- Volume 154, Issue 5, 2008

In Streptococcus pneumoniae, autolysis is considered a programmed cell-death process executed principally by the major autolysin (LytA), and the underlying mechanism causing its activation is not completely understood. It is known that autolysis is triggered by competence development at alkaline pH and regulated by a two-component system, ComDE, which senses a competence-stimulating peptide (CSP) and behaves as a quorum-sensing mechanism. In this work, we found that acidic stress triggered a LytA-mediated autolysis and, curiously, this phenomenon was regulated by a CSP-independent ComE pathway. A further analysis of a hyperactive ComD mutant revealed that ComE needs to be phosphorylated to activate acidic stress-induced lysis (ASIL). The comE transcripts were induced by acidic culture conditions, suggesting that ComE could be sensing acidic stress. We also investigated CiaRH, a two-component system whose null mutants show a comE derepression and a CSP-dependent autolysis induction at alkaline pH. By analysis of cia comE double mutants, we demonstrated that CiaRH protected cells from ASIL by a ComE-independent pathway. Here, we propose that ComE is the principal route of the signalling pathway that determines a global stress response, and clearly regulates the induction of the LytA-mediated programmed cell death in S. pneumoniae. Acidic stress may represent for S. pneumoniae an alternative condition, in addition to competence and antibiotics, to assure the release of virulence factors, DNA and cell-wall compounds by autolysis, favouring genetic exchange and contributing to its pathogenesis.

N,N′-diacetylchitobiose [(GlcNAc)2] is the main degradation product from chitin by the action of chitinases of Serratia marcescens 2170. Uptake of (GlcNAc)2 via a (GlcNAc)2-specific enzyme II permease by this bacterium has been demonstrated previously. Here, we report the contribution of chitobiase and N-acetylglucosamine (GlcNAc) uptake to the utilization of (GlcNAc)2. When S. marcescens 2170 was cultivated in a medium containing chitin, chitobiase activity was detected both inside and outside the cells; intracellular chitobiase was more abundant and suggested to be mainly located in the periplasm. Production of chitobiase was induced by GlcNAc and more effectively by (GlcNAc)2. For induction of chitobiase, uptake of (GlcNAc)2 was essential but ChiR, an essential regulator of chitinase induction, was not required. S. marcescens 2170 grew well on both GlcNAc and (GlcNAc)2 but mutants defective in either chitobiase or NagE, the GlcNAc-specific enzyme II permease, showed reduced growth on (GlcNAc)2. These results suggest that, in addition to uptake as (GlcNAc)2, a proportion of the (GlcNAc)2 is converted to GlcNAc by chitobiase, mainly in the periplasm, and incorporated into the cytoplasm by NagE. The mutant defective in chitobiase grew more slowly on (GlcNAc)2 than on GlcNAc, indicating that (GlcNAc)2 is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. Therefore, uptake as both (GlcNAc)2 and GlcNAc is important for efficient utilization of (GlcNAc)2 in S. marcescens.

A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K m=399±115 μM, k cat=48.9±8.5 s−1 and k cat/K m=1.23×105 M−1 s−1. The optimal pH and temperature for IunH were found to be pH 6 and 80 °C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89±0.23×10−2 μmol min−1 (mg dry wt)−1. These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

We previously found the class C S.ma.I2 group II (GII) intron in Serratia marcescens SCH909 inserted into the variable region of a class 1 integron within the attC site of the ant(2′′)-Ia gene cassette. Here, we demonstrate that this ant(2′′)-Ia : : S.ma.I2 gene cassette is a recombinationally active element despite the presence of the S.ma.I2 intron. In addition, S.ma.I2 is an active GII intron capable of performing self-splicing and invading specific target sites. Intron homing to a DNA target site is RecA-independent and recognizes the intron binding site (IBS)1 and IBS3 regions, formed by the 5′ TTGTT 3′ consensus sequence located within the inverse core site of attC integrons. Our results also indicate that the process for S.ma.I2 intron mobilization involves a secondary structure provided by the folding of the complete attC site. Moreover, phylogenetic analysis of the class C GII introns showed a clear divergent clade formed by introns that insert within specific sites usually associated with lateral gene transfer.

Some Vibrio anguillarum strains produce a catechol-type siderophore named vanchrobactin, whose biosynthetic pathway has not been completely elucidated. In addition to the previously described genes vabA, vabC, vabB, vabE, vabF, vabS and vabH, in the present study we have identified the genes encoding a DAHP (3-deoxy-d-arabino-heptulosonate-7-phosphate) synthetase (vabG), a phosphopantheteinyl transferase (vabD), a LysR-family transcriptional regulator (vabR) and a putative siderophore receptor (fvtA). A deletion affecting vabG or vabD greatly reduced growth under iron-limiting conditions, whereas deletion of vabR did not have significant effects. Vanchrobactin production was abolished in the vabD mutant, whereas the vabG mutant retained a residual vanchrobactin production ability. Reverse transcriptase-mediated PCR indicated that this 11-gene cluster is organized into six iron-regulated transcriptional units. Transcriptional lacZ fusions demonstrated that the ferric uptake regulator (Fur) protein is the main iron-responsive regulator of these genes. Interestingly, the vabG gene was strongly iron-repressed, but Fur was not essential for this repression. In addition, the maximal expression from the vabG promoter was achieved only in the presence of an intact copy of vabR. Analysis of the β-galactosidase activities of a fvtA : : lacZ fusion in a vabB mutant and in the presence of added vanchrobactin suggested that a ferric-vanchrobactin-dependent activator plays a positive regulatory role in transcription of the fvtA–vabD operon. This possibility is reinforced by the presence of a predicted AraC box upstream of fvtA. We propose that vanchrobactin biosynthesis is subjected to a complex regulatory circuitry aimed at adjusting vanchrobactin production for the maintenance of iron homeostasis in V. anguillarum.

