- Volume 153, Issue 6, 2007
Volume 153, Issue 6, 2007
- Review
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Aspergillus flavus: human pathogen, allergen and mycotoxin producer
More LessAspergillus infections have grown in importance in the last years. However, most of the studies have focused on Aspergillus fumigatus, the most prevalent species in the genus. In certain locales and hospitals, Aspergillus flavus is more common in air than A. fumigatus, for unclear reasons. After A. fumigatus, A. flavus is the second leading cause of invasive aspergillosis and it is the most common cause of superficial infection. Experimental invasive infections in mice show A. flavus to be 100-fold more virulent than A. fumigatus in terms of inoculum required. Particularly common clinical syndromes associated with A. flavus include chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, wound infections and osteomyelitis following trauma and inoculation. Outbreaks associated with A. flavus appear to be associated with single or closely related strains, in contrast to those associated with A. fumigatus. In addition, A. flavus produces aflatoxins, the most toxic and potent hepatocarcinogenic natural compounds ever characterized. Accurate species identification within Aspergillus flavus complex remains difficult due to overlapping morphological and biochemical characteristics, and much taxonomic and population genetics work is necessary to better understand the species and related species. The flavus complex currently includes 23 species or varieties, including two sexual species, Petromyces alliaceus and P. albertensis. The genome of the highly related Aspergillus oryzae is completed and available; that of A. flavus in the final stages of annotation. Our understanding of A. flavus lags far behind that of A. fumigatus. Studies of the genomics, taxonomy, population genetics, pathogenicity, allergenicity and antifungal susceptibility of A. flavus are all required.
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- Biochemistry And Molecular Biology
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cAMP signalling is involved in growth, germination, mycoparasitism and secondary metabolism in Trichoderma virens
More LessAn adenylate-cyclase-encoding gene, tac1, of Trichoderma virens, a soil fungus used in the biocontrol of plant pathogens, has been cloned and sequenced. The tac1 ORF spanned 7032 bp, encoding a protein of 2153 aa, which shared an identity of 65 % with the adenylate cyclase of Colletotrichum lagenarium. Deletion of tac1, through double-crossover homologous recombination, lowered the intracellular cAMP levels to below the detection limit. The mutants showed only 5–6 % of the wild-type growth rate on agar, but grew normally in shake culture. The mutants did not sporulate in darkness, and the spores failed to germinate in water. In the confrontation assay, the mutants did not overgrow the test plant pathogens Sclerotium rolfsii, Rhizoctonia solani and Pythium sp. Against Pythium sp., the mutants produced a clear zone of inhibition in the confrontation assay. HPLC analysis and bioassay showed reduced secondary metabolite production in the mutants. Using suppression subtractive hybridization (SSH), the genes that were underexpressed in the mutants were identified. Based on an array of 53 SSH library clones, 11 clones were identified as strongly downregulated in the Δtac1 mutants; of these 11 clones, nine sequences were homologous to secondary metabolism-related gene sequences. Therefore, cAMP signalling positively regulates secondary metabolism in T. virens. This is believed to be the first direct genetic study on the role of cAMP signalling in a Trichoderma sp. Tac1 is also believed to be the first regulatory protein to be identified in T. virens that is involved in growth, germination, mycoparasitism and secondary metabolism.
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The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis
In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC–comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.
