Showing papers in "Microbiology in 2008"
TL;DR: Structural and transcriptional studies of members of the LTTR family are helping to unravel a compelling paradigm that has evolved from the original observations and conclusions that were made about this family of transcriptional regulators.
Abstract: The LysR family of transcriptional regulators represents the most abundant type of transcriptional regulator in the prokaryotic kingdom. Members of this family have a conserved structure with an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. Despite considerable conservation both structurally and functionally, LysR-type transcriptional regulators (LTTRs) regulate a diverse set of genes, including those involved in virulence, metabolism, quorum sensing and motility. Numerous structural and transcriptional studies of members of the LTTR family are helping to unravel a compelling paradigm that has evolved from the original observations and conclusions that were made about this family of transcriptional regulators.
750 citations
TL;DR: The T6SS is probably a novel kind of complex multi-component secretion machine, which is often involved in interaction with eukaryotic hosts, be it a pathogenic or a symbiotic relationship.
Abstract: Several secretion systems have evolved that are widespread among Gram-negative bacteria. Recently, a new secretion system was recognized, which is named the type VI secretion system (T6SS). The T6SS components are encoded within clusters of genes initially identified as IAHP for IcmF-associated homologous proteins, since they were all found to contain a gene encoding an IcmF-like component. IcmF was previously reported as a component of the type IV secretion system (T4SS). However, with the exception of DotU, other T4SS components are not encoded within T6SS loci. Thus, the T6SS is probably a novel kind of complex multi-component secretion machine, which is often involved in interaction with eukaryotic hosts, be it a pathogenic or a symbiotic relationship. The expression of T6SS genes has been reported to be mostly induced in vivo. Interestingly, expression and assembly of T6SSs are tightly controlled at both the transcriptional and the post-translational level. This may allow a timely control of T6SS assembly and function. Two types of proteins, generically named Hcp and VgrG, are secreted via these systems, but it is not entirely clear whether they are truly secreted effector proteins or are actually components of the T6SS. The precise role and mode of action of the T6SS is still unknown. This review describes current knowledge about the T6SS and summarizes its hallmarks and its differences from other secretion systems.
364 citations
TL;DR: It is indicated that while most clinical isolates are haemolytic and agr(+), non-haemolytics and agR(-) strains are found in S. aureus infections, and that agr (+) and agg(-) variants may have a cooperative interaction in certain types of infections.
Abstract: The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator of the staphylococcal virulon, which includes secreted virulence factors and surface proteins. The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection. Although most human clinical S. aureus isolates are agr(+), there have been several reports of agr-defective mutants isolated from infected patients. Since it is well known that the agr locus is genetically labile in vitro, we have addressed the question of whether the reported agr-defective mutants were involved in the infection or could have arisen during post-isolation handling. We obtained a series of new staphylococcal isolates from local clinical infections and handled these with special care to avoid post-isolation mutations. Among these isolates, we found a number of strains with non-haemolytic phenotypes owing to mutations in the agr locus, and others with mutations elsewhere. We have also obtained isolates in which the population was continuously heterogeneous with respect to agr functionality, with agr(+) and agr(-) variants having otherwise indistinguishable chromosomal backgrounds. This finding suggested that the agr(-) variants arose by mutation during the course of the infection. Our results indicate that while most clinical isolates are haemolytic and agr(+), non-haemolytic and agr(-) strains are found in S. aureus infections, and that agr(+) and agr(-) variants may have a cooperative interaction in certain types of infections.
315 citations
TL;DR: This is the most comprehensive genetic comparison of human versus animal S. aureus isolates conducted and because it used a whole-genome approach, it could estimate the key genes with the greatest variability that are associated with host specificity.
