- Volume 152, Issue 2, 2006
Volume 152, Issue 2, 2006
- Biochemistry And Molecular Biology
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The DNA-binding domain of the Escherichia coli CpxR two-component response regulator is constitutively active and cannot be fully attenuated by fused adjacent heterologous regulatory domains
More LessTwo-component systems (TCS) based on a sensor histidine kinase and a phosphorylated cognate target regulator allow rapid responses to environmental changes. TCS are highly evolutionarily conserved, though in only a few cases are the inducing signals understood. This study focuses on the Escherichia coli CpxR response regulator that responds to periplasmic and outer-membrane stress. N-terminal deletion mutations have been isolated that render the transcription factor constitutively active, indicating that the N terminus functions, in part, to keep the C-terminal winged-helix DNA-binding effector domain in an inactive state. Analysis of truncations spanning the CpxR interdomain region revealed that mutants retaining the α5 helix significantly augment activation. Hybrid proteins obtained by fusing the CpxR effector domain to structurally similar heterologous N-terminal regulatory domains, or even GFP, failed to restore repression to the C-terminal domain. These findings shed light on the mechanism of CpxR effector domain activation and on the investigation of constitutive mutants obtained by truncation in other TCS.
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The Sinorhizobium meliloti chromosomal origin of replication
More LessThe predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.
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Production and properties of the native Chromobacterium violaceum fucose-binding lectin (CV-IIL) compared to homologous lectins of Pseudomonas aeruginosa (PA-IIL) and Ralstonia solanacearum (RS-IIL)
More LessChromobacterium violaceum is a versatile, violet pigment (violacein)-producing β-proteobacterium, confined to tropical and subtropical regions, dwelling in soil and water, like Pseudomonas aeruginosa and Ralstonia solanacearum. These three bacteria are saprophytes that occasionally become aggressive opportunistic pathogens virulently attacking animals (the first two) and plants (the third). The recent availability of their genome sequences enabled identification in the C. violaceum genome of an ORF (locus no. 1744) that is similar to those of P. aeruginosa and R. solanacearum lectins, PA-IIL and RS-IIL, respectively. A recombinant protein, CV-IIL, encoded by that ORF exhibited fucose>mannose-specific lectin activity resembling PA-IIL. This paper describes production and properties of the native CV-IIL, which, like PA-IIL and RS-IIL, is probably also a quorum-sensing-driven secondary metabolite, appearing concomitantly with violacein. Its formation is repressed in the CV026 mutant of C. violaceum, which lacks endogenous N-acylhomoserine lactone. The upstream extragenic sequence of its ORF contains a 20 bp sequence (5′-101–120) with partial similarities to the luxI-box and the related P. aeruginosa and R. solanacearum promoter boxes of quorum-sensing-controlled genes. The lectin level is augmented by addition of trehalose to the medium. The subunit size of CV-IIL (around 11·86 kDa) is similar to those of PA-IIL (11·73 kDa) and RS-IIL (11·60 kDa). Like PA-IIL, in the tetrameric form CV-IIL preferentially agglutinates α1-2 fucosylated H-positive human erythrocytes (regardless of their A, B or O type), as opposed to the Oh Bombay type, but differs from it in having no interaction with rabbit erythrocytes and in displaying stronger affinity to l-galactose than to l-fucose. The greater similarity of CV-IIL to PA-IIL than to RS-IIL might be related to the selective adaptation of both C. violaceum and P. aeruginosa to animal tissues versus the preferential homing of R. solanacearum to plants.
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SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation
More LessThe soxVW genes are located upstream of the sox gene cluster encoding the sulfur-oxidizing ability of Paracoccus pantotrophus. SoxV is highly homologous to CcdA, which is involved in cytochrome c maturation of P. pantotrophus. SoxV was shown to function in reduction of the periplasmic SoxW, which shows a CysXaaXaaCys motif characteristic for thioredoxins. From strain GBΩV, which carries an Ω-kanamycin-resistance-encoding interposon in soxV, and complementation analysis it was evident that SoxV but not the periplasmic SoxW was essential for lithoautotrophic growth of P. pantotrophus with thiosulfate. However, the thiosulfate-oxidizing activities of cell extracts from the wild-type and from strain GBΩV were similar, demonstrating that the low thiosulfate-oxidizing activity of strain GBΩV in vivo was not due to a defect in biosynthesis or maturation of proteins of the Sox system and suggesting that SoxV is part of a regulatory or catalytic system of the Sox system. Analysis of DNA sequences available from different organisms harbouring a Sox system revealed that soxVW genes are exclusively present in sox operons harbouring the soxCD genes, encoding sulfur dehydrogenase, suggesting that SoxCD might be a redox partner of SoxV. No complementation of the ccdA mutant P. pantotrophus TP43 defective in cytochrome c maturation was achieved by expression of soxV in trans, demonstrating that the high identity of SoxV and CcdA does not correspond to functional homology.