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Butane monooxygenase of ‘Pseudomonas butanovora’: purification and biochemical characterization of a terminal-alkane hydroxylating diiron monooxygenase
More LessButane monooxygenase (sBMO) has been purified to homogeneity from the Gram-negative β-proteobacterium ‘Pseudomonas butanovora’ and confirmed to be a three-component diiron monooxygenase system. The reconstituted enzyme complex oxidized C3–C6 linear and branched aliphatic alkanes, which are growth substrates for ‘P. butanovora’. The sBMO complex was composed of an iron-containing hydroxylase (BMOH), a flavo-iron sulfur-containing NADH-oxidoreductase (BMOR) and a small regulatory component protein (BMOB). The physical characteristics of sBMO were remarkably similar to the sMMO family of soluble multicomponent diiron monooxgenases. However, the catalytic properties of sBMO were quantitatively different in regard to inactivation in the presence of substrate and product distribution. BMOH was capable of ethene oxidation when supplied with H2O2 and ethene (known as the peroxide shunt), but this activity was at least three orders of magnitude less than that observed for the hydroxylase of sMMO of Methylosinus trichosporium OB3b. BMOH and BMOR were efficient in the oxidation of ethene in the absence of BMOB with regard to rate of reaction and product yield. Regiospecificity of sBMO was strongly biased towards primary hydroxylation, with ≥80 % of the hydroxylations occurring at the terminal carbon atom.
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γ-Butyrolactone autoregulator-receptor system involved in lankacidin and lankamycin production and morphological differentiation in Streptomyces rochei
More LessAn afsA homologue (srrX) and three γ-butyrolactone receptor gene homologues (srrA, srrB and srrC) are coded on the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two polyketide antibiotics, lankacidin and lankamycin. Construction of gene disruptants and their phenotypic study revealed that srrX and srrA make a γ-butyrolactone receptor system in this strain. Addition of a γ-butyrolactone fraction to an srrX-deficient mutant restored the production of lankacidin and lankamycin, indicating that the SrrX protein is not necessary for this event. In addition to a positive effect on antibiotic production, srrX showed a negative effect on morphological differentiation. The receptor gene srrA reversed both effects of srrX, while the second receptor gene homologue srrC had only a positive function in spore formation. Furthermore, disruption of the third homologue srrB greatly increased the production of lankacidin and lankamycin. Electron microscopic analysis showed that aerial mycelium formation stopped at a different stage in the srrA and srrC mutants. Overall, these results indicated that srrX, srrA, srrB and srrC constitute a complex regulatory system for antibiotic production and morphological differentiation in S. rochei.
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Three different DnaK proteins are functionally expressed in the cyanobacterium Synechocystis sp. PCC 6803
More LessMultiple dnaK genes appear to be common in cyanobacteria; the function of the encoded proteins is, however, still elusive. To characterize the dnaK gene family from the cyanobacterium Synechocystis sp. PCC 6803 in detail, genetic analyses were combined with analyses of the expression and localization patterns of the three encoded proteins. While significant expression of all three genes was found, the results obtained clearly indicate physiological differences of the three proteins in vivo, and DnaK2 seems to have a key function in Synechocystis. Expression of DnaK3 appears also to be as essential as expression of DnaK2, whereas the dnaK1 gene was deleted without resulting in any distorted phenotype. In line with a suggested privileged function, expression of DnaK2 altered most significantly after heat shock.
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The phrA gene of Rhodobacter sphaeroides encodes a photolyase and is regulated by singlet oxygen and peroxide in a σ E-dependent manner
More LessThe genome of the facultatively photosynthetic bacterium Rhodobacter sphaeroides encodes three proteins of the photolyase/cryptochrome family. This paper shows that phrA (RSP2143) encodes a functional photolyase, which is an enzyme that repairs UV radiation-induced DNA damage in a blue light dependent manner. Expression of phrA is upregulated in response to light, with no photoreceptor or the photosynthetic electron transport being involved. The results reveal that singlet oxygen and hydrogen peroxide dependent signals are transmitted by the σ E factor and the anti-σ E factor ChrR affecting phrA expression, while superoxide anions do not stimulate phrA expression. Thus, the σ E regulon is involved not only in the response to singlet oxygen but also in the hydrogen peroxide response.
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- Biodiversity And Evolution
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Differential Salmonella survival against communities of intestinal amoebae
More LessPredation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae – such as Naegleria and Tetramitus from goldfish – also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.