Abstract: Staphylococcus aureus is a commensal and pathogen of several mammalian species, particularly humans and cattle. We aimed to (i) identify S. aureus genes associated with host specificity, (ii) determine the relatedness of human and animal isolates, and (iii) identify whether human and animal isolates typically exchanged mobile genetic elements encoding virulence and resistance genes. Using a well-validated seven-strain S. aureus microarray, we compared 56 UK S. aureus isolates that caused infection in cows, horses, goats, sheep and a camel with 161 human S. aureus isolates from healthy carriers and community acquired infections in the UK. We had previously shown that human isolates are clustered into ten dominant and a few minor lineages, each with unique combinations of surface proteins predicted to bind to human proteins. We found that the animal-associated S. aureus clustered into ten lineages, with 61 % assigned to four lineages, ST151, ST771, ST130 and ST873, that were unique to animals. The majority of bovine mastitis was caused by isolates of lineage ST151, ST771 and ST97, but a few human lineages also caused mastitis. S. aureus isolated from horses were more likely to cluster into human-associated lineages, with 54 % of horse-associated S. aureus assigned to the human clusters CC1, CC8 and CC22; along with the presence of some multi-drug resistant strains, this suggests a human origin. This is the most comprehensive genetic comparison of human versus animal S. aureus isolates conducted, and because we used a whole-genome approach we could estimate the key genes with the greatest variability that are associated with host specificity. Several genes conserved in all human isolates were variable or missing in one or more animal lineages, including the well-characterized lineage specific genes fnbA, fnbB and coa. Interestingly, genes carried on mobile genetic elements (MGEs) such as chp, scn and sak were less common in animal S. aureus isolates, and bap was not found. There was a lot of MGE variation within lineages, and some evidence that exchange of MGEs such as bacteriophage and pathogenicity islands between animal and human lineages is feasible, but there was less evidence of antibiotic resistance gene transfer on the staphylococcal cassette chromosomes (SCC) or plasmids. Surprisingly, animal lineages are closely related to human lineages and only a handful of genes or gene combinations may be responsible for host specificity.
307 citations
TL;DR: Current understanding of interspecies signalling in bacteria and the signals involved is reviewed, what is known of the underlying signal transduction mechanisms and their influences on bacterial behaviour are reviewed.
Abstract: Many bacteria use cell–cell communication mediated by diffusible signal molecules to monitor their population density or confinement to niches and to modulate their behaviour in response to these aspects of their environment. Work on signalling systems within individual species has formed a platform for studies of interspecies interactions that can occur within polymicrobial communities in nature. In addition to signalling between organisms that synthesize the same or related signal molecules, it is becoming evident that bacteria can sense signal molecules that they do not synthesize, thereby eavesdropping on signalling by other organisms in their immediate environment. Furthermore, molecules such as antibiotics that are considered not to be signals for the producing species can have effects on gene expression in other bacteria that indicate a signalling function. Interspecies signalling can lead to alteration in factors contributing to the virulence or persistence of bacterial pathogens as well as influencing the development of beneficial microbial communities. Here we review our current understanding of interspecies signalling in bacteria and the signals involved, what is known of the underlying signal transduction mechanisms and their influences on bacterial behaviour.
281 citations
TL;DR: The complete genome sequence of Streptococcus mutans, a bacterial pathogen commonly associated with human dental caries, was published in 2002 and the streamlined genome (2.03 Mb) revealed an organism that is well adapted to its obligately host-associated existence in multispecies biofilms on tooth surfaces as discussed by the authors.
Abstract: The complete genome sequence of Streptococcus mutans, a bacterial pathogen commonly associated with human dental caries, was published in 2002. The streamlined genome (2.03 Mb) revealed an organism that is well adapted to its obligately host-associated existence in multispecies biofilms on tooth surfaces: a dynamic environment that undergoes rapid and substantial fluctuations. However, S. mutans lacks many of the sensing systems and alternative sigma factors that bacteria often use to coordinate gene expression in response to stress and changes in their environment. Over the past 7 years, functional genomics and proteomics have enhanced our understanding of how S. mutans has integrated the stress regulon and global transcriptional regulators to coordinate responses to environmental fluctuations with modulation of virulence in a way that ensures persistence in the oral cavity and capitalizes on conditions that are favourable for the development of dental caries. Here, we highlight advances in dissection of the stress regulon of S. mutans and its intimate interrelationship with pathogenesis.
271 citations
TL;DR: It is found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface.
Abstract: Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil α-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.
271 citations
TL;DR: Generally, it was more difficult to detach bacteria from DDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies, and Calculated attractive forces based on the DLVO theory (F(DLVO)) towards the secondary interaction minimum were higher on glass than on D DS- coated glass.
Abstract: Using a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion (F(prev)) and to detach adhering bacteria (F(det)) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air-liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air-liquid interface. F(prev) varied from 0.03 to 0.70 pN, while F(det) varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory (F(DLVO)) towards the secondary interaction minimum were higher on glass than on DDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air-liquid interface passages) to the substratum surface.
