Assuntos
Coinfecção , Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa , Infecções Estafilocócicas , Staphylococcus aureus , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Humanos , Infecções por Pseudomonas/complicações , Coinfecção/microbiologia , Infecções Estafilocócicas/complicações , Masculino , Feminino , Adulto , Doença Crônica , CriançaRESUMO
Pseudomonas aeruginosa is a robust and versatile organism capable of surviving and prospering in a diverse array of environments and is an opportunistic pathogen of humans. One reason for the success of this pathogen is the large arsenal of antimicrobial weapons that it possesses. Here we focus our attention on these antimicrobial weapons and how they give P. aeruginosa a survival edge in polymicrobial environments. We define antimicrobial weapons as components produced by P. aeruginosa that are used to kill, inhibit growth and/or subvert key cellular functions in other microbes. P. aeruginosa has a large and complex genome and encodes an armament of antimicrobial weapons that fall into two subclasses; those that are delivered directly to competing microbes using a contact-dependent method, and those that are secreted in a contact-independent manner into the environment to then be available to target neighbouring cells. This chapter provides an overview of the major antimicrobial weapons possessed by P. aeruginosa, captures recent advances in the field and discusses how these could be targeted as a therapeutic intervention, or potentially harnessed to combat infection.
Assuntos
Anti-Infecciosos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêuticoRESUMO
Recalcitrant chronic infections of implanted medical devices are often linked to the presence of biofilms. The prevention and treatment of medical device-associated infections is a major source of antibiotic use and driver of antimicrobial resistance globally. Lowering the incidence of infection in patients that receive implanted medical devices could therefore significantly improve antibiotic stewardship and reduce patient morbidity. Here we determined if modifying the design of an implantable medical device to reduce bacterial attachment, impacted the incidence of device-associated infections in clinical practice. Since the 1980s cochlear implants have provided long-term treatment of sensorineural hearing deficiency in hundreds of thousands of patients world-wide. Nonetheless, a relatively small number of devices are surgically explanted each year due to unresolvable infections. Features associated with the accumulation of bacteria on the Cochlear™ Nucleus® CI24RE™ model of cochlear implant devices were identified using both in vitro bacterial attachment assays and examination of explanted devices. Macro-scale design modifications that reduced bacterial attachment in vitro were incorporated into the design of the CI500™ and Profile™ series of Nucleus implant. Analyses of mandatory post-market vigilance data of 198,757 CI24RE and 123,084 CI500/Profile series implantation surgeries revealed that these design modifications correlated with significantly reduced infection rates. This study demonstrates that a design-centric approach aimed at mitigating bacterial attachment was a simple, and effective means of reducing infections associated with Cochlear Nucleus devices. This approach is likely to be applicable to improving the designs of other implantable medical devices to reduce device-associated infections.
RESUMO
The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Complexos Multiproteicos/genética , Ligação Proteica , Sistemas de Secreção Tipo VI/genéticaRESUMO
Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.
Assuntos
Bacteriófagos/fisiologia , Membrana Celular/virologia , Escherichia coli/virologia , Vesículas Extracelulares/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vesículas Extracelulares/genéticaRESUMO
Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Bacteriólise , Endopeptidases/genética , Endopeptidases/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Mutação , Pseudomonas aeruginosa/metabolismoRESUMO
Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa.
Assuntos
Biofilmes , Pseudomonas aeruginosa/fisiologia , Transformação Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , DNA/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/genéticaRESUMO
BACKGROUND: The use of non-physician advanced practice providers (NPAPP) has increased in the United States to offset shortages in the physician workforce. Yet there are still gaps in some locations where there is little to no access to quality health care. This study sought to identify whether physicians perceived a workforce gap and their level of interest in hiring an NPAPP with cardiopulmonary expertise to fill the perceived gap. METHODS: An American Association for Respiratory Care (AARC)-led workgroup surveyed 1,401 physicians in 6 different specialties. The survey instrument contained 32 closed-ended questions and 4 open-ended questions. RESULTS: 74% of the 1,401 physician respondents agreed or strongly agreed that there will be a future need for an NPAPP with cardiopulmonary expertise. Respondents from sleep, pediatrics, pulmonary, and critical care were most likely to indicate that there is a current need for an NPAPP. A majority of respondents perceived that the specialized NPAPP would improve efficiency and productivity (74%), patient experience (73%), and patient outcomes (72%). Interest in adding this NPAPP did not increase when participants were told to presume authority for hiring, budget, and reimbursement. CONCLUSIONS: These results indicate that there is both a need for and an interest in hiring an NPAPP with cardiopulmonary expertise. Having an NPAPP would boost physician efficiency and productivity, improve the patient care experience, and provide benefits that other clinicians are not trained to provide to persons with cardiopulmonary disease. Results suggest there should be continued efforts to develop the NPAPP role to add value for physicians and patients alike.