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Functional analysis of the competence transcription factor ComK of Bacillus subtilis by characterization of truncation variants
The competence transcription factor ComK is the master regulator of competence development in Bacillus subtilis. In the regulatory pathway, ComK is involved in different interactions: (i) protein–DNA interactions to stimulate transcription of ComK-dependent genes and (ii) protein–protein interactions, divided into interactions with other proteins and interactions between ComK proteins involving oligomerization. The fact that ComK displays different types of interactions suggests the presence of specific, distinct domains in the protein. This paper describes a search for functional domains, by constructing ComK truncation variants, which were tested for DNA binding, oligomerization and transcription activation. Truncations at the C-terminal end of ComK demonstrated the requirement of this part for transcription activation, but not for DNA binding. The C-terminal region is probably involved in oligomerization of ComK-dimers into tetramers. Surprisingly, a ComK truncation variant lacking 9 aa from the N-terminal end (ΔN9ComK) showed higher transcription activation than wild-type ComK, when expressed in Lactococcus lactis. However, in B. subtilis, transcription activation by ΔN9ComK was twofold lower than that by wild-type ComK, resulting from a five- to sixfold lower protein level of ComKΔN9. Thus, relatively, ΔN9ComK is more active in transcription activation than wild-type ComK. These results suggest that the presence of this N-terminal extension on ComK is a trade-off between high transcription activation and a thus far unidentified role in regulation of ComK.
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Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH
More LessSerratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using β-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. β-Galactosidase activity assays revealed that a lower growth temperature (28 °C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 °C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions.
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The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division
More LessThis article has been retracted - read the retraction notice here: https://doi.org/10.1099/mic.0.000110
A cluster of genes encoded by ORFs Rv0014c–Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.
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- Biodiversity And Evolution
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Metagenomic analysis of mesopelagic Antarctic plankton reveals a novel deltaproteobacterial group
More LessPhylogenetic screening of 3200 clones from a metagenomic library of Antarctic mesopelagic picoplankton allowed the identification of two bacterial 16S-rDNA-containing clones belonging to the Deltaproteobacteria, DeepAnt-1F12 and DeepAnt-32C6. These clones were very divergent, forming a monophyletic cluster with the environmental sequence GR-WP33-58 that branched at the base of the myxobacteria. Except for the possession of complete rrn operons without associated tRNA genes, DeepAnt-1F12 and DeepAnt-32C6 were very different in gene content and organization. Gene density was much higher in DeepAnt-32C6, whereas nearly one-third of DeepAnt-1F12 corresponded to intergenic regions. Many of the predicted genes encoded by these metagenomic clones were informational (i.e. involved in replication, transcription, translation and related processes). Despite this, a few putative cases of horizontal gene transfer were detected, including a transposase. DeepAnt-1F12 contained one putative gene encoding a long cysteine-rich protein, probably membrane-bound and Ca2+-binding, with only eukaryotic homologues. DeepAnt-32C6 carried some predicted genes involved in metabolic pathways that suggested this organism may be anaerobic and able to ferment and to degrade complex compounds extracellularly.
