- Volume 156, Issue 7, 2010

Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis.

DNA–protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter–RNAP interactions during transcription initiation in the σ A-dependent promoters P rrnAPCL1 , P rrnB and P gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

The Campylobacter jejuni flagellin protein is O-glycosylated with structural analogues of the nine-carbon sugar pseudaminic acid. The most common modifications in the C. jejuni 81-176 strain are the 5,7-di-N-acetylated derivative (Pse5Ac7Ac) and an acetamidino-substituted version (Pse5Am7Ac). Other structures detected include O-acetylated and N-acetylglutamine-substituted derivatives (Pse5Am7Ac8OAc and Pse5Am7Ac8GlnNAc, respectively). Recently, a derivative of pseudaminic acid modified with a di-O-methylglyceroyl group was detected in C. jejuni NCTC 11168 strain. The gene products required for Pse5Ac7Ac biosynthesis have been characterized, but those genes involved in generating other structures have not. We have demonstrated that the mobility of the NCTC 11168 flagellin protein in SDS-PAGE gels can vary spontaneously and we investigated the role of single nucleotide repeats or homopolymeric-tract-containing genes from the flagellin glycosylation locus in this process. One such gene, Cj1295, was shown to be responsible for structural changes in the flagellin glycoprotein. Mass spectrometry demonstrated that the Cj1295 gene is required for glycosylation with the di-O-methylglyceroyl-modified version of pseudaminic acid.

Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among Gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase–β-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis.

The lipid-rich cell wall of mycobacteria is essential not only for virulence but also for survival. Whilst anabolic pathways for mycobacterial lipid biosynthesis have been well studied, there has been little research looking into lipid catabolism. The genome of Mycobacterium tuberculosis encodes multiple enzymes with putative roles in the β-oxidation of fatty acids. In this report we explore the functionality of FadB2, one of five M. tuberculosis homologues of a β-hydroxybutyryl-CoA dehydrogenase, an enzyme that catalyses the third step in the β-oxidation cycle. Purified M. tuberculosis FadB2 catalysed the in vitro NAD+-dependent dehydration of β-hydroxybutyryl-CoA to acetoacetyl-CoA at pH 10. Mutation of the active-site serine-122 residue resulted in loss of enzyme activity, consistent with the function of FadB2 as a fatty acyl dehydrogenase involved in the β-oxidation of fatty acids. Surprisingly, purified FadB2 also catalysed the reverse reaction, converting acetoacetyl-CoA to β-hydroxybutyryl-CoA, albeit in a lower pH range of 5.5–6.5. Additionally, a null mutant of fadB2 was generated in Mycobacterium smegmatis. However, the mutant showed no significant differences from the wild-type strain with regard to lipid composition, utilization of different fatty acid carbon sources and tolerance to various stresses; the absence of any phenotype in the mutant strain could be due to the potential redundancy between the five M. smegmatis fadB paralogues.

In the genome of Klebsiella pneumoniae NTUH-K2044, nine fimbrial gene clusters were identified. Besides type 1 and type 3 fimbriae, the others are novel and were named Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg fimbriae. Prevalence analysis among 105 K. pneumoniae clinical isolates revealed that the kpc genes were highly associated with the K1 serotype isolates. Induced expression of the recombinant kpcABCD genes in Escherichia coli resulted in Kpc fimbriation and increased biofilm formation. A putative site-specific recombinase encoding gene kpcI and a 302 bp intergenic DNA flanked by 11 bp inverted repeats, namely kpcS, were identified in the upstream region of the kpcABCD genes. Using LacZ as the reporter, a dramatic difference in promoter activity of kpcS in two different orientations was observed and accordingly assigned as ON and OFF phase. kpcI expression was found to be able to invert kpcS in trans from phase ON to OFF and vice versa. Using the two-plasmid system, expression of kpcA, encoding the major component of the Kpc fimbriae, could be observed upon the induced expression of kpcI. These results indicate that KpcI is involved in the regulation of Kpc fimbriation in a phase-variable manner.

The TTHA1719 gene from Thermus thermophilus HB8 encodes an orthologue of the copper-sensing transcriptional repressor CsoR. X-ray crystal structure analysis of T. thermophilus CsoR indicated that it forms a homotetramer. The structures of the CsoR monomer and dimer are similar to those of Mycobacterium tuberculosis CsoR. In the absence of copper ions, T. thermophilus CsoR bound to the promoter region of the copper-sensitive operon copZ-csoR-copA, which encodes the copper chaperone CopZ, CsoR and the copper efflux P-type ATPase CopA, to repress their expression, while in the presence of approximately an equal amount of copper ion, CsoR was released from the DNA, to allow expression of the downstream genes. Both Cu(II) and Cu(I) ions could bind CsoR, and were effective for transcriptional derepression. Additionally, CsoR could also sense various other metal ions, such as Zn(II), Ag(I), Cd(II) and Ni(II), which led to transcriptional derepression. The copper-binding motif of T. thermophilus CsoR contains C-H-H, while those of most orthologues contain C-H-C. The X-ray crystal structure of T. thermophilus CsoR suggests that a histidine residue in the N-terminal domain is also involved in metal-ion binding; that is, the binding motif could be H-C-H-H, like that of Escherichia coli RcnR, which binds Ni(II)/Co(II). The non-conserved H70 residue in the metal-binding motif of T. thermophilus CsoR is important for its DNA-binding affinity and metal-ion responsiveness.

