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Comunidade bacteriana endofítica em microplantas de abacaxizeiro: estrutura, diversidade e sua influência na morfofisiologia após antibioticoterapia; Bacterial endophyte community in pineapple microplants: structure, diversity and its influence on morphophysiology after antibiotic therapy

Tarazi, Monita Fiori de Abreu
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 12/04/2010 PT
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O cultivo in vitro de plantas possibilita o controle de fatores ambientais e nutricionais, facilitando o estudo da interação planta-bactéria e a interpretação de eventuais alterações morfofisiológicas nas microplantas, decorrentes dessa interação. Entretanto, a presença de bactérias endofíticas na micropropagação é quase sempre caracterizada como contaminação microbiana prejudicial, sendo prontamente eliminada com o uso de quimioterápicos. Essa abordagem desconsidera os efeitos benéficos que bactérias endofíticas podem trazer ao desenvolvimento vegetal, aniquilando a possibilidade de se explorar esse potencial no ambiente in vitro. Em geral, devido a sua baixa culturabilidade, as comunidades bacterianas endofíticas requerem o uso de técnicas independentes de cultivo para seu estudo. Neste contexto, o presente trabalho teve como objetivo principal comprovar a presença de uma comunidade bacteriana endofítica em microplantas de abacaxizeiro (Ananas comosus (L.) Merrill) cv. IAC Gomo-de-mel consideradas axênicas e os efeitos dessa comunidade na morfofisiologia vegetal após antibioticoterapia. Para tanto, técnicas de PCR-DGGE, PCR-ARISA, clonagem, sequenciamento, análises estruturais e ultraestruturais foram utilizadas. Os resultados evidenciaram que existe uma comunidade bacteriana endofítica composta por membros de Alfa...

Análise do papel dos transportadores ABC de oligopeptídeos, poliaminas, fosfato inorgânico, glutamato e glutamina na fisiologia e patogênese de bactérias do trato gastro-intestinal.; Analysis of the role of ABC transporters of oligopeptides, polyamines, inorganic phosphate, glutamate and glutamine in the physiology and pathogenesis of gastrointestinal tract bacteria.

Lima, Roberto Nepomuceno de Souza
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 05/08/2013 PT
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Neste estudo focamos no papel de cinco transportadores da família ABC (ATP-binding cassete) envolvidos com a captação ativa de oligopeptídeos, poliaminas, fosfato inorgânico, glutamato e glutamina, em duas espécies bacterianas: Streptococcus mutans, que causa a cárie, e Escherichia coli enterohemorrágica (EHEC), responsável por diarreias e síndrome hemolítica urêmica em humanos. Com relação a inativação do sistema de transporte de oligopeptídeos de S. mutans não observamos alteração do crescimento bacteriano ou da aderência à superfícies abióticas. A deleção do sistema de captação de poliaminas não interferiu com o crescimento em meio rico, porém aumentou a resistência à ambiente ácidos. Inativação do sistema de transporte de fosfato inorgânico reduziu a aderência de S. mutans. Sobre os sistemas de transporte de glutamato e glutamina, mutantes de S. mutans apresentaram alterações nas taxas de crescimento e adesão à superfícies. Inativação da proteína OppA de EHEC não afetou a produção da toxina Stx bem como a patogenicidade in vitro e in vivo de EHEC. Em suma, o presente estudo demonstra que o papel dos transportadores ABC na fisiologia e patogenicidade de bactérias pode variar de acordo com a espécies bem como com o substrato transportado.; This study focuses on the role of five ABC (ATP-binding cassette) transport systems related to active uptake of oligopeptides...

Phosphoenolpyruvate:glycose phosphotransferase system in species of Vibrio, a widely distributed marine bacterial genus.