264 citations
TL;DR: The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists.
Abstract: The H-NS nucleoid-associated DNA-binding protein is an important global repressor of transcription in Gram-negative bacteria. Recently, H-NS has been implicated in the process of xenogeneic silencing, where it represses the transcription of foreign genes acquired by horizontal transfer. This raises interesting questions about the integration of the horizontally acquired genes into the existing gene regulatory networks of the microbe. In particular, how do bacteria derepress silenced genes in order to benefit from their expression without compromising competitive fitness through doing so inappropriately? This article reviews current knowledge about the derepression of genes that are transcriptionally silenced by H-NS. It describes a variety of anti-silencing mechanisms involving (i) protein-independent processes that operate at the level of local DNA structure, (ii) DNA-binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins related to AraC, and (iii) modulatory mechanisms in which H-NS forms heteromeric protein-protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST. The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists. There is also evidence that in a number of cases more sophisticated regulatory processes have been superimposed on these rather simple anti-silencing mechanisms, broadening the range of environmental signals to which H-NS-repressed genes respond.
249 citations
TL;DR: Results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.
Abstract: Acinetobacter baumannii forms biofilms on abiotic surfaces, a phenotype that may explain its ability to survive in nosocomial environments and to cause device-related infections in compromised patients. The biofilm proficiency of the 19606 type strain depends on the production of pili, cell-surface appendages assembled via the CsuAB-A-B-C-D-E chaperone-usher secretion system. The screening of a bank of isogenic insertion derivatives led to the identification of a biofilm-deficient derivative in which a transposon insertion disrupted a gene predicted to encode the response regulator of a two-component regulatory system. This gene, which was named bfmR, is required for the expression of the Csu pili chaperone-usher assembly system. This coding region is followed by an ORF encoding a putative sensor kinase that was named bfmS, which plays a less relevant role in biofilm formation when cells are cultured in rich medium. Further examination showed that the bfmR mutant was capable of attaching to abiotic surfaces, although to levels significantly lower than those of the parental strain, when it was cultured in a chemically defined minimal medium. Additionally, the morphology of planktonic cells of this mutant, when grown in minimal medium, was drastically affected, while adherent mutant cells were indistinguishable in shape and size from the parental strain. Together, these results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.
249 citations
TL;DR: Results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.
Abstract: The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.
TL;DR: This article summarizes the various methods that have been developed for discovering the products of cryptic biosynthetic gene clusters in microbes and gives an account of a group's discovery of the products in the model actinomycete Streptomyces coelicolor M145.
Abstract: Analyses of microbial genome sequences have revealed numerous examples of ‘cryptic’ or ‘orphan’ biosynthetic gene clusters, with the potential to direct the production of novel, structurally complex natural products. This article summarizes the various methods that have been developed for discovering the products of cryptic biosynthetic gene clusters in microbes and gives an account of my group's discovery of the products of two such gene clusters in the model actinomycete Streptomyces coelicolor M145. These discoveries hint at new mechanisms, roles and specificities for natural product biosynthetic enzymes. Our efforts to elucidate these are described. The identification of new secondary metabolites of S. coelicolor raises the question: what is their biological function? Progress towards answering this question is also summarized.
TL;DR: Results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo, however, the overall contribution of the Sap1-3 and Sap4-6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.
Abstract: A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1–10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Δsap1–3, Δsap4–6, Δefg1 and Δefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4–6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Δsap1–3, expression was compensated for by increased expression of SAP5, and in Δsap4–6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Δsap1–3 and Δsap4–6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Δefg1/cph1 and Δefg1, but SAP5 expression was only dramatically reduced in Δefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1–3 and Sap4–6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.
TL;DR: The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed 'myco-diesel'.
Abstract: An endophytic fungus, Gliocladium roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed ‘myco-diesel’. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.
TL;DR: It is demonstrated that the AI-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and suggested the existence in Ed.
Abstract: Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.
TL;DR: In this article, a review describes recent studies implicating the coexistence and intracellular adaptation of Porphyromonas gingivalis in these target host cells and highlights the putative mechanisms of persistence, intercellular dissemination and opportunism.