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Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation
Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of poor lung function in cystic fibrosis patients, and SCVs are often more resistant to antibiotics than wild-type cells. P. aeruginosa strain PA14 also contains a Pf1-like filamentous prophage, which is designated Pf5, and is highly homologous to Pf4. Since P. aeruginosa PA14 produces SCVs very efficiently in biofilms grown in static cultures, the role of Pf5 in SCV formation under these conditions was investigated. The presence of the Pf5 replicative form in total DNA from SCVs and wild-type cells was detected, but it was not possible to detect the Pf5 major coat protein by immunoblot analysis in PA14 SCV cultures. This suggests that the Pf5 filamentous phage is not present at high densities in the PA14 SCVs. Consistent with these results, we were unable to detect coaB expression in SCV cultures and SCV colonies. The SCV variants formed under static conditions were not linked to Pf5 phage activity, since Pf5 insertion mutants with decreased or no production of the Pf5 RF produced SCVs as efficiently as the wild-type strain. Finally, analysis of 48 clinical P. aeruginosa isolates showed no association between the presence of Pf1-like filamentous phages and the ability to form SCVs under static conditions; this suggests that filamentous phages are generally not involved in the emergence of P. aeruginosa SCVs.
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Hypermutability in environmental Pseudomonas aeruginosa and in populations causing pulmonary infection in individuals with cystic fibrosis
Pseudomonas aeruginosa is the pathogen most commonly associated with morbidity and mortality in cystic fibrosis (CF) patients. The host–pathogen interactions responsible for progressive CF lung diseases are complex. However, there is growing interest in the role of hypermutable P. aeruginosa (that is, those strains with an increased mutation frequency due to mutations in mismatch repair and error prevention genes), in terms of both bacterial adaptation and antimicrobial resistance. The prevalence of hypermutable P. aeruginosa in chronic CF infection has been established, and at 37 % is surprisingly high. To the authors’ knowledge, there are no reports of prevalence during the early stages of infection, in environmental pseudomonas, which are believed to be the primary source of infection, and in epidemic strains, which have emerged as a major challenge. The aim of this study was to establish the prevalence of hypermutable P. aeruginosa in these pseudomonas populations. The hypothesis was that hypermutability would be rare in early and in environmental P. aeruginosa but in contrast would explain the relatively recent emergence of epidemic strains. It was found that 10/100 (10 %) of early isolates were strong or weak mutators, suggesting that the CF lung is not the only factor influencing the existence of mutators in this group of patients. Two weak mutators (6 %) were found in 32 environmental isolates. Only two of 15 (13 %) epidemic P. aeruginosa strains were hypermutable, and although closer analysis revealed this issue to be complex, on the whole the data suggested that the atypical characteristics of these highly transmissible strains cannot solely be explained by this phenomenon. The higher than predicted prevalence of mutators in early infection, and in environmental isolates, reinforces the importance of early and aggressive treatment for P. aeruginosa infection in CF.
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- Environmental Microbiology
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Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida
More LessTranscription of the plasmid-borne phenol catabolic operon pheBA in Pseudomonas putida is activated by the LysR-family regulator CatR in the presence of the effector molecule cis,cis-muconate (CCM), which is an intermediate of the phenol degradation pathway. In addition to the positive control of the operon, several factors negatively affect transcription initiation from the pheBA promoter. First, the activation of the pheBA operon depends on the extracellular concentration of phenol. The pheBA promoter is rapidly activated in the presence of micromolar concentrations of phenol in minimal growth medium, but the initiation of transcription from this promoter is severely delayed after sudden exposure of bacteria to 2.5 mM phenol. Second, the transcriptional activation from this promoter is impeded when the growth medium of bacteria contains amino acids. The negative effects of amino acids can be suppressed either by overproducing CatR or by increasing, the intracellular amount of CCM. However, the intracellular amount of CCM is a major limiting factor for the transcriptional activation of the pheBA operon, as accumulation of CCM in a P. putida catB-defective strain, unable to metabolize CCM (but expressing CatR at a natural level), almost completely relieves the negative effects of amino acids. The intracellular amount of CCM is negatively affected by the catabolite repression control protein via downregulating at the post-transcriptional level the expression of the pheBA-encoded catechol 1,2-dioxygenase and the phenol monooxygenase, the enzymes needed for CCM production.
