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Volume 155,
Issue 5,
2009
Volume 155, Issue 5, 2009
- Review
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Strategies for acquiring the phospholipid metabolite inositol in pathogenic bacteria, fungi and protozoa: making it and taking it
More Lessmyo-Inositol (inositol) is an essential nutrient that is used for building phosphatidylinositol and its derivatives in eukaryotes and even in some eubacteria such as the mycobacteria. As a consequence, fungal, protozoan and mycobacterial pathogens must be able to acquire inositol in order to proliferate and cause infection in their hosts. There are two primary mechanisms for acquiring inositol. One is to synthesize inositol from glucose 6-phosphate using two sequentially acting enzymes: inositol-3-phosphate synthase (Ino1p) converts glucose 6-phosphate to inositol 3-phosphate, and then inositol monophosphatase (IMPase) dephosphorylates inositol 3-phosphate to generate inositol. The other mechanism is to import inositol from the environment via inositol transporters. Inositol is readily abundant in the bloodstream of mammalian hosts, providing a source from which many pathogens could potentially import inositol. However, despite this abundance of inositol in the host, some pathogens such as the bacterium Mycobacterium tuberculosis and the protist parasite Trypanosoma brucei must be able to make inositol de novo in order to cause disease (M. tuberculosis) or even grow (T. brucei). Other pathogens such as the fungus Candida albicans are equally adept at causing disease by importing inositol or by making it de novo. The role of inositol acquisition in the biology and pathogenesis of the parasite Leishmania and the fungus Cryptococcus are being explored as well. The specific strategies used by these pathogens to acquire inositol while in the host are discussed in relation to each pathogen's unique metabolic requirements.
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- Mini-Review
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LuxR-family ‘solos’: bachelor sensors/regulators of signalling molecules
More LessN-Acylhomoserine lactone (AHL) quorum-sensing (QS) signalling is the best-understood chemical language in proteobacteria. In the last 15 years a large amount of research in several bacterial species has revealed in detail the genetic, molecular and biochemical mechanisms underlying AHL signalling. These studies have revealed the role played by protein pairs of the AHL synthase belonging to the LuxI family and cognate LuxR-family AHL sensor–regulator. Proteobacteria however commonly possess a QS LuxR-family protein for which there is no obvious cognate LuxI synthase; these proteins are found in bacteria which possess a complete AHL QS system(s) as well as in bacteria that do not. Scientists are beginning to address the roles played by these proteins and it is emerging that they could allow bacteria to respond to endogenous and exogenous signals produced by their neighbours. AHL QS research thus far has mainly focused on a cell-density response involving laboratory monoculture studies. Recent findings on the role played by the unpaired LuxR-family proteins highlight the need to address bacterial behaviour and response to signals in mixed communities. Here we review recent progress with respect to these LuxR proteins, which we propose to call LuxR ‘solos’ since they act on their own without the need for a cognate signal generator. Initial investigations have revealed that LuxR solos have diverse roles in bacterial interspecies and interkingdom communication.
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- Cell And Molecular Biology Of Microbes
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An alternative methionine aminopeptidase, MAP-A, is required for nitrogen starvation and high-light acclimation in the cyanobacterium Synechocystis sp. PCC 6803
More LessMethionine aminopeptidases (MetAPs or MAPs, encoded by map genes) are ubiquitous and pivotal enzymes for protein maturation in all living organisms. Whereas most bacteria harbour only one map gene, many cyanobacterial genomes contain two map paralogues, the genome of Synechocystis sp. PCC 6803 even three. The physiological function of multiple map paralogues remains elusive so far. This communication reports for the first time differential MetAP function in a cyanobacterium. In Synechocystis sp. PCC 6803, the universally conserved mapC gene (sll0555) is predominantly expressed in exponentially growing cells and appears to be a housekeeping gene. By contrast, expression of mapA (slr0918) and mapB (slr0786) genes increases during stress conditions. The mapB paralogue is only transiently expressed, whereas the widely distributed mapA gene appears to be the major MetAP during stress conditions. A mapA-deficient Synechocystis mutant shows a subtle impairment of photosystem II properties even under non-stressed conditions. In particular, the binding site for the quinone QB is affected, indicating specific N-terminal methionine processing requirements of photosystem II components. MAP-A-specific processing becomes essential under certain stress conditions, since the mapA-deficient mutant is severely impaired in surviving conditions of prolonged nitrogen starvation and high light exposure.
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Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides
More LessExpression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.
