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1.  Intrinsic curvature associated with the coordinately regulated anthrax toxin gene promoters 
Microbiology (Reading, England)  2008;154(0 8):2501-2512.
The current model for virulence gene regulation in Bacillus anthracis involves several trans-acting factors, the most important of which appears to be the anthrax toxin activator encoded by the atxA gene. AtxA is a positive regulator of the toxin genes pagA, cya and lef, and of a number of other plasmid- and chromosome-encoded genes. The AtxA protein (56 kDa) possesses a predicted winged-helix DNA-binding domain and phosphate transferase domains, but the mechanism for positive regulation of AtxA target genes is not known. Sequence similarities in the promoter regions of AtxA-regulated genes are not apparent, and recombinant AtxA binds DNA with a high affinity in a non-specific manner. We hypothesized that the toxin genes possess common structural features or cis-acting elements that are required for positive regulation. We employed deletion analyses to determine the minimal sequences required for atXA-mediated toxin gene expression. In silico modelling and in vitro experiments using double-stranded DNA corresponding to the toxin gene promoter regions indicated significant curvature associated with these regions. These findings suggest that the structural topology of the DNA plays an important role in the control of anthrax toxin gene expression.
doi:10.1099/mic.0.2007/016162-0
PMCID: PMC4225132  PMID: 18667583
2.  Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high affinity binding site 
Microbiology (Reading, England)  2009;155(0 2):566-575.
SUMMARY
The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the −10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during group A streptococcal infection.
doi:10.1099/mic.0.024620-0
PMCID: PMC4130213  PMID: 19202105
3.  Shuttle expression plasmids for genetic studies in Streptococcus mutans 
Microbiology (Reading, England)  2008;154(0 8):2275-2282.
A set of shuttle plasmids containing four different constitutive promoters was generated to facilitate overexpression of foreign and native genes in streptococci, such as Streptococcus mutans. The four promoters that were chosen were: Pami, Pspac, P23 and Pveg. These promoters are active in many Gram-positive bacteria, and allow various levels of gene expression depending on the host bacterium. Shuttle plasmids were constructed based on two types of broad-host-range replication origins: a rolling-circle replicon (pSH71) and a theta replicon (pAMβ1). Shuttle plasmids derived from the pAMβ1 replicon were generated to avoid the structural and segregational stability problems associated with rolling-circle replication, since these problems may be encountered during large gene cloning. In a complementation assay, we used one such plasmid to express a gene in trans to show the utility of these plasmids. In addition, a series of plasmids was generated for the expression of recombinant proteins with an N-terminal 6×His tag or a C-terminal Strep-tag fusion, and, using a gene derived from S. mutans, we showed a high level of recombinant protein expression in S. mutans and Streptococcus pyogenes. Since these plasmids contain broad-host-range replication origins, and because the selected promoters are functional in many bacteria, they can be used for gene expression studies, such as complementation and recombinant protein expression.
doi:10.1099/mic.0.2008/019265-0
PMCID: PMC4110107  PMID: 18667560
4.  Early molecular-recognition events in the synthesis and export of group 2 capsular polysaccharides 
Microbiology (Reading, England)  2009;155(0 1):9-15.
The outer membrane (OM) of almost all Gram-negative bacteria is composed of phospholipids, lipopolysaccharide, proteins and capsular or loosely adherent polysaccharides that together mediate cellular interactions with diverse environments. Most OM components are synthesized intracellularly or at the inner membrane (IM) and thus require an export mechanism. This mini-review focuses on recent progress in understanding how synthesis of one kind of capsular polysaccharide (group 2) is coupled to the export apparatus located in the IM and spanning the periplasmic space, thus providing a transport channel to the cell surface. Although the model system for these investigations is the medically important extraintestinal pathogen Escherichia coli K1 and its polysialic acid capsule, the conclusions are general for other group 2 and group 2-like polysaccharides synthesized by many different bacterial species.
doi:10.1099/mic.0.023564-0
PMCID: PMC4044924  PMID: 19118341
5.  Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review 
Microbiology (Reading, England)  2011;157(0 8):2181-2195.
Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.
doi:10.1099/mic.0.050518-0
PMCID: PMC3980633  PMID: 21622526
6.  Production of a unique pneumococcal capsule serotype belonging to serogroup 6 
Microbiology (Reading, England)  2009;155(0 2):576-583.
Serogroup 6 of Streptococcus pneumoniae contains three serotypes named 6A, 6B and 6C with highly homologous capsule gene loci. The 6A and 6B capsule gene loci consistently differ from each other by only one nucleotide in the wciP gene. The 6A capsule gene locus has a galactosyl transferase, which has been replaced with a glucosyl transferase in the 6C capsule gene locus. We considered that a new serotype named “6X1” would be possible if the galactosyl transferase of the 6B capsule gene locus is replaced with the glucosyl transferase of 6C. We demonstrate that this gene transfer yields a viable pneumococcal strain and the capsular polysaccharide from this strain has the predicted chemical structure and serologic similarity to the capsular polysaccharide of the 6B serotype. The new strain (i.e., serotype 6X1) is typed as 6B by the quellung reaction but it can be distinguished from 6B strains with monoclonal antibodies to 6B polysaccharide. Reexamination of 264 pneumococcal isolates that were previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6X1. Nevertheless, this study shows this capsular polysaccharide is biochemically possible and could exist/emerge in nature.
doi:10.1099/mic.0.024521-0
PMCID: PMC3706093  PMID: 19202106
pneumococcus; serotype; vaccine
7.  Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA 
Microbiology (Reading, England)  2012;159(0 1):96-106.
The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase on R. capsulatus ctrA gene expression. These experiments indicate that ctrA expression is higher when cultures are grown in phototrophic (anaerobic) conditions compared with chemotrophic (aerobic) conditions, and is higher when grown in a minimal medium compared with a rich medium. We used several mutants to investigate possible regulatory pathways, and found that in R. capsulatus ctrA is not autoregulated but is regulated by a quorum-sensing system. The expression of ctrA increased as cell cultures moved through exponential phase and into stationary phase, with high levels of expression persisting long after culture turbidity plateaued. Although this growth phase-dependent pattern of expression was also observed in a quorum-sensing mutant, the magnitude of ctrA expression was about 50 % of the wild-type strain at all phases. Furthermore, reduction of phosphate concentration in the growth medium decreased ctrA expression in a culture density-independent manner, whereas reduction of malic acid (carbon source) or ammonium (nitrogen source) concentration had no effect. The regulation of ctrA expression in R. capsulatus appears to require the coordination of multiple pathways involved in detecting a variety of environmental conditions.
doi:10.1099/mic.0.062349-0
PMCID: PMC3673944  PMID: 23154973 CAMSID: cams2907
8.  Ribosomal Multi-Locus Sequence Typing: universal characterisation of bacteria from domain to strain 
Microbiology (Reading, England)  2012;158(Pt 4):1005-1015.
Summary
No single characterisation scheme currently encompasses all levels of bacterial diversity, from domain to strain. We propose Ribosomal Multi Locus Sequence Typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrated microbial taxonomy and typing. As with MLST, rMLST employs curated reference sequences to identify gene variants efficiently and rapidly. The rps loci are ideal targets for a universal characterization scheme as they are: (i) present in all bacteria; (ii) distributed around the chromosome; and (iii) encode proteins which are under stabilising selection for functional conservation. Collectively, the rps loci exhibit variation that resolves bacteria in to groups at all taxonomic and most typing levels providing significantly more resolution than 16S small subunit rRNA gene phylogenies. A web-accessible expandable database, comprising whole genome data from more than 1900 bacterial isolates, including 28 draft genomes assembled de novo from the EBI sequence read archive, has been assembled. The rps gene variation catalogued in this database permits rapid and computationally non-intensive identification of the phylogenetic position of any bacterial sequence at the domain, phylum, class, order, family, genus, species and strain levels. The groupings generated with rMLST data are consistent with current nomenclature schemes and independent of the clustering algorithm used. This approach is applicable to the other domains of life, potentially providing a rational and universal approach to the classification of life that is based on one of its fundamental features, the translation mechanism.