Abstract: The microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host. Porphyromonas gingivalis, an aetiological agent in severe forms of periodontitis (a chronic inflammatory disease), is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. This Gram-negative anaerobe can also exist within the host epithelium without the existence of overt disease. Gingival epithelial cells, the outer lining of the gingival mucosa, which function as an important part of the innate immune system, are among the first host cells colonized by P. gingivalis. This review describes recent studies implicating the co-existence and intracellular adaptation of the organism in these target host cells. Specifically, recent findings on the putative mechanisms of persistence, intercellular dissemination and opportunism are highlighted. These new findings may also represent an original and valuable model for mechanistic characterization of other successful host-adapted, self-limiting, persistent intracellular bacteria in human epithelial tissues.
TL;DR: The review discussed the molecular mechanisms of electron transfer to the environment in the case of the two best studied microorganisms, Shewanella oneidensis and Geobacter sulfurreducens.
Abstract: Over the last ten years, the recently discovered process of direct electron transfer from anaerobically grown microorganisms to an electrode of a fuel cell has been the object of intense study. The microorganisms responsible for such electron transport were termed electrogenic; the devices using them to generate electric current, microbial fuel cells (MFCs). The review discussed the molecular mechanisms of electron transfer to the environment in the case of the two best studied microorganisms, Shewanella oneidensis and Geobacter sulfurreducens. The discovery of bacterial conducting pili (nanowires) used for electron transfer to the electrode and between bacterial cells was sensational. In the real MFCs, which use complex substrates (industrial liquid waste), microbial associations are active, often as biofilms. The progress in MFCs design and the prospects of their practical application are considered.
TL;DR: The developments in knowledge of the application of bacteria in desulfurization processes are examined, the technical viability of this technology is assessed, and its future challenges are examined.
Abstract: Oil refineries are facing many challenges, including heavier crude oils, increased fuel quality standards, and a need to reduce air pollution emissions. Global society is stepping on the road to zero-sulfur fuel, with only differences in the starting point of sulfur level and rate reduction of sulfur content between different countries. Hydrodesulfurization (HDS) is the most common technology used by refineries to remove sulfur from intermediate streams. However, HDS has several disadvantages, in that it is energy intensive, costly to install and to operate, and does not work well on refractory organosulfur compounds. Recent research has therefore focused on improving HDS catalysts and processes and also on the development of alternative technologies. Among the new technologies one possible approach is biocatalytic desulfurization (BDS). The advantage of BDS is that it can be operated in conditions that require less energy and hydrogen. BDS operates at ambient temperature and pressure with high selectivity, resulting in decreased energy costs, low emission, and no generation of undesirable side products. Over the last two decades several research groups have attempted to isolate bacteria capable of efficient desulfurization of oil fractions. This review examines the developments in our knowledge of the application of bacteria in BDS processes, assesses the technical viability of this technology and examines its future challenges.
TL;DR: The present work is the first study, to the authors' knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronECTin- binding proteins, SsEno may contribute to the virulence of S. suis.
Abstract: Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.
TL;DR: The spatial distribution of putative soil-related AOA in certain sampling stations indicated a strong impact of the Changjiang freshwater discharge on the marine benthic microbial ecosystem.
Abstract: Ammonia-oxidizing archaea (AOA) have recently been found to be potentially important in nitrogen cycling in a variety of environments, such as terrestrial soils, wastewater treatment reactors, marine waters and sediments, and especially in estuaries, where high input of anthropogenic nitrogen is often experienced. The sedimentary AOA diversity, community structure and spatial distribution in the Changjiang Estuary and the adjacent East China Sea were studied. Multivariate statistical analysis indicated that the archaeal amoA genotype communities could be clustered according to sampling transects, and the station located in an estuarine mixing zone harboured a distinct AOA community. The distribution of AOA communities correlated significantly with the gradients of surface-water salinity and sediment sorting coefficient. The spatial distribution of putative soil-related AOA in certain sampling stations indicated a strong impact of the Changjiang freshwater discharge on the marine benthic microbial ecosystem. Besides freshwater, nutrients, organic matter and suspended particles, the Changjiang Diluted Water might also contribute to the transport of terrestrial archaea into the seawater and sediments along its flow path.
TL;DR: A large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoreGulatory pathway, independent of these regulators.
Abstract: Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 degrees C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 degrees C. Another category of genes more highly expressed at 23 degrees C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 degrees C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 degrees C. Increased expression at 23 degrees C compared to 37 degrees C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and viability at 4 degrees C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 degrees C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.