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Interplay of cellular cAMP levels, σ S activity and oxidative stress resistance in Escherichia coli
Hypochlorous acid (HOCl), the active ingredient of household bleach, functions as a powerful antimicrobial that is used not only in numerous industrial applications but also in mammalian host defence. Here we show that multicopy expression of cpdA, encoding the cAMP phosphodiesterase, leads to a dramatically increased resistance of Escherichia coli to HOCl stress as well as to the unrelated hydrogen peroxide (H2O2) stress. This general oxidative stress resistance is apparently caused by the CpdA-mediated decrease in cellular cAMP levels, which leads to the partial inactivation of the global transcriptional regulator cAMP receptor protein (CRP). Downregulation of CRP in turn causes the derepression of rpoS, encoding the alternative sigma factor σ S, which activates the general stress response in E. coli. We found that these highly oxidative stress-resistant cells have a substantially increased capacity to combat HOCl-mediated insults and to degrade reactive oxygen species. Mutational analysis revealed that the DNA-protecting protein Dps, the catalase KatE, and the exonuclease III XthA play the predominant roles in conferring the high resistance of rpoS-overexpressing strains towards HOCl and H2O2 stress. Our results demonstrate the close regulatory interplay between cellular cAMP levels, σ S activity and oxidative stress resistance in E. coli.
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Gis1 is required for transcriptional reprogramming of carbon metabolism and the stress response during transition into stationary phase in yeast
More LessTransition from growth to the stationary phase in yeast is still poorly understood. Previously, we identified a group of yeast genes that are universally upregulated upon starvation for different macronutrients. Here, we demonstrate that the Gis1 transcription factor and the Rim15 kinase are responsible for the upregulation of many of these genes. In chemostat cultures, gis1 or rim15 mutant cells are outcompeted by their wild-type parents under conditions resembling the later stages of diauxie (glucose-limiting) and post-diauxie (ethanol as a carbon source). Whilst Gis1p and Rim15p have distinct functions in gene repression, the growth defects of gis1 or rim15 deletants can be accounted for by the overlapping functions of their protein products in promoting the expression of genes involved in glutamate biosynthesis, the glyoxylate cycle, the pentose phosphate pathway and the stress response. Further, we show that the sets of GIS1- and RIM15-dependent genes and the degree of their regulation change in response to the identity of the carbon source, suggesting the likely dynamics of gene regulation exerted by Rim15p and Gis1p during different phases of the transition into stationary phase. In particular, Rim15p is required for the expression of genes involved in gluconeogenesis/glycolysis and glycerol biosynthesis only when ethanol is used as the carbon source. In agreement with this, Rim15p is shown to act in parallel with Hog1p to defend cells against osmotic stress.
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- Environmental And Evolutionary Microbiology
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Characterization of a novel air–liquid interface biofilm of Pseudomonas fluorescens SBW25
More LessPseudomonads are able to form a variety of biofilms that colonize the air–liquid (A–L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation–evolution experiments which produce robust biofilms of the physically cohesive class at the A–L interface, and which have been well characterized. In this study we describe a novel A–L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static King's B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023–0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A–L interface, surface-interaction effects involving cellulose, which reduced surface tension by ∼3.8 mN m−1, may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s−1 and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.
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Cellular chain formation in Escherichia coli biofilms
More LessIn this study we report on a novel structural phenotype in Escherichia coli biofilms: cellular chain formation. Biofilm chaining in E. coli K-12 was found to occur primarily by clonal expansion, but was not due to filamentous growth. Rather, chain formation was the result of intercellular interactions facilitated by antigen 43 (Ag43), a self-associating autotransporter (SAAT) protein, which has previously been implicated in auto-aggregation and biofilm formation. Immunofluorescence microscopy suggested that Ag43 was concentrated at or near the cell poles, although when the antigen was highly overexpressed, a much more uniform distribution was seen. Immunofluorescence microscopy also indicated that other parameters, including dimensional constraints (flow, growth alongside a surface), may also affect the final biofilm architecture. Moreover, chain formation was affected by other surface structures; type I fimbriae expression significantly reduced cellular chain formation, presumably by steric hindrance. Cellular chain formation did not appear to be specific to E. coli K-12. Although many urinary tract infection (UTI) isolates were found to form rather homogeneous, flat biofilms, three isolates, including the prototypic asymptomatic bacteriuria strain, 83972, formed highly elaborate cellular chains during biofilm growth in human urine. Combined, these results illustrate the diversity of biofilm architectures that can be observed even within a single microbial species.