doi:10.1099/mic.0.055459-0
PMCID: PMC3492749  PMID: 22282518
9.  Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum 
Microbiology (Reading, England)  2008;154(Pt 9):2689-2699.
The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL–NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein–protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.
doi:10.1099/mic.0.2008/019406-0
PMCID: PMC3484679  PMID: 18757802
10.  The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a 
Microbiology (Reading, England)  2009;155(Pt 10):3260-3269.
The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzzpHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzzpHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and WzzpHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.
doi:10.1099/mic.0.028944-0
PMCID: PMC3419750  PMID: 19556292 CAMSID: cams1141
11.  Evidence for a common gene pool and frequent recombinational exchange of the tbpBA operon in Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi 
Microbiology (Reading, England)  2010;157(Pt 1):123-135.
SUMMARY
The tbpBA operon was sequenced in 42 representative isolates of Mannheimia haemolytica (32), Mannheimia glucosida (6) and Bibersteinia trehalosi (4). A total of 27 tbpB and 20 tbpA alleles were identified whilst the tbpBA operon was represented by 28 unique alleles that could be assigned to seven classes. There were 1566 (34.8% variation) polymorphic nucleotide sites and 482 (32.1% variation) variable inferred amino acid positions among the 42 tbpBA sequences. The tbpBA operons of serotype A2 M. haemolytica isolates are, with one exception, substantially more diverse than those of the other M. haemolytica serotypes and most likely have a different ancestral origin. The tbpBA phylogeny has been severely disrupted by numerous small- and large-scale intragenic recombination events. In addition, assortative (entire gene) recombination events, involving either the entire tbpBA operon or the individual tbpB and tbpA genes, have played a major role in shaping tbpBA structure and it’s distribution in the three species. Our findings indicate that a common gene pool exists for tbpBA in M. haemolytica, M. glucosida and B. trehalosi. In particular, B. trehalosi, M. glucosida and ovine M. haemolytica isolates share a large portion of the tbpA gene and this probably reflects selection for a conserved TbpA protein that provides effective iron-uptake in sheep. Bovine and ovine serotype A2 lineages have very different tbpBA alleles. Bovine-like tbpBA alleles have been partially, or completely, replaced by ovine-like tbpBA alleles in ovine serotype A2 isolates suggesting that different transferrin receptors are required by serotype A2 isolates for optimum iron uptake in cattle and sheep. Conversely, the tbpBA alleles of bovine-pathogenic serotype A1 and A6 isolates are very similar to those of closely related ovine isolates suggesting a recent and common evolutionary origin.
doi:10.1099/mic.0.041236-0
PMCID: PMC3387554  PMID: 20884693
12.  Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain 
Microbiology  2012;158(Pt 4):1005-1015.
No single genealogical reconstruction or typing method currently encompasses all levels of bacterial diversity, from domain to strain. We propose ribosomal multilocus sequence typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrating microbial genealogy and typing. As with multilocus sequence typing (MLST), rMLST employs curated reference sequences to identify gene variants efficiently and rapidly. The rps loci are ideal targets for a universal characterization scheme as they are: (i) present in all bacteria; (ii) distributed around the chromosome; and (iii) encode proteins which are under stabilizing selection for functional conservation. Collectively, the rps loci exhibit variation that resolves bacteria into groups at all taxonomic and most typing levels, providing significantly more resolution than 16S small subunit rRNA gene phylogenies. A web-accessible expandable database, comprising whole-genome data from more than 1900 bacterial isolates, including 28 draft genomes assembled de novo from the European Bioinformatics Institute (EBI) sequence read archive, has been assembled. The rps gene variation catalogued in this database permits rapid and computationally non-intensive identification of the phylogenetic position of any bacterial sequence at the domain, phylum, class, order, family, genus, species and strain levels. The groupings generated with rMLST data are consistent with current nomenclature schemes and independent of the clustering algorithm used. This approach is applicable to the other domains of life, potentially providing a rational and universal approach to the classification of life that is based on one of its fundamental features, the translation mechanism.