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- Environmental Microbiology
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Biofilm formation in Campylobacter jejuni
More LessThe major gastrointestinal pathogen Campylobacter jejuni is shown to exist as three forms of monospecies biofilm in liquid culture. It attaches to a glass surface; forms an unattached aggregate (floc); and forms a pellicle at the liquid–gas interface. The three forms of biofilm resemble each other when examined by scanning electron microscopy. The biofilm mode of growth confers protection against environmental stress, the microaerobic bacteria in flocs surviving up to 24 days at ambient temperature and atmosphere compared to 12 days survival by planktonic bacteria. The wild-type strains C. jejuni 33106, 32799, 33084 and 31485 did not form flocs, and floc formation was reduced in strains mutant in a putative flagellar protein (FliS) and in a phosphate acetyltransferase (Cj0688). All other strains tested, including strains with mutations affecting capsular polysaccharide (kpsM), flagella (maf5), protein glycosylation (pglH) and lipo-oligosaccharide (neuB1) formed flocs. Similarly, all strains tested formed a pellicle and attached to glass except the aflagellate mutant maf5; pellicle formation was reduced in fliS and cj0688 mutants. Different mechanisms, therefore, may control formation of different forms of biofilm. It is proposed that these poorly characterized forms of growth are important for the persistence of C. jejuni in the environment and may in part explain the high incidence of Campylobacter-associated food borne disease.
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Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing
More LessDeterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic–aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6·6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.
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- Genes And Genomes
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Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis
More LessRsmA is a posttranscriptional regulatory protein in Pseudomonas aeruginosa that works in tandem with a small non-coding regulatory RNA molecule, RsmB (RsmZ), to regulate the expression of several virulence-related genes, including the N-acyl-homoserine lactone synthase genes lasI and rhlI, and the hydrogen cyanide and rhamnolipid biosynthetic operons. Although these targets of direct RsmA regulation have been identified, the full impact of RsmA on cellular activities is not as yet understood. To address this issue the transcriptome profiles of P. aeruginosa PAO1 and an isogenic rsmA mutant were compared. Loss of RsmA altered the expression of genes involved in a variety of pathways and systems important for virulence, including iron acquisition, biosynthesis of the Pseudomonas quinolone signal (PQS), the formation of multidrug efflux pumps, and motility. Not all of these effects can be explained through the established regulatory roles of RsmA. This study thus provides both a first step towards the identification of further genes under RsmA posttranscriptional control in P. aeruginosa and a fuller understanding of the broader impact of RsmA on cellular functions.
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Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: application to studies of the minimal gene complement
More LessMycoplasma genitalium has been proposed as a suitable model for an in-depth understanding of the biology of a free-living organism. This paper reports that the expression of the aminoglycoside resistance gene aac(6′)-aph(2″), the only selectable marker hitherto available for M. genitalium genetic studies, correlates with a growth impairment of the resistant strains. In light of this finding, a tetM438 construction based on the tetracycline resistance gene tetM was developed; it can be used efficiently in M. genitalium and confers multiple advantages when compared to aac(6′)-aph(2″). The use of tetM438 significantly improves transformation efficiency and generates visible colonies faster. Finally, the improvements in the pMTnTetM438 construction made it possible to obtain insertions in genes which have not been previously considered to be dispensable under laboratory growth conditions.
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The structural proteome of Pseudomonas aeruginosa bacteriophage ϕKMV
The structural proteome of ϕKMV, a lytic bacteriophage infecting Pseudomonas aeruginosa, was analysed using two approaches. In one approach, structural proteins of the phage were fractionated by SDS-PAGE for identification by liquid chromatography-mass spectrometry (LC-MS). In a second approach, a whole-phage shotgun analysis (WSA) was applied. WSA uses trypsin digestion of whole phage particles, followed by reversed-phase HPLC and gas-phase fractionation of the complex peptide mixture prior to MS. The results yield a comprehensive view of structure-related proteins in ϕKMV and suggest subtle structural differences from phage T7.
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- Molecular Biology Of The Pneumococcus
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Innate immunity and the pneumococcus
More LessThe innate immune system provides a non-specific first line of defence against microbes and is crucial both in the development and effector stages of subsequent adaptive immune responses. Consistent with its importance, study of the innate immune system is a broad and fast-moving field. Here we provide an overview of the recent key advances made in this area with relation to the important pathogen Streptococcus pneumoniae (the pneumococcus).
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Versatility of pneumococcal surface proteins
More LessSurface-exposed proteins are key players during the infectious process of pathogenic bacteria. The cell surface of the Gram-positive human pathogen Streptococcus pneumoniae is decorated not only by typical Gram-positive surface proteins, but also by a family of proteins that recognizes the phosphorylcholine of the lipoteichoic and teichoic acids, namely the choline-binding proteins, and by non-classical surface proteins that lack a leader peptide and membrane-anchor motif. A comprehensive understanding of how microbial proteins subvert host immunity or host protein functions is a prerequisite for the development of novel therapeutic strategies to combat pneumococcal infections. This article reviews recent progress in the investigation of the versatility and sophistication of the virulence functions of surface-exposed pneumococcal proteins.
