- Volume 156, Issue 7, 2010
Volume 156, Issue 7, 2010
- Mini-Review
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Using comparative genome analysis to identify problems in annotated microbial genomes
More LessGenome annotation is a tedious task that is mostly done by automated methods; however, the accuracy of these approaches has been questioned since the beginning of the sequencing era. Genome annotation is a multilevel process, and errors can emerge at different stages: during sequencing, as a result of gene-calling procedures, and in the process of assigning gene functions. Missed or wrongly annotated genes differentially impact different types of analyses. Here we discuss and demonstrate how the methods of comparative genome analysis can refine annotations by locating missing orthologues. We also discuss possible reasons for errors and show that the second-generation annotation systems, which combine multiple gene-calling programs with similarity-based methods, perform much better than the first annotation tools. Since old errors may propagate to the newly sequenced genomes, we emphasize that the problem of continuously updating popular public databases is an urgent and unresolved one. Due to the progress in genome-sequencing technologies, automated annotation techniques will remain the main approach in the future. Researchers need to be aware of the existing errors in the annotation of even well-studied genomes, such as Escherichia coli, and consider additional quality control for their results.
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Siderophore-mediated iron acquisition in Bacillus anthracis and related strains
More LessRecent 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.
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- Comment
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- Cell And Molecular Biology Of Microbes
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Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites
Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM H2O2 for 9 h Entamoeba histolytica presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for H2O2-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H2O2-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca2+ and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the H2O2-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca2+ chelators EGTA and BAPTA/AM, and the Ca2+ influx inhibitor verapamil prior to H2O2 exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca2+-dependent calpain-like cysteine proteases in PCD of E. histolytica. A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed E. histolytica trophozoites.
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Comparison of promoter-specific events during transcription initiation in mycobacteria
More LessDNA–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.
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Modification of the Campylobacter jejuni flagellin glycan by the product of the Cj1295 homopolymeric-tract-containing gene
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.
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Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria
More LessType 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.
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Characterization of a β-hydroxybutyryl-CoA dehydrogenase from Mycobacterium tuberculosis
More LessThe 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.
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Regulation of the Klebsiella pneumoniae Kpc fimbriae by the site-specific recombinase KpcI
More LessIn 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.
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Structural and functional characterization of the transcriptional repressor CsoR from Thermus thermophilus HB8
More LessThe 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.
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The respiratory chain is the cell's Achilles' heel during UVA inactivation in Escherichia coli
More LessSolar 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.
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- Environmental And Evolutionary Microbiology
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Ribulose-1,5-bisphosphate carboxylase/oxygenase genes as a functional marker for chemolithoautotrophic halophilic sulfur-oxidizing bacteria in hypersaline habitats
More LessThe presence and diversity of the cbb genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (a key enzyme of the Calvin–Benson cycle of autotrophic CO2 assimilation) were investigated in pure cultures of seven genera of halophilic chemolithoautotrophic sulfur-oxidizing bacteria (SOB) and in sediments from a hypersaline lake in which such bacteria have been recently discovered. All of the halophilic SOB strains (with the exception of Thiohalomonas nitratireducens) possessed the cbbL gene encoding RuBisCO form I, while the cbbM gene encoding RuBisCO form II was detected only in some of the pure cultures. The general topologies of the CbbL/CbbM trees and the 16S rRNA gene tree were different, but both markers showed that the halophilic SOB genera formed independent lineages in the Gammaproteobacteria. In some cases, such as with several strains of the genus Thiohalospira and with Thioalkalibacter halophilus, the cbbL clustering was incongruent with the positions of these strains on the ribosomal tree. In the cbbM tree, the clustering of Thiohalospira and Thiohalorhabdus strains was incongruent with their branching in both cbbL and 16S rRNA gene trees. cbbL and cbbM genes related to those found in the analysed halophilic SOB were also detected in a sediment from a hypersaline lake in Kulunda Steppe (Russia). Most of the cbbL and cbbM genes belonged to members of the genus Thiohalorhabdus. In the cbbL clone library, sequences related to those of Halothiobacillus and Thiohalospira were detected as minor components. Some of the environmental cbbM sequences belonged to as yet unknown phylotypes, representing deep lineages of halophilic autotrophs.
