strains representing different subspecies of S. enterica. These results showed that the genes studied have undergone frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica.

The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper a lon mutant of the α-proteobacterium Agrobacterium tumefaciens C58 has been characterized. Unlike lon mutants of Escherichia coli, the lon mutant of A. tumefaciens grows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease of A. tumefaciens shares high homology with its counterparts in E. coli and in Sinorhizobium meliloti, and functionally complements an E. coli lon mutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of LonAt corresponding to the ATP-binding site and the active site serine of the E. coli Lon protease abolish complementation of phenotypes of the A. tumefaciens and E. coli lon mutants. The nucleotide sequence upstream of A. tumefaciens lon contains an element similar to the consensus σ 32 heat-shock promoter of E. coli. Northern and Western blot analyses indicated that expression of lon is induced by elevated temperature, albeit to a much lower level than that of groEL. The lon mutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system.

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

Inspection of the genomic DNA sequence of the oral anaerobe Porphyromonas gingivalis reveals that the micro-organism possesses the peroxide-sensing transcription activator OxyR, but not the superoxide-sensing transcription factor SoxR. Investigatation of oxidative-stress-responsive proteins in P. gingivalis by two-dimensional gel electrophoresis showed that two proteins were predominantly upregulated in oxidative conditions. In a P. gingivalis oxyR mutant these two proteins were not induced by treatment with hydrogen peroxide under aerobic conditions. By N-terminal amino acid sequencing, the two proteins were found to be superoxide dismutase and alkyl hydroperoxide reductase, encoded by sod and ahpC, respectively. Northern blot and lacZ fusion analyses revealed that P. gingivalis sod and ahpC were positively regulated by OxyR. Primer extension analysis located the promoter regions of sod and ahpC, and putative −35 boxes of these promoters were found immediately adjacent to their putative OxyR-binding sequences. Moreover, the promoter regions of sod and ahpC had the ability to bind P. gingivalis OxyR protein. These results demonstrate that P. gingivalis sod is one of the OxyR regulons, suggesting that OxyR functions as an intracellular redox sensor rather than a peroxide sensor in this organism. A sod gene of Bacteroides fragilis, which is taxonomically related to P. gingivalis, is inducible by redox stresses but not controlled by its OxyR. A DNA fragment including the B. fragilis sod promoter region could bind the P. gingivalis OxyR protein; however, a putative OxyR binding sequence within the DNA fragment was 14 bases distant from a putative −35 box of its promoter.