Solar disinfection (SODIS) is used as an effective and inexpensive tool to improve the microbiological quality of drinking water in developing countries where no other means are available. Solar UVA light is the agent that inactivates bacteria during the treatment. Damage to bacterial membranes plays a crucial role in the inactivation process. This study showed that even slightly irradiated cells (after less than 1 h of simulated sunlight) were strongly affected in their ability to maintain essential parts of their energy metabolism, in particular of the respiratory chain (activities of NADH oxidase, succinate oxidase and lactate oxidase were measured). The cells' potential to generate ATP was also strongly inhibited. Many essential enzymes of carbon metabolism (glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase) and defence against oxidative stress (catalases and glutathione-disulfide reductase) were reduced in their activity during SODIS. The work suggests that damage to membrane enzymes is a likely cause of membrane dysfunction (loss of membrane potential and increased membrane permeability) during UVA irradiation. In this study, the first targets on the way to cell death were found to be the respiratory chain and F1F0 ATPase.

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

Lactobacillus sanfranciscensis is a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh, gyrA, mapA, nox, pgmA and pta) was developed for this study. PFGE with the restriction enzyme ApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population in L. sanfranciscensis. The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in-depth examination of the strain diversity and evolution of this species.

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) lung infections are responsible for much of the mortality in cystic fibrosis (CF). However, little is known about the ecological interactions between these two, often co-infecting, species. This study provides what is believed to be the first report of the intra- and interspecies bacteriocin-like inhibition potential of Pa and Bcc strains recovered from CF patients. A total of 66 strains were screened, and shown to possess bacteriocin-like inhibitory activity (97 % of Pa strains and 68 % of Bcc strains showed inhibitory activity), much of which acted across species boundaries. Further phenotypic and molecular-based assays revealed that the source of this inhibition differs for the two species. In Pa, much of the inhibitory activity is due to the well-known S and RF pyocins. In contrast, Bcc inhibition is due to unknown mechanisms, although RF-like toxins were implicated in some strains. These data suggest that bacteriocin-based inhibition may play a role in governing Pa and Bcc interactions in the CF lung and may, therefore, offer a novel approach to mediating these often fatal infections.

The hindgut of wood-feeding lower termites is densely colonized by a multitude of symbiotic micro-organisms. While it is well established that the eukaryotic flagellates play a major role in the degradation of lignocellulose, much less is known about the identity and function of the prokaryotic symbionts associated with the flagellates. Our ultrastructural investigations of the gut flagellate Joenia annectens (from the termite Kalotermes flavicollis) revealed a dense colonization of this flagellate by diverse ecto- and endosymbiotic bacteria. Phylogenetic analysis of the small-subunit rRNA gene sequences combined with fluorescence in situ hybridization allowed us to identify and localize the different morphotypes. Furthermore, we could show that K. flavicollis harbours two phylotypes of J. annectens that could be distinguished not only by their small-subunit rRNA gene sequences, but also by differences in their assemblages of bacterial symbionts. Each of the flagellate populations hosted phylogenetically distinct ectosymbionts from the phylum Bacteroidetes, one of them closely related to the ectosymbionts of other termite gut flagellates. A single phylotype of ‘Endomicrobia’ was consistently associated with only one of the host phylotypes, although not all individuals were colonized, corroborating that ‘Endomicrobia’ symbionts do not always cospeciate with their host lineages. Flagellates from both populations were loosely associated with a single phylotype of Spirochaetales attached to their cell surface in varying abundance. Current evidence for the involvement of Bacteroidales and ‘Endomicrobia’ symbionts in the nitrogen metabolism of the host flagellate is discussed.

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

The successful nitrogen-fixing symbiosis between the Gram-negative soil bacterium Sinorhizobium meliloti and its leguminous plant host alfalfa (Medicago sativa) requires the bacterial exopolysaccharide succinoglycan. Succinoglycan and flagellum production, along with the ability to metabolize more than 20 different carbon sources and control the expression of a large number of S. meliloti genes, is regulated by the ExoR–ExoS/ChvI signalling pathway. The ExoR protein interacts with and suppresses the sensing activities of ExoS, the membrane-bound sensor of the ExoS/ChvI two-component regulatory system. Here we show that exoR expression is clearly upregulated in the absence of any functional ExoR protein. This upregulation was suppressed by the presence of the wild-type ExoR protein but not by a mutated ExoR protein lacking signal peptide. The levels of exoR expression could be directly modified in real time by changing the levels of total ExoR protein. The expression of exoR was also upregulated by the constitutively active sensor mutation exoS96, and blocked by two single mutations, exoS* and exoSsupA , in the ExoS sensing domain. Presence of the wild-type ExoS protein further elevated the levels of exoR expression in the absence of functional ExoR protein, and reversed the effects of exoS96, exoS* and exoSsupA mutations. Altogether, these data suggest that ExoR protein autoregulates exoR expression through the ExoS/ChvI system, allowing S. meliloti cells to maintain the levels of exoR expression based on the amount of total ExoR protein.