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The surface physicochemistry and adhesiveness of Shewanella are affected by their surface polysaccharides
More LessShewanella strains have previously been studied with regard to their cell surface ultrastructure and LPS composition. They have now been further characterized with respect to their surface physicochemistry and ability to adhere to haematite. The surfaces of the Shewanella strains were found to be electronegative and hydrophilic, and these properties could be correlated with LPS composition or the presence of capsular polysaccharides. Strains expressing rough LPS with no capsule were more hydrophobic and electronegative than those possessing smooth LPS or capsules. By combining different approaches, such as contact-angle measurement, hydrophilic/hydrophobic chromatography, microelectrophoresis, adhesion assays and calculation of interaction energies, it was shown that electrostatic interactions predominate over hydrophobic interactions at the cell–iron oxide interface. Bacterial adhesion to haematite was significantly reduced in strains expressing smooth LPS or a capsule. These findings remained true for Shewanella strains grown under either aerobic or anaerobic conditions, although the surfaces of anaerobic cells appeared to be less electronegative and more hydrophilic than those of aerobic cells.
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- Genes And Genomes
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Optical maps distinguish individual strains of Escherichia coli O157 : H7
More LessOptical maps of 11 Escherichia coli O157 : H7 strains have been generated by the assembly of contiguous sets of restriction fragments across their entire 5.3 to 5.6 Mbp chromosomes. Each strain showed a distinct, highly individual configuration of 500–700 BamHI fragments, yielding a map resembling a DNA ‘bar code’. The accuracy of optical mapping was assessed by comparing directly the in silico restriction maps of two wholly sequenced reference genomes of E. coli O157 : H7, i.e. EDL933 and the Sakai isolate (RIMD 0509952), with the optical maps of the same strains. The optical maps of nine other E. coli O157 : H7 strains were compared similarly, using the sequence-based maps of the Sakai and EDL933 strains as references. A total of 91 changes at 28 loci were positioned and sized; these included complex chromosomal inversions, insertions, deletions, substitutions, as well as a number of simple RFLPs. The optical maps defined unique genome landmarks in each of the strains and demonstrated the ability of optical mapping to distinguish and differentiate, at the individual level, strains of this important pathogen.
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Cloning and characterization of the genes encoding the high-affinity iron-uptake protein complex Fet3/Ftr1 in the basidiomycete Phanerochaete chrysosporium
More LessMCO1, a multicopper oxidase from Phanerochaete chrysosporium exhibiting strong ferroxidase activity, has recently been described. This enzyme shows biochemical and structural similarities with the yeast Fet3p, a type I membrane glycoprotein that efficiently oxidizes Fe(II) to Fe(III) for its subsequent transport to the intracellular compartment by the iron permease Ftr1p. The genome database of P. chrysosporium was searched to verify whether it includes a canonical fet3 in addition to mco1, and single copies of fet3 and ftr1 orthologues were found, separated by a divergent promoter. Pc-fet3 encodes a 628 aa protein that exhibits overall identities of about 40 % with other reported Fet3 proteins. In addition to a secretion signal, it has a C-terminal transmembrane domain, characteristic of these cell-surface-attached ferroxidases. Structural modelling of Pc-Fet3 revealed that the active site has all the residues known to be essential for ferroxidase activity. Pc-ftr1 encodes a 393 aa protein that shows about 38 % identity with several Ftr1 proteins from ascomycetes. Northern hybridization studies showed that the mRNA levels of both genes are reduced upon supplementation of the growth medium with iron, supporting the functional coupling of Fet3 and Ftr1 proteins in vivo.
