- Volume 155, Issue 7, 2009

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant ΔafsA. A-factor was added at a concentration of 25 ng ml−1 to mutant ΔafsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Pneumococci that have developed the competent state kill and lyse non-competent sister cells and members of closely related species during co-cultivation in vitro. The key component in this process, called fratricide, is the product of the late competence gene cbpD. In addition, the peptidoglycan hydrolases LytA and LytC are required for efficient lysis of target cells. Here, we have investigated the relative contribution and possible role of each of the proteins mentioned above. Previous studies have shown that CbpD is produced exclusively by competent cells, whereas LytA and LytC can be provided by the competent attackers as well as the non-competent target cells. By using an improved assay to compare the effect of cis- versus trans-acting LytA and LytC, we were able to show that target cells are lysed much more efficiently when LytA and LytC are provided in cis, i.e. by the target cells themselves. Western analysis demonstrated that considerable amounts of LytC are present in the growth medium. In contrast, we were not able to detect any extracellular LytA. This finding indicates that LytA- and LytC-mediated fratricide represent different processes. In the absence of LytA and LytC, only a tiny fraction of the target cells were lysed, demonstrating that CbpD does not function efficiently on its own. However, in the presence of 1 mM EDTA, the fraction of target cells lysed directly by CbpD increased dramatically, indicating that divalent cations are involved in the regulation of fratricide under natural conditions.

In Synechocystis sp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS in Escherichia coli indicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi availability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Pi availability.

Pseudomonas aeruginosa exotoxin A (ETA) production depends on the virulence-factor regulator Vfr. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS also enhances ETA production through the ETA-regulatory gene regA. Mutants defective in vfr, regA and pvdS, plasmids that overexpress these genes individually and lacZ transcriptional/translational fusion plasmids were utilized to examine the relationship between vfr, regA and pvdS in regulating P. aeruginosa ETA production. ETA concentration and regA expression were reduced significantly in PAOΔvfr, but pvdS expression was not affected. Overexpression of Vfr produced a limited increase in ETA production in PAOΔpvdS, but not PAOΔregA. Additionally, overexpression of either RegA or PvdS did not enhance ETA production in PAOΔvfr. RT-PCR analysis showed that iron did not affect the accumulation of vfr mRNA in PAO1. These results suggest that: (i) Vfr enhances toxA expression in PAO1 both directly and indirectly through regA, but not through pvdS; (ii) vfr expression is not regulated by iron; and (iii) both Vfr and PvdS cooperate in the presence of RegA to achieve a maximum level of toxA expression.

Numerous strategies allowing bacteria to detect and respond to oxidative conditions depend on the cell redox state. Here we examined the ability of Lactococcus lactis to survive aerobically in the presence of the reducing agent dithiothreitol (DTT), which would be expected to modify the cell redox state and disable the oxidative stress response. DTT inhibited L. lactis growth at 37 °C in aerobic conditions, but not in anaerobiosis. Mutants selected as DTT resistant all mapped to the pstFEDCBA locus, encoding a high-affinity phosphate transporter. Transcription of pstFEDCBA and a downstream putative regulator of stress response, phoU, was deregulated in a pstA strain, but amounts of major oxidative stress proteins were unchanged. As metals participate in oxygen radical formation, we compared metal sensitivity of wild-type and pstA strains. The pstA mutant showed approximately 100-fold increased resistance to copper and zinc. Furthermore, copper or zinc addition exacerbated the sensitivity of a wild-type L. lactis strain to DTT. Inactivation of pstA conferred a more general resistance to oxidative stress, alleviating the oxygen- and thermo-sensitivity of a clpP mutant. This study establishes a role for the pst locus in metal homeostasis, suggesting that pst inactivation lowers intracellular reactivity of copper and zinc, which would limit bacterial sensitivity to oxygen.

Poly-γ-glutamic acid (γ-PGA) is an extracellular polymer produced by various strains of Bacillus. Ιt was first described as the component of the capsule in Bacillus anthracis, where it plays a relevant role in virulence. γ-PGA is also a distinctive component of ‘natto’, a traditional Japanese food consisting of soybean fermented by Bacillus subtilis (natto). Domesticated B. subtilis strains do not synthesize γ-PGA although they possess the functional biosynthetic pgs operon. In the present work we explore the correlation between the genetic determinants, swrAA and degU, which allow a derivative of the domestic strain JH642 to display a mucoid colony morphology on LB agar plates due to the production of γ-PGA. Full activation of the pgs operon requires the co-presence of SwrAA and the phosphorylated form of DegU (DegU∼P). The presence of either DegU∼P or SwrAA alone has only marginal effects on pgs operon transcription and γ-PGA production. Although SwrAA was identified as necessary for swarming and full swimming motility together with DegU, we show that motility is not involved in γ-PGA production. Activation of γ-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU∼P display a cooperative effect.

