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Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vectorvertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct hostpathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.
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Proteínas de Bactérias , Borrelia burgdorferi , Complemento C1q , Evasão da Resposta Imune , Lipoproteínas , Doença de Lyme , Proteínas de Bactérias/imunologia , Borrelia burgdorferi/imunologia , Complemento C1q/imunologia , Humanos , Imunoglobulinas/imunologia , Lipoproteínas/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Proteoma/imunologiaRESUMO
Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.
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Toxinas Bacterianas , Criptosporidiose , Cryptosporidium , Escherichia coli Enteropatogênica , Probióticos , Anticorpos de Domínio Único , Humanos , Antígenos de Superfície , Vermelho Congo , Flagelina , Sistemas de Secreção Tipo III , Fatores de Virulência/genéticaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.
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Escherichia coli Êntero-Hemorrágica/metabolismo , Prófagos/metabolismo , Ativação Viral/fisiologia , Animais , Bacteriófagos/metabolismo , Bacteriófagos/patogenicidade , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/microbiologia , Feminino , Mucosa Intestinal/microbiologia , Lisogenia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Resposta SOS em Genética/fisiologia , Toxina Shiga II/genética , Toxina Shiga II/metabolismoRESUMO
Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate.IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.
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Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , RNA Bacteriano/biossíntese , Esporos Bacterianos/fisiologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Etanol/farmacologia , Genoma Bacteriano , Temperatura Alta , Mutação , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Radioisótopos de Fósforo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/efeitos da radiaçãoRESUMO
Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.
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[This corrects the article DOI: 10.3389/fnut.2023.1230061.].
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Introduction: The safety of novel forms of iron in healthy, iron-replete adults as might occur if used in population-based iron supplementation programs was examined. We tested the hypotheses that supplementation with nanoparticulate iron hydroxide adipate tartrate (IHAT), an iron-enriched Aspergillus oryzae product (ASP), or ferrous sulphate heptahydrate (FS) are safe as indicated by erythrocyte susceptibility to malarial infection, bacterial proliferation, and gut inflammation. Responses to FS administered daily or weekly, and with or without other micronutrients were compared. Methods: Two phases of randomized, double-blinded trials were conducted in Boston, MA. Phase I randomized 160 volunteers to six treatments: placebo, IHAT, ASP, FS, and FS plus a micronutrient powder (MNP) administrated daily at 60 mg Fe/day; and FS administered as a single weekly dose of 420 mg Fe. Phase II randomized 86 volunteers to IHAT, ASP, or FS administered at 120 mg Fe/day. Completing these phases were 151 and 77 participants, respectively. The study was powered to detect effects on primary endpoints: susceptibility of participant erythrocytes to infection by Plasmodium falciparum, the proliferation potential of selected pathogenic bacteria in sera, and markers of gut inflammation. Secondary endpoints for which the study was not powered included indicators of iron status and gastrointestinal symptoms. Results: Supplementation with any form of iron did not affect any primary endpoint. In Phase I, the frequency of gastrointestinal symptoms associated with FS was unaffected by dosing with MNP or weekly administration; but participants taking IHAT more frequently reported abdominal pain (27%, p < 0.008) and nausea (4%, p = 0.009) than those taking FS, while those taking ASP more frequently reported nausea (8%, p = 0.009). Surprisingly, only 9% of participants taking IHAT at 120 mg Fe/day (Phase II) reported abdominal pain and no other group reported that symptom. Discussion: With respect to the primary endpoints, few differences were found when comparing these forms of iron, indicating that 28 days of 60 or 120 mg/day of IHAT, ASP, or FS may be safe for healthy, iron-replete adults. With respect to other endpoints, subjects receiving IHAT more frequently reported abdominal pain and nausea, suggesting the need for further study. Clinical Trial Registration: ClinicalTrials.gov, NCT03212677; registered: 11 July 2017.
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Primary productivity in the ocean's oligotrophic regions is often limited by phosphorus (P) availability. In low phosphate environments, the prevalence of many genes involved in P acquisition is elevated, suggesting that the ability to effectively access diverse P sources is advantageous for organisms inhabiting these regions. Prochlorococcus, the numerically dominant primary producer in the oligotrophic ocean, encodes high-affinity P transporters, P regulatory proteins and enzymes for organic phosphate utilization, but its ability to use reduced P compounds has not been previously demonstrated. Because Prochlorococcus strain MIT9301 encodes genes similar to phnY and phnZ, which constitute a novel marine bacterial 2-aminoethylphosphonate (2-AEPn) utilization pathway, it has been suggested that this organism might use 2-AEPn as an alternative P source. We show here that although MIT9301 was unable to use 2-AEPn as a sole P source under standard culture conditions, it was able to use phosphite. Phosphite utilization by MIT9301 appears to be mediated by an NAD-dependent phosphite dehydrogenase encoded by ptxD. We show that phosphite utilization genes are present in diverse marine microbes and that their abundance is higher in low-P waters. These results strongly suggest that phosphite represents a previously unrecognized component of the marine P cycle.