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Plasmids derived from Gifsy-1/Gifsy-2, lambdoid prophages contributing to the virulence of Salmonella enterica serovar Typhimurium: implications for the evolution of replication initiation proteins of lambdoid phages and enterobacteria
More LessGifsy-1 and Gifsy-2 are lambdoid prophages which contribute to the virulence of Salmonella enterica serovar Typhimurium. The nucleotide sequence of the replication region of both prophages is identical, and similar in organization to the replication region of bacteriophage λ. To investigate the replication of the Gifsy phages and the relationship between Gifsy and host chromosome replication, a plasmid which contained all the genes and regulatory sequences required for autonomous replication in bacterial cells was constructed. This plasmid, pGifsy, was stably maintained in Escherichia coli cells. The helicase loader of the Gifsy phages is very similar to the DnaC protein of the host, a feature characteristic of a large group of prophages common in the sequenced genomes of pathogenic enterobacteria. This DnaC-like protein showed no similarity to the helicase loader of bacteriophage λ and closely related phages. Interestingly, unlike plasmids derived from bacteriophage λ (λ plasmids), pGifsy did not require a gene encoding the putative helicase loader for replication, although deletion of this gene resulted in a decrease in plasmid copy number. Under these conditions, it was shown that the plasmid utilized the helicase loader coded by the host. On the other hand, the viral protein could not substitute for DnaC in bacterial chromosome replication. The results of the current study support the hypothesis that the enterobacterial helicase loader is of viral origin. This hypothesis explains why the gene for DnaC, the protein central to both replication initiation and replication restart in E. coli, is present in the genomes of Escherichia, Shigella, Salmonella and Buchnera, but not in the genomes of related enterobacteria.
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Characterization of a catalase gene from Aeromonas veronii, the digestive-tract symbiont of the medicinal leech
More LessThe catalase gene katA of the medicinal leech symbiont Aeromonas veronii bv. sobria was cloned, sequenced, and functionally characterized. Southern hybridization, using an A. veronii katA-specific hybridization probe, suggested the presence of a single gene copy in many Aeromonas species. A. veronii katA consisted of 1446 nt encoding a protein with a high degree of similarity to the small-subunit group III bacterial catalases. A catalase-null mutant (JG186) was constructed through gene-replacement mutagenesis. In the parent strain (HM21R), catalase activity was only detected in extracts of cells grown to early exponential phase following H2O2 induction, in which the ability to induce activity was inversely related to optical density. In contrast, induced JG186 cells were very sensitive to oxidative stress, with survival being affected even at low H2O2 concentrations. In contrast to the findings of previous reports of other symbiotic systems, the catalase mutant was not defective in its ability to competitively colonize or persist within its host, in both co-inoculation and sole-colonization assays. This body of evidence suggests either that oxidative stress, in the form of H2O2 exposure, is not encountered by the microbial partner under the examined symbiotic conditions or that compensatory mechanisms exist. The data suggest that although many colonization factors reoccur, each symbiotic system has also evolved specific mechanisms that affect symbiont–host dynamics.
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- Pathogens And Pathogenicity
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Phenotypic and functional characterization of Bacillus anthracis biofilms
Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.
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Phase-variable expression of the biofilm-associated protein (Bap) in Staphylococcus aureus
A process of phase variation is described that affects the expression of Bap (biofilm-associated protein) in Staphylococcus aureus. Upon subculture of the Bap-positive S. aureus strain V329 on Congo red agar, spontaneous smooth biofilm-negative colonies appeared at a low frequency (5×10−4). Northern blot analysis of these variants with a bap-specific gene probe showed that transcription of the bap gene did not occur. However, DNA typing, Southern blot hybridization and DNA sequencing did not show any differences between the parent V329 strain and the biofilm-negative variants. The biofilm-negative phenotype reverted to wild-type at a similar frequency upon subculture of Bap-negative variants in liquid media. Experimental infection of ovine mammary glands with Bap-negative variants showed that phase variation occurred in vivo, because Bap-expressing, biofilm-positive revertants were isolated from infected mammary glands. The absence of Bap correlated with increased adherence to fibrinogen and fibronectin. It is possible that S. aureus can detach from a biofilm by switching to a Bap-negative state.