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Differential expression of key pneumococcal virulence genes in vivo
More LessFew studies have examined in vivo virulence gene expression in Streptococcus pneumoniae. In this study, expression of key pneumococcal virulence genes cbpA, pspA, ply, psaA, cps2A, piaA, nanA and spxB in the nasopharynx, lungs and bloodstream of mice was investigated, following intranasal challenge with the serotype 2 strain D39. Bacterial RNA was extracted, linearly amplified and assayed by real-time RT-PCR. At 72 h, cbpA mRNA was present at higher levels in the nasopharynx and lungs than in the blood. At this time-point, the mRNAs for PspA and PiaA were most abundant in the nasopharynx, whereas no significant difference in gene expression between niches was observed for ply, psaA and cps2A. Both nanA and spxB mRNAs were present in higher amounts in the nasopharynx than in the lungs or blood. These findings illustrate the dynamic nature of pneumococcal virulence gene expression in vivo.
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Zinc metalloproteinase genes in clinical isolates of Streptococcus pneumoniae: association of the full array with a clonal cluster comprising serotypes 8 and 11A
Pneumococci display large zinc metalloproteinases on the surface, including the IgA protease, which cleaves human IgA1 in the hinge region, the ZmpC proteinase, which cleaves human matrix metalloproteinase 9 (MMP-9), and two other proteinases, ZmpB and ZmpD, whose substrates have not yet been identified. Surface metalloproteinases are antigenic and have been linked to virulence. The genes encoding these proteinases reside in three distinct loci: two loci specific for zmpB and zmpC, and a third, the iga locus, containing iga and zmpD. Data obtained by this and other groups have shown that pneumococcal metalloproteinase genes are transcribed and yield mature and enzymatically active proteins. Since the presence of the four proteinase genes is variable in the pneumococcal strains whose genomes have been sequenced, the presence of these genes in a collection of 218 pneumococcal isolates, mostly from invasive disease, was investigated. The data showed that zmpB and iga were present in all the isolates examined, while zmpC and zmpD were present in a variable proportion of the isolates (in 18 and 49 %, respectively). Interestingly, isolates carrying both zmpC and zmpD were found to belong mainly to two serotypes (sts), 8 and 11A. By molecular typing, st 8 and st 11A isolates appeared to belong to the same clonal cluster. The presence of these two additional metalloproteinases could contribute to the fitness of particular pneumococcal clones.
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Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system
More LessNatural competence for genetic transformation in Streptococcus pneumoniae is controlled by the ComCDE signal-transduction pathway. Together, ComD, a membrane histidine kinase, and ComE, its cognate response regulator, constitute a typical two-component regulatory system involved in sensing the comC-encoded competence-stimulating peptide (CSP). The comCDE operon is strongly upregulated when CSP reaches a critical threshold, probably to coordinate competence induction throughout the population. During a study of the early regulation of the comCDE operon, a mutation which resulted in increased β-galactosidase production from a comC : : lacZ fusion was isolated. This mutation, which was characterized as a G→T change in the transcription terminator of the tRNAArg located immediately upstream of comCDE, is suggested to destabilize the terminator and to allow transcriptional readthrough of comCDE. Here, it is shown that, quite unexpectedly, the mutation confers reduced transformability. A series of experiments undertaken with the aim of understanding this surprising phenotype is described. Evidence is presented that increased basal-level expression of comDE impedes both spontaneous and CSP-induced competence in S. pneumoniae. There is a discussion of how an increased concentration of ComD and/or ComE could affect competence development.