Meadow, N D; Revuelta, R; Chen, V N; Colwell, R R; Roseman, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1987 EN
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The genus Vibrio is one of the most common and widely distributed groups of marine bacteria. Studies on the physiology of marine Vibrio species were initiated by examining 15 species for the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS). All species tested contained a PTS analogous to the glucose-specific (IIGlc) system in enteric bacteria. Crude extracts of the cells showed immunological cross-reactivity with antibodies to enzyme I, HPr, and IIIGlc from Salmonella typhimurium when assayed by the rocket-line method. Toluene-permeabilized cells of 11 species were tested and were active in phosphorylating methyl alpha-D-glucoside with phosphoenolpyruvate but not ATP as the phosphoryl donor. Membranes from 10 species were assayed, and they phosphorylated methyl alpha-D-glucoside when supplemented with a phospho-IIIGlc-generating system composed of homogeneous proteins from enteric bacteria. Toluene-permeabilized cells and membranes of seven species were assayed, as were phosphorylated fructose and 2-deoxyglucose. IIIGlc was isolated from Vibrio fluvialis and was active in phosphorylating methyl alpha-D-glucoside when supplemented with a phospho-HPr-generating system composed of homogeneous proteins from Escherichia coli and membranes from either E. coli or V. fluvialis. These results show that the bacterial PTS is widely distributed in the marine environment and that it is likely to have a significant role in marine bacterial physiology and in the marine ecosystem.

Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

McAllister, Kelly A.; Peery, Robert B.; Zhao, Genshi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2006 EN
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Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally...

Hopanoids Are Not Essential for Growth of Streptomyces scabies 87-22▿

Seipke, Ryan F.; Loria, Rosemary
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Hopanoids are triterpenoic, pentacyclic compounds that are structurally similar to sterols, which are required for normal cell function in eukaryotes. Hopanoids are thought to be an important component of bacterial cell membranes because they control membrane fluidity and diminish passive diffusion of ions, and a few taxons modulate their hopanoid content in response to environmental stimuli. However, to our knowledge, mutational studies to assess the importance of hopanoids in bacterial physiology have never been performed. Genome sequencing of the potato scab pathogen, Streptomyces scabies 87-22, revealed a hopanoid biosynthetic gene cluster (HBGC) that is predicted to synthesize hopene and aminotrihydroxybacteriohopane products. Hopene was produced by fully sporulated cultures of S. scabies on solid ISP4 (International Streptomyces Project 4) medium as well as by submerged mycelia grown in liquid minimal medium. The elongated hopanoid aminotrihydroxybacteriohopane was not detected under either growth condition. Transcription of the S. scabies HBGC was upregulated during aerial growth, which suggests a link between hopanoid production and morphological development. Functional analysis of the S. scabies Δhop615-1 and Δhop615-7 mutant strains...

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia▿†

Schmerk, Crystal L.; Bernards, Mark A.; Valvano, Miguel A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2011 EN
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Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Δshc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Δshc mutant produced only very small amounts of the hopanoid peak. The Δshc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Δshc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics...

Using a Molecular-Genetic Approach to Investigate Bacterial Physiology in a Continuous, Research-Based, Semester-Long Laboratory for Undergraduates †

Ault, Jeremiah Foster; Renfro, Betsey Marie; White, Andrea Kirsten
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 01/12/2011 EN
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Designing investigative laboratory exercises that encourage critical thinking, problem solving, and independent thought for upper-division biology courses is a difficult but worthwhile task. In an effort to do so, we developed a semester-long, continuous, research-based investigative laboratory that integrates numerous genetic and molecular biology methods into the investigation of a bacterial physiological process. In this lab, students use random Tn5 transposon mutagenesis to create prodigiosin pigment mutants in the bacterium, Serratia marcescens. This is followed by phenotypic characterization, cloning, and sequencing the Tn insertion site to identify genes involved in pigment biosynthesis. During this lab, students gain ample experience performing basic lab techniques while learning about — and applying — methods for elucidating gene function. The approach to the laboratory and the outcomes are intimately integrated into the teaching of many fundamental physiological processes underlying prodigiosin production in bacteria. The result is a cohesive course that integrates the theory and application of molecular genetic techniques with the study of bacterial physiology. Assessments of student learning objectives demonstrated that students greatly improved their understanding of both physiological processes and the genetic techniques used to investigate them. In addition...