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Biofilm exclusion of uropathogenic bacteria by selected asymptomatic bacteriuria Escherichia coli strains
More LessMany bacterial infections are associated with biofilm formation. In the urinary tract bacterial biofilms develop on both living surfaces and artificial implants, producing chronic and often intractable infections. Escherichia coli is the most common organism associated with urinary tract infections. In contrast to uropathogenic E. coli (UPEC), which cause symptomatic urinary tract infection, asymptomatic bacteriuria (ABU) strains are associated with essentially symptom-free infections. Here the biofilm-forming capacity on abiotic surfaces of selected E. coli ABU strains and UPEC strains in human urine was investigated. It was found that there is a strong bias for biofilm formation by the ABU strains. Not only were the ABU strains significantly better biofilm formers than UPEC strains, they were also able to out-compete UPEC strains as well as uropathogenic strains of Klebsiella spp. during biofilm formation. The results support the notion of bacterial prophylaxis employing selected ABU strains to eliminate UPEC strains and other pathogens in patients prone to recalcitrant infections.
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TccP2-mediated subversion of actin dynamics by EPEC 2 – a distinct evolutionary lineage of enteropathogenic Escherichia coli
Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott–Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspFU (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H−), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck−/− cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades.
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Characterization of two distinct phospholipase C enzymes from Burkholderia pseudomallei
Burkholderia pseudomallei is a serious bacterial pathogen that can cause a lethal infection in humans known as melioidosis. In this study two of its phospholipase C (PLC) enzymes (Plc-1 and Plc-2) were characterized. Starting with a virulent strain, two single mutants were constructed, each with one plc gene inactivated, and one double mutant with both plc genes inactivated. The single plc mutants exhibited decreased extracellular PLC activity in comparison to the wild-type strain, thereby demonstrating that the two genes encoded functional extracellular PLCs. Growth comparisons between the wild-type and PLC mutants in egg-yolk-supplemented medium indicated that both PLCs contributed to egg-yolk phospholipid utilization. Both PLCs hydrolysed phosphatidylcholine and sphingomyelin but neither was haemolytic for human erythrocytes. Experimental infections of eukaryotic cells demonstrated that Plc-1 itself had no effect on plaque-forming efficiency but it had an additive effect on increasing the efficiency of Plc-2 to form plaques. Only Plc-2 had a significant role in host cell cytotoxicity. In contrast, neither Plc-1 nor Plc-2 appeared to play any role in multinucleated giant cell (MNGC) formation or induction of apoptotic death in the cells studied. These data suggested that PLCs contribute, at least in part, to B. pseudomallei virulence and support the view that Plc-1 and Plc-2 are not redundant virulence factors.
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Involvement of minor components associated with the FimA fimbriae of Porphyromonas gingivalis in adhesive functions
The FimA fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. In this study, the four open reading frames (ORF1, ORF2, ORF3 and ORF4) downstream of the fimbrilin gene (fimA) in strain ATCC 33277 were examined. ORF2, ORF3 and ORF4 were demonstrated to encode minor components of the fimbriae and were therefore renamed fimC, fimD and fimE, respectively. Immunoblotting analyses revealed that inactivation of either fimC or fimD by an ermF-ermAM insertion, but not inactivation of ORF1, was accompanied by concomitant loss of the products from the downstream genes, raising the possibility that fimC, fimD and fimE constitute a transcription unit. The fimE mutant produced FimC and FimD, but fimbriae purified from it contained neither protein, suggesting that FimE is required for the assembly of FimC and FimD onto the fimbrilin (FimA) fibre. The fimC, fimD and fimE mutants lost autoaggregation abilities. Fimbriae purified from these three mutants showed attenuated binding activities to glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis and to two extracellular matrix proteins, fibronectin and type I collagen. These results suggest that FimE, as well as FimC and FimD, play critical roles in the adhesive activities of the mature FimA fimbriae in P. gingivalis.