Sulphur and nitrogen catabolic repressions are regulations that have long been recognized in fungi, but whose molecular bases remain largely elusive. This paper shows that catabolic repression of a protease-encoding gene correlates with the modulation of a phosphatidylethanolamine (PE)-specific phospholipase D (PLD) activity in the pathogenic fungus Botrytis cinerea. Our results first demonstrate that the ACP1 gene is subject to sulphur catabolic repression, with sulphate and cysteine inhibiting its expression. Sulphate and cysteine also cause a decrease of the total cellular PLD activity and, reciprocally, the two PLD inhibitors AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride] and curcumin negatively affect ACP1 expression in vivo. Cysteine moreover inhibits the PE-specific PLD activity in cell extracts. ACP1 is regulated by nitrogen, but here we show that this regulation does not rely on the proximal AREA binding site in its promoter, and that glutamine does not play a particular role in the process. A decrease in the total cellular PLD activity is also observed when the cells are fed ammonia, but this effect is smaller than that produced by sulphur. RNA-interference experiments finally suggest that the enzyme responsible for the PE-specific PLD activity is encoded by a gene that does not belong to the known HKD gene family of PLDs.

One potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and FST , and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.

The bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby, Macropus eugenii, was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments, indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing <97 % homology to a known bacterial species or phylotype was novel. Thus, despite the close proximity of the openings of the urogenital and anal tracts within the cloaca, the two sites retained a diverse range of distinct bacteria, with only a small percentage of overlapping species.

Recent data from microarray analysis have shown that integrated prophages are the most frequent sources of genomic variation between different strains of Salmonella enterica serovar Typhimurium (S. Typhimurium). This led us to hypothesize that PCR detection of the integrated prophages might be an efficient typing tool that could be used as an alternative to PFGE. In this study, we optimized four triplex PCRs specific for 12 target sequences of mostly prophage origin, and tested them in 102 field strains. The same set of strains was also characterized by PFGE. Among the strains, 22 different multiplex PCR, and 25 different PFGE profiles, were identified. Despite the fact that the PFGE was slightly more discriminatory, multiplex PCR typing, owing to its simplicity and potential of simple data sharing between laboratories, represents an interesting user-friendly alternative to PFGE typing of S. Typhimurium.

Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 % similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.

A proteomic survey of the halophilic archaeon Haloferax volcanii was performed by comparative two-dimensional gel electrophoresis in order to determine the molecular effects of salt stress on the organism. Cells were grown under optimal (2.1 M) and high (3.5 M) NaCl conditions. From this analysis, over 44 protein spots responsive to these conditions were detected. These spots were excised, digested in-gel with trypsin, subjected to QSTAR tandem mass spectrometry (LC/MS/MS) analysis, and identified by comparing the MS/MS-derived peptide sequence to that deduced from the H. volcanii genome. Approximately 40 % of the proteins detected (18 in total) displayed differential abundance based on the detection of at least two peptide fragments per protein and overall MOWSE scores of ≥75 per protein. All of these identified proteins were either uniquely present or 2.3- to 26-fold higher in abundance under one condition compared to the other. The majority of proteins identified in this study were preferentially displayed under optimal salinity and primarily involved in translation, transport and metabolism. However, one protein of interest whose transcript levels were confirmed in these studies to be upregulated under high salt conditions was identified as a homologue of the phage shock protein PspA. The pspA gene belongs to the psp stress-responsive regulon commonly found among Gram-negative bacteria where its transcription is stimulated by a wide variety of stressors, including heat shock, osmotic shock and prolonged stationary-phase incubation. Homologues of PspA are also found among the genomes of cyanobacteria, higher plants and other Archaea, suggesting that this protein may retain some aspects of functional conservation across the three domains of life. Given its integral role in sensing a variety of membrane stressors in bacteria, these results suggest that PspA may play an important role in hypersaline adaptation in H. volcanii.

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na+ and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H2 and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5–10, and grew in soda brines containing 1–4 M total Na+ (optimum at 1.0–2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1T and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6T from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov.

dsDNA bacteriophages use the dual system endolysin–holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His6-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His6-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C4–C18), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C16 and C18). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 °C and pH 7.5–8.0. Activity was increased in the presence of Ca2+ and Mn2+. To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

IgaA is a Salmonella enterica membrane protein that attenuates the response of the RcsCDB signalling system to envelope stress. This protein is essential unless the RcsCDB system is inactivated, suggesting that IgaA may constantly adjust the magnitude of the response. Such a functional link is also supported by the concurrence of the igaA and rcsD-rcsB-rcsC loci in genomes of enteric bacteria and the selection of spontaneous mutations in the RcsCDB system following IgaA deprivation. However, the exact nature of the spontaneous mutations rendering IgaA dispensable remains undefined. In this work, we examined how the transduction of an igaA null allele affects the status of the RcsCDB system. Loss of RcsCDB response was registered in ∼90 % of the IgaA-defective clones, which failed to produce the capsule material positively regulated by this system. About half of these non-mucoid clones suppressed the loss of IgaA with large deletions encompassing variable regions of the rcsD-rcsB-rcsC locus. Unexpectedly, mucoid transductants were also reproducibly obtained and indicated the capacity of S. enterica to retain a functional RcsCDB system in the absence of IgaA. Decreased levels of either RcsC or RcsD were shown in ‘mucoid’ clones lacking IgaA and displaying low responsiveness to stimuli. Taken together, these data demonstrate that the stability and responsiveness of the RcsCDB system relies on its attenuator IgaA. The type of suppressions found also support a model with IgaA controlling the level of signal flowing through RcsC and RcsD.

The Flavescence dorée phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.