Effects of Antibiotics on Bacterial Species Composition and Metabolic Activities in Chemostats Containing Defined Populations of Human Gut Microorganisms

Newton, Dorothy F.; Macfarlane, Sandra; Macfarlane, George T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2013 EN
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The composition and metabolic activities of the human colonic microbiota are modulated by a number of external factors, including diet and antibiotic therapy. Changes in the structure and metabolism of the gut microbiota may have long-term consequences for host health. The large intestine harbors a complex microbial ecosystem comprising several hundreds of different bacterial species, which complicates investigations on intestinal physiology and ecology. To facilitate such studies, a highly simplified microbiota consisting of 14 anaerobic and facultatively anaerobic organisms (Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium adolescentis, Clostridium butyricum, C. perfringens, C. bifermentans, C. innocuum, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus) was used in this investigation. Ampicillin [9.2 μg (ml culture)−1] was added to two chemostats operated at different dilution rates (D; 0.10 h−1 and 0.21 h−1), and metronidazole [76.9 μg (ml culture)−1] was added to a third vessel (D = 0.21 h−1). Perturbations in bacterial physiology and metabolism were sampled over a 48-h period. Lactobacillus acidophilus and C. bifermentans populations did not establish in the fermentors under the imposed growth conditions. Ampicillin resulted in substantial reductions in bacteroides and C. perfringens populations at both dilution rates. Metronidazole strongly affected bacteroides communities but had no effect on bifidobacterial communities. The bacteriostatic effect of ampicillin on bifidobacterial species was growth rate dependent. Several metabolic activities were affected by antibiotic addition...

A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide

Stern, Andrew M.; Liu, Binbin; Bakken, Lars R.; Shapleigh, James P.; Zhu, Jun
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2013 EN
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Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress.

The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity

Parry, Bradley R.; Surovtsev, Ivan V.; Cabeen, Matthew T.; O’Hern, Corey S.; Dufresne, Eric R.; Jacobs-Wagner, Christine
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.

CRISPR-Cas systems: new players in gene regulation and bacterial physiology

Sampson, Timothy R.; Weiss, David S.
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 04/04/2014 EN
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CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries...

Phenotypic Signatures Arising from Unbalanced Bacterial Growth

Tan, Cheemeng; Smith, Robert Phillip; Tsai, Ming-Chi; Schwartz, Russell; You, Lingchong
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 07/08/2014 EN
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Fluctuations in the growth rate of a bacterial culture during unbalanced growth are generally considered undesirable in quantitative studies of bacterial physiology. Under well-controlled experimental conditions, however, these fluctuations are not random but instead reflect the interplay between intra-cellular networks underlying bacterial growth and the growth environment. Therefore, these fluctuations could be considered quantitative phenotypes of the bacteria under a specific growth condition. Here, we present a method to identify “phenotypic signatures” by time-frequency analysis of unbalanced growth curves measured with high temporal resolution. The signatures are then applied to differentiate amongst different bacterial strains or the same strain under different growth conditions, and to identify the essential architecture of the gene network underlying the observed growth dynamics. Our method has implications for both basic understanding of bacterial physiology and for the classification of bacterial strains.

A brief history of bacterial growth physiology

Schaechter, Moselio
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 21/04/2015 EN
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Arguably, microbial physiology started when Leeuwenhoek became fascinated by observing a Vorticella beating its cilia, my point being that almost any observation of microbes has a physiological component. With the advent of modern microbiology in the mid-19th century, the field became recognizably distinctive with such discoveries as anaerobiosis, fermentation as a biological phenomenon, and the nutritional requirements of microbes. Soon came the discoveries of Winogradsky and his followers of the chemical changes in the environment that result from microbial activities. Later, during the first half of the 20th century, microbial physiology became the basis for much of the elucidation of central metabolism. Bacterial physiology then became a handmaiden of molecular biology and was greatly influenced by the discovery of cellular regulatory mechanisms. Microbial growth, which had come of age with the early work of Hershey, Monod, and others, was later pursued by studies on a whole cell level by what became known as the “Copenhagen School.” During this time, the exploration of physiological activities became coupled to modern inquiries into the structure of the bacterial cell. Recent years have seen the development of a further phase in microbial physiology...