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hetR and patS, two genes necessary for heterocyst pattern formation, are widespread in filamentous nonheterocyst-forming cyanobacteria
More LessHeterocysts, cells specialized in N2 fixation in cyanobacteria, appeared at near to 2.1 Ga. They constitute one of the oldest forms of differentiated cells in evolution, and are thus an interesting model for studies on evolutionary-developmental biology. How heterocysts arose during evolution remains unknown. In Anabaena PCC 7120, heterocyst development requires, among other genes, hetR for the initiation of heterocyst differentiation, and patS, encoding a diffusible inhibitor of heterocyst formation. In this study, we report that both hetR and patS are widespread among filamentous cyanobacteria that do not form heterocysts or fix N2. hetR and patS are found in proximity on the chromosome in several cases, such as Arthrospira platensis, in which the level of HetR increased following nitrogen deprivation. The hetR gene of A. platensis could complement a hetR mutant of Anabaena PCC 7120, and patS of A. platensis could suppress heterocyst differentiation in Anabaena PCC 7120. Thus, key regulatory genes, including hetR and patS, involved in heterocyst development may have evolved before heterocysts appeared, suggesting that their function was not limited to heterocyst differentiation.
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Evolution of colicin BM plasmids: the loss of the colicin B activity gene
More LessColicins, a class of antimicrobial compounds produced by bacteria, are thought to be important mediators of intra- and interspecific interactions, and are a significant factor in maintaining microbial diversity. Colicins B and M are among the most common colicins produced by Escherichia coli, and are usually encoded adjacently on the same plasmid. In this study, the characterization of a collection of E. coli isolated from Australian vertebrates revealed that a significant fraction of colicin BM strains lack an intact colicin B activity gene. The colicin B and M gene region was sequenced in 60 strains and it was found (with one exception) that all plasmids lacking an intact colicin B activity gene have an identical colicin gene structure, possessing a complete colicin B immunity gene and a 130 bp remnant of the B activity gene. A phylogenetic analysis of the colicin M and B operons and characterization of the plasmids suggested that ColBM plasmids with a truncated B activity gene have evolved on at least three separate occasions. Colicin B immunity was found to be non-functional in strains that have lost colicin B activity, and colicin M was still produced despite the absence of the SOS box believed to regulate its production in colicin BM strains. The presence of a remnant of the microcin V operon next to the truncated colicin B activity gene indicated that these plasmids evolved as a consequence of gene transfer between colicin BM and microcin V plasmids. We suggest that these transfer events most likely involved the transfer of some microcin V genes and associated virulence factors onto ColBM plasmids.
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Multiple acquisitions of CTX-M plasmids in the rare D2 genotype of Escherichia coli provide evidence for convergent evolution
Over the last decade, CTX-M enzymes have become the most prevalent extended-spectrum β-lactamases (ESBLs) worldwide, mostly in Escherichia coli, causing a major health problem. An epidemiological relationship has been established between a rare genotype of E. coli, the D2 genotype, and the presence of CTX-M genes. We investigated this striking association by exploring the genetic backgrounds of 18 D2 genotype CTX-M-producing strains and of the plasmids encoding CTX-M enzymes. The 18 strains had different genetic backgrounds, as assessed by multilocus sequence and O typing, and were associated with various plasmids bearing diverse CTX-M genes. The region encompassing the genetic marker of the D2 genotype (TSPE4.C2) was not correlated with the presence of CTX-M genes. CTX-M-producing D2 strains had far fewer virulence factors than a control group of 8 non-ESBL-producing D2 strains, and an inverse relationship was found between the number of co-resistances associated with the CTX-M gene and the number of virulence factors found in the strain. These findings provide evidence for multiple acquisitions of plasmids carrying CTX-M genes in different D2 genotype strains. They strongly suggest that convergent evolution has occurred, and indicate that there has been selection for the association of a specific genetic background of the strain and the CTX-M gene. This fine-tuning of the relationship between the D2 genotype and CTX-M genes presumably increases the fitness of the strain, indicating a role for the host cell in the acquisition and dissemination of CTX-M genes.
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- Genes And Genomes
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Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays
The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter–operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.
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Identification of resistance and virulence factors in an epidemic Enterobacter hormaechei outbreak strain
Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment.
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Mitochondrial nucleoids from the yeast Candida parapsilosis: expansion of the repertoire of proteins associated with mitochondrial DNA
More LessMolecules of mitochondrial DNA (mtDNA) are packed into nucleic acid–protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analysed mt-nucleoids of the yeast Candida parapsilosis, which harbours a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated the protein composition of mt-nucleoids isolated from C. parapsilosis mitochondria, determined N-terminal amino acid sequences of 14 proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components with additional candidates identified in the complete genome sequence of C. parapsilosis as homologues of Saccharomyces cerevisiae mt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components that expand the repertoire of proteins associated with these cytological structures. In particular, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits structural features in common with the mtDNA packaging protein Abf2 from S. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activity in vitro, and is able to stabilize mtDNA in the S. cerevisiae Δabf2 mutant, all of which points to a role in the maintenance of the C. parapsilosis mitochondrial genome. Importantly, in contrast to Abf2p, in silico analysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.
