- Volume 154, Issue 9, 2008

Staphylococcal biofilm formation depends on the transcription factor σ B. We further investigated the role of σ B in biofilm formation and persistence in vitro and in vivo in a subcutaneous rat model. As expected, expression of all σ B operon genes was transiently higher in the first 6 h of biofilm formation compared to planktonic bacteria, concurrent with a temporary upregulation of icaA and aap expression. However, we also observed a second upregulation of sigB expression in biofilm more than 2 days old without upregulation of icaA or aap. Biofilm formation by Staphylococcus epidermidis strains 8400 and 1457 was compared to that of isogenic mutants with inactivation of rsbU, of rsbUVW and of the entire σ B operon. Both wild-type strains and the constitutively sigB-expressing rsbUVW mutant showed a strong biofilm-positive phenotype. The rsbUVW mutant biofilm was, however, thinner and more evenly spread than the wild-type biofilm. Inactivation of SigB in the rsbUVWsigB mutant or mutation of the positive regulator RsbU reduced both the number of sessile bacteria and polysaccharide intercellular adhesin (PIA) synthesis. These differences between the wild-types and their respective mutants appeared after 6 h in in vitro biofilms but only after 4 days in in vivo biofilms. Our results provide additional evidence for a role for σ B in biofilm formation. They also suggest a role for σ B in biofilm maturation and stability that is independent of PIA or accumulation-associated protein (Aap) and point to significant differences in the temporal development between in vitro and in vivo biofilms.

Plague is characterized by geographical foci from which it re-emerges after decades of silence, a fact currently explained by enzootic and epizootic cycles between plague-susceptible and plague-resistant rodents. To assess the potential role of soil in plague epidemiology, we experimentally investigated whether Yersinia pestis could persist alive and virulent in soil. Sterilized soil inoculated with virulent Y. pestis biotype Orientalis was regularly sampled for 40 weeks in duplicate. Each sample was observed by acridine orange staining and immunofluorescence using an anti-Y. pestis polyclonal antibody, and DNA was extracted for PCR amplification and sequencing of the Y. pestis ureD, caf1 and pla genes. All samples were inoculated onto selective agar, and samples from soil that had been incubated for 10, 60, 165, 210 and 280 days were also inoculated into each of two BALB/c female mice. The mouse experiment was performed in triplicate. Non-inoculated, sterilized soil samples were used as negative controls. Micro-organisms fluorescing orange and detected by immunofluorescence were identified as Y. pestis in all samples. They were recovered in pure agar cultures for up to 30 weeks but thereafter were contaminated with Pseudomonas spp. Soil that had been inoculated with Y. pestis proved to be fully virulent in mice, which died with Y. pestis septicaemia and multiple organ involvement. Negative control mice showed no signs of disease. These data indicate that Y. pestis biotype Orientalis can remain viable and fully virulent after 40 weeks in soil. This study is a first step on which to base further investigations of a potential telluric reservoir for Y. pestis, which could represent an alternative mechanism for the maintenance of plague foci.

We have developed a system for the induction of marker-free mutation of Bacillus subtilis. The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the araR gene, which encodes the repressor for the arabinose operon (ara) of B. subtilis. The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene (neo) fused to the ara promoter. First, the chromosomal araR locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by araR on the selective marker cassette, represses the expression of neo in the absence of l-arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of araR. The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb iolS–csbC region and the 41.8 kb hutM–csbC region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.

The genome of Mycoplasma gallisepticum strain Rlow has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMIori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMIori disrupted the vlhA1.2 gene, which has 97 % DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.

The RpoN–RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN–RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.

Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.

We identified and attempted to disrupt the Cryptococcus neoformans homoserine and/or threonine biosynthetic genes encoding aspartate kinase (HOM3), homoserine kinase (THR1) and threonine synthase (THR4); however, each gene proved recalcitrant to disruption. By replacing the endogenous promoters of HOM3 and THR1 with the copper-repressible CTR4-1 promoter, we showed that HOM3 and THR1 were essential for the growth of C. neoformans in rich media, when ammonium was the nitrogen source, or when threonine was supplied as an amino acid instead of a dipeptide. Moreover, the severity of the growth defect associated with HOM3 or THR1 repression increased with increasing incubation temperature. We believe this to be the first demonstration of threonine biosynthetic genes being essential in a fungus. The necessity of these genes for C. neoformans growth, particularly at physiologically relevant temperatures, makes threonine biosynthetic genes ideal anti-cryptococcal drug targets.