Assuntos
Micronutrientes/metabolismo , Fosfitos/metabolismo , Prochlorococcus/fisiologia , Oligoelementos/metabolismo , Bactérias/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oceanos e Mares , Fósforo/metabolismo , Prochlorococcus/genética , Prochlorococcus/metabolismoRESUMO
The formation of attaching and effacing (A/E) lesions on intestinal epithelium, combined with Shiga toxin production, are hallmarks of enterohemorrhagic Escherichia coli (EHEC) infection that can lead to lethal hemolytic uremic syndrome. Although an animal infection model that fully recapitulates human disease remains elusive, mice orally infected with Citrobacter rodentium(ÏStx2dact), a natural murine pathogen lysogenized with an EHEC-derived Shiga toxin 2-producing bacteriophage, develop intestinal A/E lesions and toxin-dependent systemic disease. This model has facilitated investigation of how: (A) phage gene expression and prophage induction contribute to disease and are potentially triggered by antibiotic treatment; (B) virulence gene expression is altered by microbiota and the colonic metabolomic milieu; and (C) innate immune signaling is affected by Stx. Thus, the model provides a unique tool for accessing diverse aspects of EHEC pathogenesis.
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Bacteriófagos , Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Animais , Bacteriófagos/metabolismo , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/metabolismo , Feminino , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/patologia , Humanos , Mucosa Intestinal/metabolismo , Masculino , CamundongosRESUMO
Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.
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Bacteriófagos , Citrobacter rodentium , Colite , Hemorragia Gastrointestinal , Síndrome Hemolítico-Urêmica , Mucosa Intestinal , Lisogenia , Toxinas Shiga , Escherichia coli Shiga Toxigênica , Animais , Bacteriófagos/genética , Bacteriófagos/metabolismo , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Citrobacter rodentium/patogenicidade , Citrobacter rodentium/virologia , Colite/genética , Colite/metabolismo , Colite/microbiologia , Modelos Animais de Doenças , Hemorragia Gastrointestinal/genética , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/microbiologia , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Toxinas Shiga/biossíntese , Toxinas Shiga/genéticaRESUMO
Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.
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DNA Bacteriano/genética , Desoxirribodipirimidina Fotoliase/biossíntese , Óperon , Prochlorococcus/efeitos da radiação , Pirofosfatases/biossíntese , Deleção de Sequência , Raios Ultravioleta , Proteínas de Bactérias/biossíntese , Sequência de Bases , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Viabilidade Microbiana/efeitos da radiação , Dados de Sequência Molecular , Nudix HidrolasesRESUMO
T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.
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Bacteriófago T4/genética , Cianobactérias/virologia , Ácidos Cetoglutáricos/metabolismo , Myoviridae/genética , Compostos de Amônio Quaternário/metabolismo , Água do Mar/virologia , Bacteriófago T4/classificação , Composição de Bases , Evolução Molecular , Variação Genética , Genoma Viral , Metagenômica , Dados de Sequência Molecular , Myoviridae/classificação , Nitrogênio/metabolismo , Oceanos e Mares , Prochlorococcus/virologia , Água do Mar/microbiologia , Análise de Sequência de DNA , Synechococcus/virologia , Proteínas do Core Viral/genética , Proteínas da Cauda Viral/genética , Microbiologia da ÁguaRESUMO
As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.
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Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.
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Bacteriófagos/ultraestrutura , Prochlorococcus/ultraestrutura , Prochlorococcus/virologia , Ligação ViralRESUMO
Rifalazil is a potent second-generation ansamycin that kills bacterial cells by inhibiting the beta subunit of RNA polymerase. Rifalazil has several improved properties compared with rifampicin, but retains rifampicin's propensity to develop resistant mutants at high frequency. To explore strategies to overcome resistance development, we studied the effects of rifalazil in combination with several different antibiotics in an in vitro time-kill model, against both log phase and stationary phase Staphylococcus aureus cells. Experiments were carried out at high initial cell density so that the frequency and proliferation of resistant mutants could be monitored. We found that each combination was advantageous in terms of enhanced killing and the suppression of mutants, compared with each drug used alone. None of the three combinations was effective against stationary phase cells.