doi:10.1099/mic.0.055459-0
PMCID: PMC3492749  PMID: 22282518
13.  Characterization of two in vivo-expressed methyltransferases of the Mycobacterium tuberculosis complex: antigenicity and genetic regulation 
Microbiology (Reading, England)  2008;154(Pt 4):1059-1067.
Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli.
doi:10.1099/mic.0.2007/014548-0
PMCID: PMC3145104  PMID: 18375799
14.  Effect of FliK mutation on the transcriptional activity of the σ54 sigma factor RpoN in Helicobacter pylori 
Microbiology (Reading, England)  2009;155(Pt 6):1901-1911.
Helicobacter pylori is a motile Gram-negative bacterium that colonizes and persists in the human gastric mucosa. The flagellum gene regulatory circuitry of H. pylori is unique in many aspects compared with the Salmonella/Escherichia coli paradigms, and some regulatory checkpoints remain unclear. FliK controls the hook length during flagellar assembly. Microarray analysis of a fliK-null mutant revealed increased transcription of genes under the control of the σ54 sigma factor RpoN. This sigma factor has been shown to be responsible for transcription of the class II flagellar genes, including flgE and flaB. No genes higher in the flagellar hierarchy had altered expression, suggesting specific and localized FliK-dependent feedback on the RpoN regulon. FliK thus appears to be involved in three processes: hook-length control, export substrate specificity and control of RpoN transcriptional activity.
doi:10.1099/mic.0.026062-0
PMCID: PMC3145110  PMID: 19383688
15.  Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1 
Microbiology (Reading, England)  2010;156(Pt 7):2205-2215.
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.
doi:10.1099/mic.0.036145-0
PMCID: PMC2923034  PMID: 20395269
16.  Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester 
Microbiology (Reading, England)  2007;153(Pt 5):1435-1444.
The low level of available iron in vivo is a major obstacle for microbial pathogens and is a stimulus for the expression of virulence genes. In this study, Mycobacterium tuberculosis H37Rv was grown aerobically in the presence of limited iron availability in chemostat culture to determine the physiological response of the organism to iron-limitation. A previously unidentified wax ester accumulated under iron-limited growth, and changes in the abundance of triacylglycerol and menaquinone were also observed between iron-replete and iron-limited chemostat cultures. DNA microarray analysis revealed differential expression of genes involved in glycerolipid metabolism and isoprenoid quinone biosynthesis, providing some insight into the underlying genetic changes that correlate with cell-wall lipid profiles of M. tuberculosis growing in an iron-limited environment.
doi:10.1099/mic.0.2006/004317-0
PMCID: PMC3123377  PMID: 17464057
17.  Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues 
Microbiology (Reading, England)  2004;150(Pt 5):1215-1224.
The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N1-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.
PMCID: PMC3109667  PMID: 15133083
18.  Induction of Salmonella Pathogenicity Island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro 
Microbiology (Reading, England)  2009;156(Pt 4):1120-1133.