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Phenotypic and genotypic characterization of a new fish-virulent Vibrio vulnificus serovar that lacks potential to infect humans
More LessVibrio vulnificus is a bacterial species that is virulent for humans and fish. Human isolates are classified into biotypes 1 and 3 (BT1 and BT3) and fish isolates into biotype 2 (BT2). However, a few human infections caused by BT2 isolates have been reported worldwide (zoonosis). These BT2 human isolates belong to serovar E (SerE), which is also present in diseased fish. The aim of the present work was to characterize a new BT2 serovar [serovar A (SerA)], which emerged in the European fish-farming industry in 2000, by means of phenotypic, serological and genetic [plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD)] methodologies. The results confirmed that SerA constitutes a homogeneous O-serogroup within the species that shares plasmidic information with SerE. Like SerE, this new serogroup was resistant to fresh fish serum, as well as being highly virulent for fish. In contrast, it was sensitive to human serum and avirulent for mice, even after pretreatment with iron. The two serovars presented different biochemical profiles as well as specific patterns by ribotyping and RAPD analysis. In conclusion, SerA seems to constitute a different clonal group that has recently emerged within the species V. vulnificus, with pathogenic potential for fish but not for humans.
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Effect of host fatty acid-binding protein and fatty acid uptake on growth of Chlamydia trachomatis L2
More LessChlamydia trachomatis is an obligate intracellular bacterium and acquires both building blocks and energy from host cells for growth. The fatty acid-binding protein (FABP) plays an important role in uptake of long-chain fatty acids (LCFA) and energy metabolism by eukaryotic cells. The roles of FABP and LCFA in chlamydial infection were evaluated. Infection of liver cells with chlamydial organisms promoted fatty acid uptake by the infected cells, suggesting that LCFA may benefit chlamydial growth. Introduction of FABP into the liver cells not only enhanced fatty acid uptake, but also increased chlamydial intravacuolar replication and maturation. The FABP-enhanced chlamydial intracellular growth was dependent on the host cell uptake of fatty acids. These results have demonstrated that C. trachomatis can productively infect liver cells and utilize FABP-transported LCFA for its own biosynthesis.
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Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis
More LessSalmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic enteric pathogen of worldwide importance and pigs are a significant reservoir of human infection. Signature-tagged transposon mutagenesis (STM) was used to identify genes required by S. Typhimurium to colonize porcine intestines. A library of 1045 signature-tagged mutants of S. Typhimurium ST4/74 NalR was screened following oral inoculation of pigs in duplicate. A total of 119 attenuating mutations were identified in 95 different genes, many of which encode known or putative secreted or surface-anchored molecules. A large number of attenuating mutations were located within Salmonella pathogenicity islands (SPI)-1 and -2, confirming important roles for type III secretion systems (T3SS)-1 and -2 in intestinal colonization of pigs. Roles for genes encoded in other pathogenicity islands and islets, including the SPI-6-encoded Saf atypical fimbriae, were also identified. Given the role of secreted factors and the protection conferred against other pathogens by vaccination with extracellular and type III secreted proteins, the efficacy of a secreted protein vaccine from wild-type S. Typhimurium following intramuscular vaccination of pigs was evaluated. Serum IgG responses against type III secreted proteins were induced following vaccination and a significant reduction in faecal excretion of S. Typhimurium was observed in the acute phase of infection compared to mock-vaccinated animals. Vaccination with secreted proteins from an isogenic S. Typhimurium prgH mutant produced comparable levels of protection to vaccination with the preparation from the parent strain, indicating that protection was not reliant on T3SS-1 secreted proteins. The data provide valuable information for the control of Salmonella in pigs.
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EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD
More LessEdwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the ‘translocon’ of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by coils. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of ΔescC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB–LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.