TL;DR: The comprehensive identification and the biochemical and structural characterization of the nutrient transporters of M. tuberculosis will not only promote the understanding of the physiology of this important human pathogen, but might also be exploited to improve tuberculosis chemotherapy.
Abstract: The growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis more than a century ago. However, the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite a wealth of genomic data and the availability of sophisticated genetic tools. Recently, considerable progress has been made in recognizing that two lipid permeability barriers have to be overcome in order for a nutrient molecule to reach the cytoplasm of mycobacteria. Uptake processes are discussed by comparing M. tuberculosis with Mycobacterium smegmatis. For example, M. tuberculosis has only five recognizable carbohydrate transporters in the inner membrane, while M. smegmatis has 28 such transporters at its disposal. The specificities of inner-membrane transporters for sulfate, phosphate and some amino acids have been determined. Outer-membrane channel proteins in both organisms are thought to contribute to nutrient uptake. In particular, the Msp porins have been shown to be required for uptake of carbohydrates, amino acids and phosphate by M. smegmatis. The set of porins also appears to be different for M. tuberculosis and M. smegmatis. These differences likely reflect the lifestyles of these mycobacteria and the availability of nutrients in their natural habitats: the soil and the human body. The comprehensive identification and the biochemical and structural characterization of the nutrient transporters of M. tuberculosis will not only promote our understanding of the physiology of this important human pathogen, but might also be exploited to improve tuberculosis chemotherapy.
TL;DR: It is shown that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.
Abstract: In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (<32 degrees C), but not at 37 degrees C. We show that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.
TL;DR: 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, and showed a high level of recombinant protein expression in S. mutans and StrePTococcus pyogenes.
Abstract: 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: P(ami), P(spac), P(23) and P(veg). 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 (pAMbeta1). Shuttle plasmids derived from the pAMbeta1 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 6xHis 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.
TL;DR: It is concluded that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.
Abstract: Members of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii #21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.
TL;DR: It is shown that the availability of iron greatly influences UTI strains' ability to form biofilm, and the FyuA/yersiniabactin system is known to be important for virulence.
Abstract: Urinary tract infection (UTI) is the most common infection in patients with indwelling urinary catheters, and bacterial biofilm formation is a major problem in this type of infection. Escherichia coli is responsible for the large majority of UTIs. Free iron is strictly limited in the human urinary tract and there is fierce competition between the host and infectious bacteria for this essential metal. Urinary tract infectious E. coli have highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, encoding the yersiniabactin receptor, is one of the most upregulated genes in biofilm; it was upregulated 63-fold in the E. coli UTI strain VR50. FyuA was found to be highly important for biofilm formation in iron-poor environments such as human urine. Mutants in fyuA show aberrant biofilm formation and the cells become filamentous; a VR50fyuA mutant showed a 92 % reduction in biofilm formation in urine flow-cell chambers compared with the wild-type. The FyuA/yersiniabactin system is known to be important for virulence. Here we demonstrate a direct link between FyuA and biofilm formation in iron-poor environments. We also show that the availability of iron greatly influences UTI strains' ability to form biofilm.
TL;DR: The substantial overlap between genes regulated by RpoS and RPON provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through Rpo S.
Abstract: The RpoN–RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN–RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.
TL;DR: It is proposed that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.
Abstract: The mechanism by which Bacillus cereus causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of B. cereus, but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an alpha-helix bundle and a unique subdomain containing a hydrophobic beta-hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.
TL;DR: Using quantitative reverse-transcription PCR, it is found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with me pA overexpression predominating.
Abstract: Biocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified. The effect of exposure to low concentrations of biocides and dyes on the expression of specific pump genes has not been evaluated. Using quantitative reverse-transcription PCR we found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequently associated with promoter-region or regulatory protein mutations. Mutants having significant increases in MICs of common pump substrates but no changes in expression of studied pump genes were also observed; in these cases changes in expression of as-yet-unidentified MDR pump genes may have occurred. Strains of S. aureus that exist in relatively protected environments and are repeatedly exposed to sublethal concentrations of biocides can develop efflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin and moxifloxacin.
TL;DR: It is concluded that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.
Abstract: A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1–SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1–SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1–SAP3 or the SAP4–SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Δ mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p–Sap6p are not required for invasion of RHE by C. albicans.