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An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans
More LessSome 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.
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Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses
More LessLactobacillus 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.
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Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice
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.
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Role of bacteriocins in mediating interactions of bacterial isolates taken from cystic fibrosis patients
More LessPseudomonas 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.
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Identification and localization of the multiple bacterial symbionts of the termite gut flagellate Joenia annectens
More LessThe 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.
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Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella
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.
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Autoregulation of Sinorhizobium meliloti exoR gene expression
More LessThe 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.
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- Genes And Genomes
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Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control
More LessWe generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNACys, which lacks an encoded CCA 3′ end. We observed that shorter tRNACys species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 3′ ends produced in the various mutant tRNACys species to detect maturation and decay intermediates and observed that decay of the tRNACys occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNACys at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.
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Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue
A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third α-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG.
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Use of optical mapping to sort uropathogenic Escherichia coli strains into distinct subgroups
Optical maps were generated for 33 uropathogenic Escherichia coli (UPEC) isolates. For individual genomes, the NcoI restriction fragments aligned into a unique chromosome map for each individual isolate, which was then compared with the in silico restriction maps of all of the sequenced E. coli and Shigella strains. All of the UPEC isolates clustered separately from the Shigella strains as well as the laboratory and enterohaemorrhagic E. coli strains. Moreover, the individual strains appeared to cluster into distinct subgroups based on the dendrogram analyses. Phylogenetic grouping of these 33 strains showed that 32/33 were the B2 subgroup and 1/33 was subgroup A. To further characterize the similarities and differences among the 33 isolates, pathogenicity island (PAI), haemolysin and virulence gene comparisons were performed. A strong correlation was observed between individual subgroups and virulence factor genes as well as haemolysis activity. Furthermore, there was considerable conservation of sequenced-strain PAIs in the specific subgroups. Strains with different antibiotic-resistance patterns also appeared to sort into separate subgroups. Thus, the optical maps distinguished the UPEC strains from other E. coli strains and further subdivided the strains into distinct subgroups. This optical mapping procedure holds promise as an alternative way to subgroup all E. coli strains, including those involved in infections outside of the intestinal tract and epidemic strains with distinct patterns of antibiotic resistance.
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Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism
Ralstonia eutropha H16 is probably the best-studied ‘Knallgas’ bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB−4 and ΔphaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB−4 and (iv) cells from the transition phase of R. eutropha ΔphaC1 with those from the transition phase of R. eutropha PHB−4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB−4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB−4. In strain ΔphaC1, phaA and phaB1 were highly induced compared with PHB−4. Additionally, the results of this study suggest that mutant strain PHB−4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB−4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB−4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16ΔphaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.
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The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5′ termini
As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5′ ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element.
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- Microbial Pathogenicity
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Could insect phagocytic avoidance by entomogenous fungi have evolved via selection against soil amoeboid predators?
More LessThe entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana are ubiquitously distributed in soils. As insect pathogens they adhere to the insect cuticle and penetrate through to the insect haemocoel using a variety of cuticle-hydrolysing enzymes. Once in the insect haemocoel they are able to survive and replicate within, and/or evade, phagocytic haemocyte cells circulating in the haemolymph. The mechanism by which these soil fungi acquire virulence factors for insect infection and insect immune avoidance is unknown. We hypothesize that insect phagocytic cell avoidance in M. anisopliae and B. bassiana is the consequence of a survival strategy against soil-inhabiting predatory amoebae. Microscopic examination, phagocytosis assays and amoeba mortality assays showed that these insect pathogenic fungi are phagocytosed by the soil amoeba Acanthamoeba castellanii and can survive and grow within the amoeba, resulting in amoeba death. Mammalian fungal and bacterial pathogens, such as Cryptococcus neoformans and Legionella pneumophila, respectively, show a remarkable overlap between survival against soil amoebae and survival against human macrophages. The insect immune system, particularly phagocytic haemocytes, is analogous to the mammalian macrophage. Our data suggest that the ability of the fungal insect pathogens M. anisopliae and B. bassiana to survive insect phagocytic haemocytes may be a consequence of adaptations that have evolved in order to avoid predation by soil amoebae.