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Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli
In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01–04, and have molecular sizes of 1815, 3524, 8881 and 65 037 bp, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the blast search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.
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Molecular cloning and functional characterization of two CYP619 cytochrome P450s involved in biosynthesis of patulin in Aspergillus clavatus
Patulin is an acetate-derived tetraketide mycotoxin produced by several fungal species, especially Aspergillus, Penicillium and Byssochlamys species. The health risks due to patulin consumption by humans have led many countries to regulate it in human food. Previous studies have shown the involvement of cytochrome P450 monooxygenases in the hydroxylation of two precursors of patulin, m-cresol and m-hydroxybenzylalcohol. In the present study, two cytochrome P450 genes were identified in the genome sequence of Aspergillus clavatus, a patulin-producing species. Both mRNAs were strongly co-expressed during patulin production. CYP619C2, encoded by the first gene, consists of 529 aa, while the second cytochrome, CYP619C3, consists of 524 aa. The coding sequences were used to perform the heterologous expression of functional enzymes in Saccharomyces cerevisiae. The bioconversion assays showed that CYP619C3 catalysed the hydroxylation of m-cresol to yield m-hydroxybenzyl alcohol. CYP619C2 catalysed the hydroxylation of m-hydroxybenzyl alcohol and m-cresol to gentisyl alcohol and 2,5-dihydroxytoluene (toluquinol), respectively. Except for the last compound, all enzyme products are known precursors of patulin. Taken together, these data strongly suggest the involvement of CYP619C2 and CYP619C3 in the biosynthesis of patulin. CYP619C2 and CYP619C3 are located near to two other genes involved in patulin biosynthesis, namely the 6-methylsalicylic acid synthase (6msas) and isoepoxydon dehydrogenase (idh) genes. The current data associated with an analysis of the sequence of A. clavatus suggest the presence of a cluster of 15 genes involved in patulin biosynthesis.
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- Microbial Pathogenicity
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Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida
Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.
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A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1
More LessSeveral genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the ΔpdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.
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Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models
More LessStaphylococcus aureus produces a variety of virulence factors that allow it to cause a wide range of infections in humans and animals. In the latter, S. aureus is a leading cause of intramammary infections. The contribution of catalase (KatA), an enzyme implicated in oxidative stress resistance, and beta-toxin (Hlb), a haemolysin, to the pathogenesis of S. aureus is poorly characterized. To investigate the role of these enzymes as potential virulence factors in S. aureus, we examined the intracellular survival of ΔkatA, Δhlb and ΔkatA Δhlb mutants in murine macrophages (J774A.1) and bovine mammary epithelial cells (MAC-T), and their virulence in different murine and ovine models. Catalase was not required for the survival of S. aureus within either J774A.1 or MAC-T cells. However, it was necessary for the intracellular proliferation of the bacterium after invasion of MAC-T cells. In addition, catalase was not needed for the full virulence of S. aureus in mice. Deletion of the hlb gene had no effect on the intracellular survival of S. aureus in J774A.1 cells but did cause a slight increase in survival in MAC-T cells. Furthermore, like catalase, beta-toxin was not required for complete virulence of S. aureus in murine models. Unexpectedly, the ΔkatA Δhlb mutant showed a notably increased persistence in both cell lines, and was significantly less virulent for mice than were the wild-type strain and single mutants. Most interestingly, it was also markedly attenuated in intramammary and subcutaneous infections in ewes and lambs.
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Galleria mellonella as an alternative infection model for Yersinia pseudotuberculosis
We report that larvae of the wax moth (Galleria mellonella) are susceptible to infection with the human enteropathogen Yersinia pseudotuberculosis at 37 °C. Confocal microscopy demonstrated that in the initial stages of infection the bacteria were taken up into haemocytes. To evaluate the utility of this model for screening Y. pseudotuberculosis mutants we constructed and tested a superoxide dismutase C (sodC) mutant. This mutant showed increased susceptibility to superoxide, a key mechanism of killing in insect haemocytes and mammalian phagocytes. It showed reduced virulence in the murine yersiniosis infection model and in contrast to the wild-type strain IP32953 was unable to kill G. mellonella. The complemented mutant regained all phenotypic properties associated with SodC, confirming the important role of this metalloenzyme in two Y. pseudotuberculosis infection models.
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Volumes and issues
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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