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LuxS impacts on LytA-dependent autolysis and on competence in Streptococcus pneumoniae
More LessThe ubiquitous protein LuxS with S-ribosylhomocysteinase activity is involved in S-adenosyl methionine detoxification, C-1 unit recycling and the production of autoinducers that allow the cell to sense and respond to cell density. Independent reports describe the impact of LuxS deficiency on Streptococcus pneumoniae virulence in the mouse. In vitro, LuxS deficiency confers discrete phenotypes. A combined approach using genetic dissection and mixed-culture experiments allowed the involvement of LuxS in the developmental physiology of S. pneumoniae to be investigated. Functional LuxS was found to be related on the one hand to down-regulation of competence, and on the other hand to attenuation of autolysis in cultures entering stationary phase. The competence phenotype of luxS mutant bacteria was complemented by media conditioned by competence-defective ComAB0 bacteria, but not by BSA. The autolytic phenotype was complemented by BSA, but not by conditioned supernatants. It is suggested that the impact of LuxS on competence, but not on autolysis, involves cell–cell communication. The phenotype of luxS mutant strains reveals a hierarchy in the competence regulatory networks of S. pneumoniae.
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Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae
In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, PM, separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of PM can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.
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To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae
More LessIn past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous β-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.
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Distribution of epidemic antibiotic-resistant pneumococcal clones in Scottish pneumococcal isolates analysed by multilocus sequence typing
More LessSequence types of pneumococci isolated in Scotland between 1996 and 2003 were compared with those of globally prevalent antibiotic-resistant clones. Multilocus sequence typing was performed on 252 invasive pneumococcal isolates referred to the Scottish Meningococcus and Pneumococcus Reference Laboratory. Isolates were not preselected for antimicrobial resistance, patient age or disease caused. Sequence types were compared with globally significant antimicrobial-resistant clones identified by the Pneumococcal Molecular Epidemiology Network (PMEN). Sequence types identical with three of the 26 PMEN clones were present in the Scottish collection; the clones were the Spain9V-3 clone (sequence type 156, seven isolates), the England14-9 clone (sequence type 9, eight isolates) and the Utah35B-24 clone (sequence type 377, one isolate). Many Scottish isolates related to PMEN clones had lower antimicrobial MICs than those described for the corresponding PMEN type strain. A number of single- (SLVs) and double-locus variants (DLVs) were present. Fifteen SLVs related to PMEN sequence types 37, 67, 90, 81, 156, 236 and 377 were detected. The collection contained 10 DLVs related to PMEN sequence types 37, 156, 173 and 338. The majority of SLVs and DLVs were penicillin- or erythromycin-sensitive variants of the resistant PMEN type strains. Capsule switching in isolates related to the PMEN clones was also detected. The highest levels of penicillin resistance were detected in sequence type 320 (serotype 19F), which is not a PMEN clone. These data suggest that PMEN clones are not widely distributed in disease-causing isolates in Scotland.
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Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres
The authors aimed to get insights into the population structure of non-(sero)typable pneumococci (NTPn), a specific group of natural atypical pneumococci whose identification is often difficult, and which has remained insufficiently studied. A total of 265 presumptive NTPn, isolated between 1997 and 2003 from the nasopharynx of children, were characterized. Strains were confirmed to be pneumococci on the basis of bile solubility, and PCR detection or Southern blotting hybridization of lytA and psaA, genes ubiquitous in this species. Multilocus sequence typing (MLST) was used to exclude two isolates that gave ambiguous results. Non-typability was confirmed by the Quellung reaction using Omniserum. A total of 213 isolates were considered to be true NTPn. The molecular analysis of the true NTPn by PFGE and MLST showed that this population was genetically diverse, although a dominant cluster, accounting for 66 % of the isolates, was identified. Antimicrobial resistance was observed in most genetic backgrounds, and multidrug resistance to penicillin, erythromycin, clindamycin, tetracycline and sulfamethoxazole-trimethoprim was associated with strains belonging to the dominant cluster. Comparison with PFGE fingerprints and sequence types of large collections of serotypable strains showed that the genetic backgrounds of all but two NTPn were different from those found in serotypable strains. In addition, we found that NTPn strains with similar genetic backgrounds to those identified in our study had been isolated from disease sources in other countries. These observations seem to indicate that NTPn have diverse genetic backgrounds and may have evolved as a distinct group of pneumococcal isolates.