Summary
Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella Pathogenicity Island 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here we show that, culture condition affects the frequency of Salmonella Pathogenicity Island 1 induced bacteria and therefore invasion efficiency and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-log bacteria were more invasive and this was associated with a greater frequency of Salmonella Pathogenicity Island 1 induced, motile bacteria as revealed by single-cell analysis of gene expression. Culture condition also affected the ability of Salmonella to adapt to the intracellular environment since it caused marked differences in intracellular replication. These findings show that induction of Salmonella Pathogenicity Island 1 under different pre-invasion growth conditions can impact the ability of Salmonella to interact with eukaryotic host cells.
doi:10.1099/mic.0.032896-0
PMCID: PMC2848694  PMID: 20035008
epithelial; flagella; invasion; motility; Salmonella-containing vacuole; transcriptome
19.  Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella–host cell interactions in vitro 
Microbiology  2010;156(Pt 4):1120-1133.
Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella pathogenicity island (SPI) 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here, we show that culture conditions affect the frequency, and therefore invasion efficiency, of SPI1-induced bacteria and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-exponential-phase bacteria were more invasive and this was associated with a greater frequency of SPI1-induced, motile bacteria, as revealed by single-cell analysis of gene expression. Culture conditions also affected the ability of Salmonella to adapt to the intracellular environment, since they caused marked differences in intracellular replication. These findings show that induction of SPI1 under different pre-invasion growth conditions can affect the ability of Salmonella to interact with eukaryotic host cells.
doi:10.1099/mic.0.032896-0
PMCID: PMC2848694  PMID: 20035008
20.  Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2Δ) mutants is influenced by the carbon source and rapamycin 
Microbiology (Reading, England)  2009;156(Pt 3):929-939.
SUMMARY
The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and C. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that C. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only four hours of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and C. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α–ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in C. albicans than S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the TOR inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased C. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but nonculturable-like state in this species, while C. albicans ilv1Δ and ilv2Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the C. albicans cidal phenotype.
doi:10.1099/mic.0.034348-0
PMCID: PMC2841795  PMID: 20019084
21.  Variants of phosphoribosylpyrophosphate synthetase (PrsA) alter cellular pools of ribose-5-phosphate and influence thiamine synthesis in S. enterica 
Microbiology (Reading, England)  2009;156(Pt 3):950-959.
Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA are present in Salmonella enterica independent of PurF. Here we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C. 2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation, and allowed non-enzymatic formation of PRA due to a buildup of ribose-5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.
doi:10.1099/mic.0.033050-0
PMCID: PMC2889433  PMID: 19959576
22.  Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2Δ) mutants is influenced by the carbon source and rapamycin 
Microbiology  2010;156(Pt 3):929-939.
The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased Ca. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Δ and ilv2 Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.
doi:10.1099/mic.0.034348-0
PMCID: PMC2841795  PMID: 20019084
23.  Phosphoribosylpyrophosphate synthetase (PrsA) variants alter cellular pools of ribose 5-phosphate and influence thiamine synthesis in Salmonella enterica 
Microbiology  2010;156(Pt 3):950-959.
Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C. 2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.
doi:10.1099/mic.0.033050-0
PMCID: PMC2889433  PMID: 19959576
24.  Crystal structure of the transcriptional repressor PagR of Bacillus anthracis 
Microbiology (Reading, England)  2009;156(Pt 2):385-391.
PagR is a transcription repressor in Bacillus anthracis, that controls the chromosomal Slayer genes eag, sap and down-regulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate resistance genes in numerous bacteria. The crystal structure of PagR was solved using MAD techniques and was refined with 1.8Å resolution diffraction data. The PagR molecules form dimers as observed in all SmtB/ArsR repressor family of proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model building studies suggest that the dimer binds to a DNA duplex with a bend of around 40º.
doi:10.1099/mic.0.033548-0
PMCID: PMC2828352  PMID: 19926656
PagR; Bacillus anthracis; anthrax
25.  Crystal structure of the transcriptional repressor PagR of Bacillus anthracis 
Microbiology  2010;156(Pt 2):385-391.
PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 Å resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 4 °.
doi:10.1099/mic.0.033548-0
PMCID: PMC2828352  PMID: 19926656

Results 1-25 (138)