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- Physiology
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NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-γ-stimulated J774.2 macrophages
More LessOver the past decade, the flavohaemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse micro-organisms, particularly pathogenic bacteria. Its expression in enterobacteria is dramatically increased on exposure to NO and other agents of nitrosative stress as a result of transcriptional regulation of hmp gene expression, mediated by (at least) four regulators. One such regulator, NsrR, has recently been shown to be responsible for repression of hmp transcription in the absence of NO in Escherichia coli and Salmonella, but the roles of other members of this regulon in Salmonella, particularly in surviving nitrosative stresses in vitro and in vivo, have not been elucidated. This paper demonstrates that an nsrR mutant of Salmonella enterica Serovar Typhimurium expresses high levels of Hmp both aerobically and anaerobically, exceeding those that can be elicited in vitro by supplementing media with S-nitrosoglutathione (GSNO). Elevated transcription of ytfE, ygbA, hcp and hcp is also observed, but no evidence was obtained for tehAB upregulation. The hyper-resistance to GSNO of an nsrR mutant is attributable solely to Hmp, since an nsrR hmp double mutant has a wild-type phenotype. However, overexpression of NsrR-regulated genes other than hmp confers some resistance of respiratory oxygen consumption to NO. The ability to enhance, by mutating NsrR, Hmp levels without recourse to exposure to nitrosative stress was used to test the hypothesis that control of Hmp levels is required to avoid oxidative stress, Hmp being a potent generator of superoxide. Within IFN-γ-stimulated J774.2 macrophages, in which high levels of nitrite accumulated (indicative of NO production) an hmp mutant was severely compromised in survival. Surprisingly, under these conditions, an nsrR mutant (as well as an nsrR hmp double mutant) was also disadvantaged relative to the wild-type bacteria, attributable to the combined oxidative effect of the macrophage oxidative burst and Hmp-generated superoxide. This explanation is supported by the sensitivity in vitro of an nsrR mutant to superoxide and peroxide. Fur has recently been confirmed as a weak repressor of hmp transcription, and a fur mutant was also compromised for survival within macrophages even in the absence of elevated NO levels in non-stimulated macrophages. The results indicate the critical role of Hmp in protection of Salmonella from nitrosative stress within and outside macrophages, but also the key role of transcriptional regulation in tuning Hmp levels to prevent exacerbation of the oxidative stress encountered in macrophages.
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Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter
This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K s) of a reference strain was about 15 μM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h−1, 0.14 h−1 and 0.20 h−1). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (v max) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (v max) and apparent affinity constants () at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.
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Changes in the redox state and composition of the quinone pool of Escherichia coli during aerobic batch-culture growth
Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.
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Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria
More LessModified β-cyclodextrins have been shown previously to enhance sterol conversion to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by growing Mycobacterium spp. The enhancement effect was mainly attributed to steroid solubilization by the formation of inclusion complexes with modified cyclodextrins. In this work, the influence of randomly methylated β-cyclodextrin (MCD) on the growth, AD- and ADD-producing activity, cell wall (CW) composition and ultrastructure of sterol-transforming Mycobacterium sp. VKM Ac-1816D was studied. The specific growth rate of the strain on glycerol increased in the presence of MCD (20–100 mM). Washed cells grown in the presence of MCD (20–40 mM) expressed 1.6-fold higher ADD-producing activity than did the cells grown without MCD, and their adhesiveness differed. Electron microscopy showed MCD-mediated CW exfoliation and accumulation of membrane-like structures outside the cells, while preserving cells intact. The analysis of CW composition revealed both a decrease in the proportion of extractable lipids and a considerable shift in fatty acid profile resulting from MCD action. The MCD-mediated enhancement of mycolic and fatty acids content was observed outside the cells. The total secreted protein level rose 2.4-fold, and the extracellular 3-hydroxysteroid oxidase activity 3.2-fold. The composition of the CW polysaccharide was not altered, while the overall proportion of the carbohydrates in the CW of the MCD-exposed mycobacteria increased. The results showed that the multiple mechanisms of MCD-mediated intensification of sterol to AD(D) conversion by mycobacteria include not only solubilization of steroids, but also the increase of CW permeability for both steroids and soluble nutrients, disorganization of the lipid bilayer and the release of steroid-transforming enzymes weakly associated with the CW.
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