In order to adapt to changing environments, bacteria have evolved two-component systems (TCSs) that are able to sense and respond to environmental stimuli. The signal perception relies on a sensor protein whose activation allows rapid adaptation through transcriptional regulation achieved by the regulatory protein. The ability to adhere to and grow on the surface of human host cells is an absolute requirement for many pathogens, including Neisseria meningitidis, in order to colonize new hosts and to disseminate inside their host. Among the four TCSs encoded in the meningococcus genome, only the PhoQ (MisS)/PhoP (MisR) system has been shown to constitute a functional signal transduction circuit. To investigate the involvement of this TCS in the adaptation process requisite for host cell colonization, we have tested the ability to grow on host cells of a mutant inactivated for the sensor of the TCS. Our results demonstrate the involvement of the TCS in the adaptation of the meningococcus to growth on host cells. We show that the expression of the PhoQ (MisS)/PhoP (MisR) TCS is cell-contact controlled. Furthermore, this TCS controls the regulation of a group of genes, the REP2 regulon, previously shown to be cell-contact regulated and to encode functions crucial for the adaptation of the bacterium to host cell colonization. Thus, we provide evidence that one of the four TCSs existing in N. meningitidis contributes to the adaptation of the pathogen to growth on host cells.

Vibrio tubiashii is a re-emerging pathogen of molluscs that secretes a variety of extracellular products (ECPs), including a metalloprotease and a cytolysin/haemolysin. Previously, we reported that the V. tubiashii haemolysin locus consists of two ORFs (vthB and vthA), similar to that of the homologous haemolysin genes (vvhB and vvhA) found in Vibrio vulnificus. Here, we demonstrate that the concomitant expression of both V. tubiashii genes resulted in significantly higher haemolytic activity than the vthA gene alone. In addition, we created a VthAB− mutant strain of V. tubiashii that was virtually devoid of haemolytic activity in liquid media. Interestingly, significant production of an additional haemolysin(s) was observed on blood plates. Moreover, we have previously reported that in V. tubiashii, proteolytic and haemolytic activities are inversely produced during bacterial growth. Here, we study this correlation in more detail and present evidence that the VtpA metalloprotease inhibits haemolytic activity in culture supernatants, based on the following evidence: (i) loss of metalloprotease activity by either mutation or EDTA inhibition resulted in increased haemolytic activity; (ii) overexpression of the vtpA gene resulted in decreased haemolytic activity; (iii) purified VtpA metalloprotease directly diminished haemolytic activity by purified VthA haemolysin. Importantly, we found not only that vthAB gene expression remained high throughout growth but also that there were no dramatic differences in vthAB gene expression between the parent and VtpA− mutant strains. Thus, our results strongly suggest that the V. tubiashii metalloprotease directly targets its haemolysin.

To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTP's final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, β-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTP's final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.

The gene encoding major adhesin protein P1 of Mycoplasma pneumoniae, MPN141, contains two DNA sequence stretches, designated RepMP2/3 and RepMP4, which display variation among strains. This variation allows strains to be differentiated into two major P1 genotypes (1 and 2) and several variants. Interestingly, multiple versions of the RepMP2/3 and RepMP4 elements exist at other sites within the bacterial genome. Because these versions are closely related in sequence, but not identical, it has been hypothesized that they have the capacity to recombine with their counterparts within MPN141, and thereby serve as a source of sequence variation of the P1 protein. In order to determine the variation within the RepMP2/3 and RepMP4 elements, both within the bacterial genome and among strains, we analysed the DNA sequences of all RepMP2/3 and RepMP4 elements within the genomes of 23 M. pneumoniae strains. Our data demonstrate that: (i) recombination is likely to have occurred between two RepMP2/3 elements in four of the strains, and (ii) all previously described P1 genotypes can be explained by inter-RepMP recombination events. Moreover, the difference between the two major P1 genotypes was reflected in all RepMP elements, such that subtype 1 and 2 strains can be differentiated on the basis of sequence variation in each RepMP element. This implies that subtype 1 and subtype 2 strains represent evolutionarily diverged strain lineages. Finally, a classification scheme is proposed in which the P1 genotype of M. pneumoniae isolates can be described in a sequence-based, universal fashion.

Using microarray-based comparative genome hybridization (mCGH), the genomic content of Wolbachia pipientis wMel from Drosophila melanogaster was compared to the closely related Wolbachia from D. innubila (wInn), D. santomea (wSan), and three strains from D. simulans (wAu, wRi, wSim). A large number of auxiliary genes are identified in these five strains, with most absent/divergent genes being unique to a given strain. Each strain caused an average of ∼60 genes to be removed from the core genome. As such, these organisms do not appear to have the streamlined genomes expected of obligate intracellular bacteria. Prophage, hypothetical and ankyrin repeat genes are over-represented in the absent/divergent genes, with 21–87 % of absent/divergent genes coming from prophage regions. The only wMel region absent/divergent in all five query strains is that containing WD_0509 to WD_0511, including a DNA mismatch repair protein MutL-2, a degenerate RNase, and a conserved hypothetical protein. A region flanked by the two portions of the WO-B prophage in wMel is found in four of the five Wolbachia strains as well as on a plasmid of a rickettsial endosymbiont of Ixodes scapularis, suggesting lateral gene transfer between these two obligate intracellular species. Overall, these insect-associated Wolbachia have highly mosaic genomes, with lateral gene transfer playing an important role in their diversity and evolution.