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Acetamidas/farmacologia , Antibacterianos/farmacologia , Levofloxacino , Mupirocina/farmacologia , Ofloxacino/farmacologia , Oxazolidinonas/farmacologia , Rifamicinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Farmacorresistência Bacteriana , Quimioterapia Combinada , Linezolida , Testes de Sensibilidade Microbiana , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Rifalazil is a novel rifamycin that, like other members of this class, inhibits bacterial transcription by targeting the beta subunit of prokaryotic DNA-dependent RNA polymerase. To address the high-frequency resistance seen with rifamycins, we assessed the ability of rifalazil, alone and in combination with vancomycin, to both kill cells and to suppress the appearance of resistant mutants in log and stationary phase Staphylococcus aureus cultures, using high cell densities in an in vitro kill curve model. We found that (1) rifalazil alone killed log-phase cultures more rapidly than rifampicin, but both drugs quickly selected for resistant mutants, (2) co-treatment of log phase cultures with rifalazil and vancomycin increased bacterial killing by about 3-Log10 over either drug used alone and delayed the appearance of rifamycin-resistant mutants, (3) rifalazil and vancomycin in combination killed stationary phase cultures
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Antibacterianos/farmacologia , Rifamicinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/farmacologia , Resistência a Medicamentos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/crescimento & desenvolvimento , Fatores de TempoRESUMO
Heterologous biosynthesis of natural products is meant to enable access to the vast array of valuable properties associated with these compounds. Often motivated by limitations inherent in native production hosts, the heterologous biosynthetic process begins with a candidate host regarded as technically advanced relative to original producing organisms. Given this requirement, E. coli has been a top choice for heterologous biosynthesis attempts as associated recombinant tools emerged and continue to develop. However, success requires overcoming challenges associated with natural product formation, including complex biosynthetic pathways and the need for metabolic support. These two challenges have been heavily featured in cellular engineering efforts completed to position E. coli as a viable surrogate host. This chapter outlines steps taken to engineer E. coli with an emphasis on genetic manipulations designed to support the heterologous production of polyketide, nonribosomal peptide, and similarly complex natural products.
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Produtos Biológicos/metabolismo , Vias Biossintéticas , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Policetídeos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Óperon , Recombinases Rec A/genética , Recombinases Rec A/metabolismoRESUMO
One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.
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Biblioteca Genômica , Metagenômica/métodos , Consórcios Microbianos/genética , DNA Bacteriano , Vetores GenéticosRESUMO
Viruses that infect marine cyanobacteria-cyanophages-often carry genes with orthologs in their cyanobacterial hosts, and the frequency of these genes can vary with habitat. To explore habitat-influenced genomic diversity more deeply, we used the genomes of 28 cultured cyanomyoviruses as references to identify phage genes in three ocean habitats. Only about 6-11% of genes were consistently observed in the wild, revealing high gene-content variability in these populations. Numerous shared phage/host genes differed in relative frequency between environments, including genes related to phosphorous acquisition, photorespiration, photosynthesis and the pentose phosphate pathway, possibly reflecting environmental selection for these genes in cyanomyovirus genomes. The strongest emergent signal was related to phosphorous availability; a higher fraction of genomes from relatively low-phosphorus environments-the Sargasso and Mediterranean Sea-contained host-like phosphorus assimilation genes compared with those from the N. Pacific Gyre. These genes are known to be upregulated when the host is phosphorous starved, a response mediated by pho box motifs in phage genomes that bind a host regulatory protein. Eleven cyanomyoviruses have predicted pho boxes upstream of the phosphate-acquisition genes pstS and phoA; eight of these have a conserved cyanophage-specific gene (PhCOG173) between the pho box and pstS. PhCOG173 is also found upstream of other shared phage/host genes, suggesting a unique regulatory role. Pho boxes are found upstream of high light-inducible (hli) genes in cyanomyoviruses, suggesting that this motif may have a broader role than regulating phosphorous-stress responses in infected hosts or that these hlis are involved in the phosphorous-stress response.
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Bacteriófagos/classificação , Ecossistema , Variação Genética , Fósforo/metabolismo , Prochlorococcus/virologia , Água do Mar/virologia , Estresse Fisiológico/genética , Bacteriófagos/genética , Frequência do Gene , Genes Virais/genética , Mar Mediterrâneo , Metagenoma , Filogenia , Água do Mar/microbiologiaRESUMO
The marine cyanobacterium Prochlorococcus, the smallest and most abundant oxygenic phototroph, has an extremely streamlined genome and a high rate of protein evolution. High-light adapted strains of Prochlorococcus in particular have seemingly inadequate DNA repair systems, raising the possibility that inadequate repair may lead to high mutation rates. Prochlorococcus mutation rates have been difficult to determine, in part because traditional methods involving quantifying colonies on solid selective media are not straightforward for this organism. Here we used a liquid dilution method to measure the approximate number of antibiotic-resistant mutants in liquid cultures of Prochlorococcus strains previously unexposed to antibiotic selection. Several antibiotics for which resistance in other bacteria is known to result from a single base pair change were used. The resulting frequencies of antibiotic resistance in Prochlorococcus cultures allowed us to then estimate maximum spontaneous mutation rates, which were similar to those in organisms such as E. coli (â¼5.4 × 10(-7) per gene per generation). Therefore, despite the lack of some DNA repair genes, it appears unlikely that the Prochlorcoccus genomes studied here are currently being shaped by unusually high mutation rates.