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Adler, Conrado; Corbalán, Natalia S.; Seyedsayamdost, Mohammad; Pomares, María Fernanda; de Cristóbal, Ricardo E.; Clardy, Jon C.; Kolter, Roberto Guillermo; Vincent, Paula A.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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Background: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. Methods and Principal Findings: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast...

Central role of manganese in regulation of stress responses, physiology, and metabolism in Streptococcus pneumoniae

Ogunniyi, A.; Mahdi, L.; Jennings, M.; McEwan, A.; McDevitt, C.; Van der Hoek, M.; Bagley, C.; Hoffmann, P.; Gould, K.; Paton, J.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2010 EN
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The importance of Mn2+ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn2+ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn2+ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn2+ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn2+-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn2+-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn2+ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn2+-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn2+ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn2+ transport by PsaA in pneumococcal competence...

The evolution of a conjugative plasmid and its ability to increase bacterial fitness.

Dionisio, F.; Conceição, I.C.; Marques, A.C.R.; Fernandes, L.; Gordo, I.
Fonte: Evolutionary Ecology Publicador: Evolutionary Ecology
Tipo: Artigo de Revista Científica
Publicado em 22/06/2005 ENG
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Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.

The Highly Conserved Bacterial RNase YbeY Is Essential in Vibrio cholerae, Playing a Critical Role in Virulence, Stress Regulation, and RNA Processing

Vercruysse, Maarten; Köhrer, Caroline; Davies, Bryan W.; Arnold, Markus F. F.; Mekalanos, John J.; RajBhandary, Uttam L.; Walker, Graham C.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 3′ end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 3′ end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably...

Pseudomonas-Saccharomyces Interactions: Influence of Fungal Metabolism on Bacterial Physiology and Survival

Romano, Julia D.; Kolter, Roberto
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2005 EN
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Fungal-bacterial interactions are ubiquitous, yet their molecular basis is only poorly understood. In this study, a novel beneficial interaction between a strain of Pseudomonas putida and the fungus Saccharomyces cerevisiae was identified. When the bacteria were incubated alone in grape juice or in synthetic medium containing various concentrations of glucose, they lost viability rapidly during stationary phase. However, when the bacteria were incubated in these media in the presence of the fungus, their stationary phase survival improved dramatically. On agar plates containing glucose, the beneficial effects of the fungus were manifested in robust bacterial growth and exopolysaccharide production that led to visible mucoidy. In contrast, bacteria grew poorly and were nonmucoid in such media in the absence of the fungus. By using the available S. cerevisiae deletion library, yeast mutants that were unable to mediate this beneficial interaction were identified. These mutants revealed that the beneficial effect on bacterial physiology and survival was mediated by the ability of the fungus to metabolize the available glucose and consequent effects on the medium's pH. In natural environments where the concentration of glucose is high, it is likely that the presence of fungi has had profound beneficial effects on the physiology and survival of certain P. putida strains throughout their natural history.

Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology

Raz, Assaf; Tanasescu, Ana-Maria; Zhao, Anna M.; Serrano, Anna; Alston, Tricia; Sol, Asaf; Bachrach, Gilad; Fischetti, Vincent A.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/10/2015 EN
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Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment...

Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

Raju, Ravikiran M.; Jedrychowski, Mark P.; Wei, Jun-Rong; Pinkham, Jessica T.; Park, Annie S.; O'Brien, Kathryn; Rehren, German; Schnappinger, Dirk; Gygi, Steven P.; Rubin, Eric J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.