Loss of a type II restriction–modification (RM) gene complex, such as EcoRI, from a bacterial cell leads to death of its descendent cells through attack by residual restriction enzymes on undermethylated target sites of newly synthesized chromosomes. Through such post-segregational host killing, these gene complexes impose their maintenance on their host cells. This finding led to the rediscovery of type II RM systems as selfish mobile elements. The host prokaryote cells were found to cope with such attacks through a variety of means. The RecBCD pathway of homologous recombination in Escherichia coli repairs the lethal lesions on the chromosome, whilst it destroys restricted non-self DNA. recBCD homologues, however, appear very limited in distribution among bacterial genomes, whereas homologues of the RecFOR proteins, responsible for another pathway, are widespread in eubacteria, just like the RM systems. In the present work, therefore, we examined the possible contribution of the RecFOR pathway to cell survival after loss of an RM gene complex. A recF mutation reduced survival in an otherwise rec-positive background and, more severely, in a recBC sbcBC background. We also found that its effect is prominent in the presence of specific non-null mutant forms of the RecBCD enzyme: the resistance to killing seen with recC1002, recC1004, recC2145 and recB2154 is severely reduced to the level of a null recBC allele when combined with a recF, recO or recR mutant allele. Such resistance was also dependent on RecJ and RecQ functions. UV resistance of these non-null recBCD mutants is also reduced by recF, recJ or recQ mutation. These results demonstrate that the RecFOR pathway of recombination can contribute greatly to resistance to RM-mediated host killing, depending on the genetic background.

Bacteria belonging to the genus Acinetobacter are ubiquitous in soil and water. Only a few species, including Acinetobacter baumannii, and the unnamed Acinetobacter genomic species (gen. sp.) 3 and 13TU, which together with the soil organism Acinetobacter calcoaceticus are combined in the A. calcoaceticus–A. baumannii (Acb) complex, have been recognized as important nosocomial infectious agents. The ecology, epidemiology and pathology of most species are not yet well established. Lack of practical and accurate methods limits routine identification of clinical isolates and thus hampers precise identification of those of the Acb complex and other Acinetobacter species of possible clinical significance. We previously identified a 350 bp highly variable zone on the rpoB gene which appeared to be a promising target for rapid molecular identification. In the present study, we validated this method for accuracy on a collection of reference strains belonging to A. calcoaceticus (5 strains), Acinetobacter gen. sp. 3 (29 strains), A. gen. sp. 13TU (18 strains), A. baumannii (30 strains) and one strain each of A. radioresistens, A. gen. sp. 15TU, A. gen. sp. 10, A. gen. sp. 11, A. gen. sp. ‘between 1 and 3’ and A. gen. sp. 14TU=13BJ. This represents the largest analysis to date that compares a large number of well-identified strains of the Acb complex to assess the intra- and interspecies variation within this complex. All were correctly identified with 98.9–100 % intraspecies relatedness based on partial rpoB sequence analysis. We then applied this tool to identify 99 Acinetobacter clinical isolates from four public hospitals in Marseille, France. All isolates could easily be identified to species as they were separated into 13 species sequence types with a sequence variance of 0–2.6 % from their respective type strains. Of these 99 isolates, 10 were A. haemolyticus, 52 were A. baumannii, 27 were A. gen. sp. 3, 5 were A. schindleri, 1 was A. lwoffii, and 1 was A. gen. sp. 13TU. Three were provisionally identified as A. gen. sp. 9. This is the first work to identify all specimens of a set of clinical Acinetobacter isolates at species level using rpoB sequence analysis. Our data emphasize the recognition of A. schindleri as an emerging cause of Acinetobacter-related infection and confirm that A. gen. sp. 3 is the second most commonly isolated Acinetobacter species after A. baumannii in patients.

Expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators. The staphylococcal-specific SarA family of transcription regulators control large numbers of target genes involved in virulence, autolysis, biofilm formation, stress responses and metabolic processes, and are recognized as potential therapeutic targets. Expression of some of these important regulators has been examined, mostly in laboratory strains, while the pattern of expression of these genes in other strains, especially clinical isolates, is largely unknown. In this report, a comparative analysis of 10 sarA-family genes was conducted in six different S. aureus strains, including two laboratory (RN6390, SH1000) and four clinical (MW2, Newman, COL and UAMS-1) strains, by Northern and Western blot analyses. Transcription of most of the sarA-family genes showed a strong growth phase-dependence in all strains tested. Among these genes, no difference was observed in expression of the sarA, sarV, sarT and sarU genes, while a major difference was observed in expression of the sarX gene only in strain RN6390. Expression of mgrA, rot, sarZ, sarR and sarS was observed in all strains, but the level of expression varied from strain to strain.