Assuntos
Cardiopatias , Pneumopatias , Médicos , Cuidados Críticos , Humanos , Qualidade da Assistência à Saúde , Inquéritos e Questionários , Estados Unidos , Recursos HumanosRESUMO
Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3'-5'-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa.
Assuntos
Biofilmes/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/metabolismo , DNA Bacteriano , Interações Hospedeiro-Patógeno , Movimento/efeitos dos fármacos , Mucinas/farmacologia , Oligopeptídeos/farmacologia , Infecções por Pseudomonas/microbiologia , Deleção de Sequência , Albumina Sérica/farmacologia , Transdução de Sinais , Fatores de Virulência/metabolismoRESUMO
Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of research.
Assuntos
Microbiota , Sistemas de Secreção Tipo VI/fisiologia , Animais , Antibiose , Proteínas de Bactérias/fisiologia , Homeostase , Interações entre Hospedeiro e Microrganismos , HumanosRESUMO
The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of ß-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.
Assuntos
Sistemas de Secreção Bacterianos/fisiologia , Pseudomonas aeruginosa/fisiologia , Sistemas de Secreção Tipo VI/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Periplasma/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Virulência/metabolismo , beta-Lactamas/metabolismoRESUMO
We report the complete genome of Pseudomonas aeruginosa strain PAK, a strain which has been instrumental in the study of a range of P. aeruginosa virulence and pathogenesis factors and has been used for over 50 years as a laboratory reference strain.
RESUMO
Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
Assuntos
Flagelos/fisiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Animais , Biofilmes , Doença Crônica , Flagelos/genética , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/microbiologia , VirulênciaRESUMO
Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.
Assuntos
Biofilmes , Fímbrias Bacterianas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Fímbrias Bacterianas/ultraestrutura , Flagelos/genética , Genes Bacterianos , Genômica , Locomoção/genética , Microscopia Eletrônica de Transmissão , Mutagênese , Pseudomonas aeruginosa/ultraestrutura , Fatores de Virulência/genéticaRESUMO
The type VI secretion system (T6SS) is a weapon of bacterial warfare and host cell subversion. The Gram-negative pathogen Pseudomonas aeruginosa has three T6SSs involved in colonization, competition, and full virulence. H1-T6SS is a molecular gun firing seven toxins, Tse1-Tse7, challenging survival of other bacteria and helping P. aeruginosa to prevail in specific niches. The H1-T6SS characterization was facilitated through studying a P. aeruginosa strain lacking the RetS sensor, which has a fully active H1-T6SS, in contrast to the parent. However, study of H2-T6SS and H3-T6SS has been neglected because of a poor understanding of the associated regulatory network. Here we performed a screen to identify H2-T6SS and H3-T6SS regulatory elements and found that the posttranscriptional regulator RsmA imposes a concerted repression on all three T6SS clusters. A higher level of complexity could be observed as we identified a transcriptional regulator, AmrZ, which acts as a negative regulator of H2-T6SS. Overall, although the level of T6SS transcripts is fine-tuned by AmrZ, all T6SS mRNAs are silenced by RsmA. We expanded this concept of global control by RsmA to VgrG spike and T6SS toxin transcripts whose genes are scattered on the chromosome. These observations triggered the characterization of a suite of H2-T6SS toxins and their implication in direct bacterial competition. Our study thus unveils a central mechanism that modulates the deployment of all T6SS weapons that may be simultaneously produced within a single cell.
Assuntos
Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Análise por Conglomerados , Deleção de Genes , Redes Reguladoras de Genes , Sistema Imunitário , Óperon Lac , Mutagênese , Plasmídeos/metabolismo , RNA Bacteriano/genética , Transcrição Gênica , Sistemas de Secreção Tipo VI/genética , Virulência/genéticaRESUMO
BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa. RESULTS: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system. CONCLUSIONS: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furthermore, eATP produced by P. aeruginosa biofilms and by damaged epithelial cells may play a role in P. aeruginosa pathogenesis by inducing inflammatory damage and fibrosis. Our findings have significant implications in the development and pathogenesis of P. aeruginosa biofilm infections.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Fímbrias Bacterianas/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Fímbrias Bacterianas/fisiologia , Expressão Gênica , Movimento/efeitos dos fármacos , Movimento/fisiologia , Pseudomonas aeruginosa/fisiologia , Transdução de SinaisRESUMO
Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.
Assuntos
Biofilmes , DNA Bacteriano/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMO
Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/fisiologia , Pseudomonas aeruginosa/fisiologia , Supressão Genética , Sequência de Aminoácidos , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Genoma Bacteriano , Fenótipo , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.