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Localization and characterization of Xylella fastidiosa haemagglutinin adhesins
More LessXylella fastidiosa is a Gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell–cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.
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Role of vimA in cell surface biogenesis in Porphyromonas gingivalis
The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.
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Two regulatory elements required for enhancing ospA expression in Borrelia burgdorferi grown in vitro but repressing its expression during mammalian infection
More LessDuring cycling between the tick vector and a mammal, the Lyme disease spirochaete Borrelia burgdorferi must coordinate expression of outer-surface proteins (Osps) A and B to quickly respond to environmental changes. The pathogen abundantly produces OspA/B in the tick, but represses their expression during mammalian infection. This paper reports a regulatory structure, consisting of two sequences flanking the ospAB promoter, that is required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. Deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing its expression during murine infection. Taken together, the results demonstrate that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle.
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Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1
Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C–X–C and C–C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.
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Biofilm formation is not required for early-phase transmission of Yersinia pestis
Early-phase transmission (EPT) is a recently described model of plague transmission that explains the rapid spread of disease from flea to mammal host during an epizootic. Unlike the traditional blockage-dependent model of plague transmission, EPT can occur when a flea takes its first blood meal after initially becoming infected by feeding on a bacteraemic host. Blockage of the flea gut results from biofilm formation in the proventriculus, mediated by the gene products found in the haemin storage (hms) locus of the Yersinia pestis chromosome. Although biofilms are required for blockage-dependent transmission, the role of biofilms in EPT has yet to be determined. An artificial feeding system was used to feed Xenopsylla cheopis and Oropsylla montana rat blood spiked with the parental Y. pestis strain KIM5(pCD1)+, two different biofilm-deficient mutants (ΔhmsT, ΔhmsR), or a biofilm-overproducer mutant (ΔhmsP). Infected fleas were then allowed to feed on naïve Swiss Webster mice for 1–4 days after infection, and the mice were monitored for signs of infection. We also determined the bacterial loads of each flea that fed upon naïve mice. Biofilm-defective mutants transmitted from X. cheopis and O. montana as efficiently as the parent strain, whereas the EPT efficiency of fleas fed the biofilm-overproducing strain was significantly less than that of fleas fed either the parent or a biofilm-deficient strain. Fleas infected with a biofilm-deficient strain harboured lower bacterial loads 4 days post-infection than fleas infected with the parent strain. Thus, defects in biofilm formation did not prevent flea-borne transmission of Y. pestis in our EPT model, although biofilm overproduction inhibited efficient EPT. Our results also indicate, however, that biofilms may play a role in infection persistence in the flea.
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Reduced synthesis of the Ybt siderophore or production of aberrant Ybt-like molecules activates transcription of yersiniabactin genes in Yersinia pestis
Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a Δpgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal.
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- Physiology And Biochemistry
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Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa
Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginate-producing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstA- and LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells.
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Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans
More LessSteroid compounds have many important physiological activities in higher organisms. Testosterone and related steroids are important environmental contaminants that disrupt the endocrine systems of animals. The degradation of steroids, especially under anoxic conditions, is challenging because of their complex chemical structure. A denitrifying γ-proteobacterium, Steroidobacter denitrificans, able to grow anaerobically on a variety of steroids as the sole carbon and energy source was adopted as a model organism to study the anoxic degradation of testosterone. We identified the initial intermediates involved in the anoxic testosterone degradation pathway of S. denitrificans. We demonstrated that under anoxic conditions, S. denitrificans initially oxidizes testosterone to 1-dehydrotestosterone, which is then transformed to androsta-1,4-diene-3,17-dione. In addition, it seems that androst-4-en-3,17-dione can also be directly produced from testosterone by S. denitrificans cells. In general, the initial steps of anoxic testosterone degradation by S. denitrificans are similar to those of the oxic pathway demonstrated in Comamonas testosteroni.
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Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586
Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H2S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H2S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H2S from l-cysteine (∼30 %) than Fn1220. The Fn0625 protein degraded a variety of substrates containing βC–S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from l-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of l-cysteine with Fn1220 produced H2S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except l-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H2S in distinct manners: Fn0625 carries out β-elimination of l-cysteine to produce H2S, pyruvate and ammonia, whereas Fn1220 catalyses the β-replacement of l-cysteine to produce H2S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.
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