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Dynamics of pneumococcal colonization in healthy Dutch children
A recent study of pneumococcal colonization in 3198 healthy children of 1–19 years of age in The Netherlands showed pneumococcal colonization in 19 % of the children, with a peak incidence of 55 % at the age of 2 years; an age-related serotype distribution was also found. In the present study, the genetic background and resistance profiles of 578 pneumococcal isolates from the latter study were characterized by means of chromosomal genotyping and susceptibility testing. In contrast to the age-related serotype distribution observed previously, the genetic background of the strains was not age related. Few strains were found showing close homology (>95 %) with the international clones Spain9V-3 (ten isolates showed homology), England14-9 (four isolates), Tennessee23F-4 (two isolates), CSR14-10 (one isolate) and Sweden15A-25 (one isolate). In total, 19 % of strains showed resistance to one or more antibiotics. Resistance to cotrimoxazole, tetracycline, erythromycin and penicillin was found in 12·9, 5·6, 5·0 and 2·7 % of isolates, respectively. Multidrug resistance was found in 1·9 % of strains. In conclusion, pneumococcal colonization isolates from healthy Dutch children represent a heterogeneous, mostly antibiotic susceptible, genetic population.
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- Pathogens And Pathogenicity
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Phenol/water extract of Treponema socranskii subsp. socranskii as an antagonist of Toll-like receptor 4 signalling
More LessTreponema socranskii is one of the most frequently found oral spirochaetes in periodontitis and endodontic infections. LPS or glycolipids from bacteria are potent stimulators of innate immune and inflammatory systems. In this study the bioactivity of a phenol/water extract from T. socranskii subsp. socranskii (TSS-P) was analysed. TSS-P showed minimal endotoxicity and no inducing potential for proinflammatory cytokines (TNF-α and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1β. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4–MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coli LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.
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MfLIP1, a gene encoding an extracellular lipase of the lipid-dependent fungus Malassezia furfur
More LessMalassezia furfur is a dimorphic fungus and a member of the normal cutaneous microflora of humans. However, it is also a facultative pathogen, associated with a wide range of skin diseases. One unusual feature of M. furfur is an absolute dependency on externally provided lipids which the fungus hydrolyses by lipolytic activity to release fatty acids necessary for both growth and pathogenicity. In this study, the cloning and characterization of the first gene encoding a secreted lipase of M. furfur possibly associated with this activity are reported. The gene, MfLIP1, shows high sequence similarity to other known extracellular lipases, but is not a member of a lipase gene family in M. furfur. MfLIP1 consists of 1464 bp, encoding a protein with a molecular mass of 54·3 kDa, a conserved lipase motif and an N-terminal signal peptide of 26 aa. By using a genomic library, two other genes were identified flanking MfLIP1, one of them encoding a putative secreted catalase, the other a putative amine oxidase. The cDNA of MfLIP1 was expressed in Pichia pastoris and the biochemical properties of the recombinant lipase were analysed. MfLip1 is most active at 40 °C and the pH optimum was found to be 5·8. The lipase hydrolysed lipids, such as Tweens, frequently used as the source of fatty acids in M. furfur media, and had minor esterase activity. Furthermore, the lipase is inhibited by different bivalent metal ions. This is the first molecular description of a secreted lipase from M. furfur.
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The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells
More LessThe means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-κB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.
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Transcriptional and translational expression patterns associated with immobilized growth of Campylobacter jejuni
Although Campylobacter jejuni is a leading cause of food-borne illness, little is known about the mechanisms by which this pathogen mediates prolonged environmental survival or host cell virulence. Although these behaviours represent distinct phenotypes, they share a common requirement of an immobilized state. In order to understand the cellular mechanisms that facilitate a surface-associated lifestyle, transcriptional and translational expression profiles were determined for sessile and planktonic C. jejuni. These investigations indicate that the immobilized bacteria undergo a shift in cellular priorities away from metabolic, motility and protein synthesis capabilities towards emphasis on iron uptake, oxidative stress defence and membrane transport. This pattern of expression partially overlaps those reported for Campylobacter during host colonization, as well as for other species of bacteria involved in biofilms, highlighting common adaptive responses to the conserved challenges within each of these phenotypes. The adaptation of Campylobacter to immobilized growth may represent a quasi-differentiated state that functions as a foundation for further specialization towards phenotypes such as biofilm formation or host cell virulence.
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- Physiology
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Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs
More LessMethionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designated met26. The met26 mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from Sac. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into Sch. pombe Δmet26 cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Δmet26 cells was higher than in other methionine auxotrophs and that introduction of the Sac. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in the met26 mutant.
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