LysR-type transcriptional regulators (LTTRs) are one of the key players that help bacteria adapt to different environments. We have designated STM0952, a putative LTTR in Salmonella enterica serovar Typhimurium (S. Typhimurium), as hydrogen peroxide resistance gene (hrg). A hrg knockout mutant of S. Typhimurium was sensitive to oxidative products of the respiratory burst, specifically to H2O2. The hrg mutant is profoundly attenuated in a murine model of infection and showed decreased intracellular proliferation in macrophages. It also induced increased amounts of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild-type. A strain overexpressing the hrg gene showed a survival advantage over the wild-type Salmonella under H2O2-induced stress. Microarray analysis suggested the presence of an Hrg regulon, which is required for resistance to the toxic oxidative products of the reticuloendothelial system.

Copper homeostasis is tightly regulated in all living cells as a result of the necessity and toxicity of this metal in free cationic form. In Gram-negative bacteria CPx-type ATPases (e.g. CopA in Escherichia coli) and heavy-metal efflux RND proteins (e.g. CusA in E. coli) play an important role in transport of copper across the cytoplasmic and outer membrane. We investigated the expression of CusA- and CopA-like proteins in Shewanella oneidensis MR1 and Shewanella strain MB4, a Mn(IV)-reducing isolate from a metal-polluted harbour sediment. Q-PCR analysis of total mRNA extracted from cultures grown under aerobic conditions with 25 μM copper showed significantly increased expression of cusA (Student's t-test: MR1, P<0.0001; MB4, P=0.0006). This gene was also induced in the presence of 100 μM copper and 10 or 25 μM cadmium in both tested strains. In the absence of oxygen, with fumarate as final electron acceptor and 100 μM copper, a prolonged lag phase (5 h) was observed and general fitness decreased as evidenced by twofold lower copy numbers of 16S rRNA compared to aerobic conditions. cusA expression in cells grown under these conditions remained at comparable levels (MR1) or was slightly decreased (MB4), compared to aerobic copper challenges. A gene homologous to the copA gene of S. oneidensis was not detected in strain MB4. Although low copA copy numbers were observed in strain MR1 under conditions with 25 and 100 μM copper, copA was not detected in mRNA from cultures grown in the presence of 10 μM cadmium, or in the absence of added heavy metals. However, copA was highly induced under anaerobic conditions with 100 μM copper (P=0.0011). These results suggest essentially different roles for the two proteins CopA and CusA in the copper response in S. oneidensis MR1, similar to findings in more metal-resistant bacteria such as Escherichia coli and Cupriavidus metallidurans.

Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.

The alternative sigma factor SigF of Mycobacterium tuberculosis has been characterized in detail as a general-stress, stationary-phase sigma factor involved in the virulence of the bacterium. While a homologous gene has been annotated in the genome of the fast-growing Mycobacterium smegmatis, little experimental evidence is available on the function of this gene. Here, we demonstrate that SigF of M. smegmatis is required for resistance to hydrogen peroxide, heat shock and acidic pH, but not for survival in human neutrophils. No difference in sensitivity to isoniazid was observed between the wild-type strain and the ΔsigF mutant, suggesting that SigF-mediated resistance to hydrogen peroxide was via a pathway independent of KatG or AhpC. RT-PCR and 5′-RACE (rapid amplification of cDNA ends) analyses showed that sigF of M. smegmatis was co-transcribed with rsbW (thought to encode an anti-sigma factor for SigF) and MSMEG_1802 (unknown function) and was expressed from two promoters, one upstream of MSMEG_1802 and the second upstream of rsbW. Analysis of transcriptional lacZ fusion constructs in the sigF-deletion background revealed that the MSMEG_1802 promoter was dependent on SigF for expression. Moreover, MSMEG_1802-lacZ was induced twofold upon entry into stationary phase, while exposure of exponentially growing cultures to various stress conditions (e.g. heat, cold, ethanol, hydrogen peroxide or different pH values) did not lead to induction of MSMEG_1802-lacZ. Expression of rsbW-lacZ was independent of SigF and remained constant throughout the growth cycle and under various stress conditions unless the bacteria were challenged with d-cycloserine.