RESUMO
Staphylococcus epidermidis CSF41498 is amenable to genetic manipulation and has been used to study mechanisms of biofilm formation. We report here the whole-genome sequence of this strain, which contains 2,427 protein-coding genes and 82 RNAs within its 2,481,008-bp-long genome, as well as three plasmids.
RESUMO
Staphylococcal infections involving biofilms represent a significant challenge in the treatment of patients with device-related infections. Staphylococcus aureus biofilms have been shown to be SaeRS regulated and dependent on the coagulase-catalyzed conversion of fibrinogen into fibrin on surfaces coated with human plasma. Here we investigated the treatment of staphylococcal biofilm device-related infections by digesting the fibrin biofilm matrix with and without existing antimicrobials. The fibrinolytic agents plasmin, streptokinase, and nattokinase, and TrypLE, a recombinant trypsin-like protease, were used to digest and treat S. aureus biofilms grown in vitro using in vivo-like static biofilm assays with and without antimicrobials. Cytotoxicity, the potential to induce a cytokine response in whole human blood, and the risk of induction of tolerance to fibrinolytic agents were investigated. A rat model of intravascular catheter infection was established to investigate the efficacy of selected fibrinolytic agents in vivo Under biomimetic conditions, the fibrinolytic agents effectively dispersed established S. aureus biofilms and, in combination with common antistaphylococcal antimicrobials, effectively killed bacterial cells being released from the biofilm. These fibrinolytic agents were not cytotoxic and did not affect the host immune response. The rat model of infection successfully demonstrated the activity of the selected fibrinolytic agents alone and in combination with antimicrobials on established biofilms in vivo TrypLE and nattokinase most successfully removed adherent cells from plasma-coated surfaces and significantly improved the efficacy of existing antimicrobials against S. aureus biofilms in vitro and in vivo These biofilm dispersal agents represent a viable future treatment option for S. aureus device-related infections.
Assuntos
Antibacterianos/farmacologia , Infecções Relacionadas a Cateter/tratamento farmacológico , Infecções Relacionadas a Cateter/microbiologia , Fibrinolíticos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Linhagem Celular , Coagulase/metabolismo , Humanos , Testes de Sensibilidade Microbiana/métodos , Ratos , Ratos Sprague-Dawley , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Staphylococcus aureus is a leading cause of healthcare-associated infections. The ability of S. aureus to attach and subsequently accumulate on the surfaces of implanted medical devices and in host tissues makes infections caused by this pathogen difficult to treat. Current treatments have been shown to have limited effect on surface-associated S. aureus, and may be enhanced by the addition of a dispersal agent. This study assessed the enzymatic agents dispersin B, lysostaphin, alpha amylase, V8 protease and serrapeptase, alone and in combination with vancomycin and rifampicin, against biofilms formed by meticillin-resistant and -susceptible strains of S. aureus. The efficacy of both antibiotics was enhanced when combined with any of the dispersal agents. Lysostaphin and serrapeptase were the most effective dispersal agents against all strains tested. These data indicate that combinations of biofilm dispersal agents and antibiotics may extend the therapeutic options for the treatment of S. aureus biofilm-associated infections.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Enzimas/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Adulto , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/fisiologiaRESUMO
Staphylococci are a leading cause of catheter-related infections (CRIs) due to biofilm formation. CRIs are typically managed by either device removal or systemic antibiotics, often in combination with catheter lock solutions (CLSs). CLSs provide high concentrations of the antimicrobial agent at the site of infection. However, the most effective CLSs against staphylococcal biofilm-associated infections have yet to be determined. The purpose of this study was to evaluate the efficacy and suitability of two newly described antimicrobial agents, ML:8 and Citrox, as CLSs against Staphylococcus aureus biofilms. ML:8 (1% [vol/vol]) and Citrox (1% [vol/vol]), containing caprylic acid and flavonoids, respectively, were used to treat S. aureus biofilms grown in vitro using newly described static and flow biofilm assays. Both agents reduced biofilm viability >97% after 24 h of treatment. Using a rat model of CRI, ML:8 was shown to inactivate early-stage S. aureus biofilms in vivo, while Citrox inactivated established, mature in vivo biofilms. Cytotoxicity and hemolytic activity of ML:8 and Citrox were equivalent to those of other commercially available CLSs. Neither ML:8 nor Citrox induced a cytokine response in human whole blood, and exposure of S. aureus to either agent for 90 days was not associated with any increase in resistance. Taken together, these data reveal the therapeutic potential of these agents for the treatment of S. aureus catheter-related biofilm infections.
Assuntos
Antibacterianos/farmacologia , Caprilatos/farmacologia , Infecções Relacionadas a Cateter/tratamento farmacológico , Flavonoides/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Ratos Sprague-Dawley , Staphylococcus aureus/patogenicidadeRESUMO
Infection of intravascular catheters by Staphylococcus aureus is a significant risk factor within the health care setting. To treat these infections and attempt salvage of an intravascular catheter, antimicrobial lock solutions (ALSs) are being increasingly used. However, the most effective ALSs against these biofilm-mediated infections have yet to be determined, and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacies of antibiotics and antiseptics in current clinical use against biofilms produced by reference and clinical isolates of S. aureus Static and flow biofilm assays were developed using newly described in vivo-relevant conditions to examine the effect of each agent on S. aureus within the biofilm matrix. The antibiotics daptomycin, tigecycline, and rifampin and the antiseptics ethanol and Taurolock inactivated established S. aureus biofilms, while other commonly used antistaphylococcal antibiotics and antiseptic agents were less effective. These findings were confirmed by live/dead staining of S. aureus biofilms formed and treated within a flow cell model. The results from this study demonstrate the most effective clinically used agents and their concentrations which should be used within an ALS to treat S. aureus-mediated intravascular catheter-related infections.
Assuntos
Anti-Infecciosos/farmacologia , Infecções Relacionadas a Cateter/microbiologia , Biofilmes/efeitos dos fármacos , Daptomicina/farmacologia , Etanol/farmacologia , Testes de Sensibilidade Microbiana , Minociclina/análogos & derivados , Minociclina/farmacologia , Rifampina/farmacologia , Staphylococcus aureus , TigeciclinaRESUMO
Staphylococci, in particular Staphylococcus aureus and Staphylococcus epidermidis, are a leading cause of healthcare-associated infections. Patients who have a medical device inserted are at particular risk of an infection with these organisms as staphylococci possess a wide range of immune evasion mechanisms, one of which being their ability to form biofilm. Once embedded in a biofilm, bacteria are inherently more resistant to treatment with antibiotics. Despite advances in our understanding of the pathogenesis of staphylococcal biofilm formation, medical devices colonised with biofilms frequently require removal. New and novel approaches to prevent and treat biofilm infections are urgently required. In recent years, progress has been made on approaches that include antiadhesive strategies to prevent surface adhesion or production of bacterial adhesins, dissolution of already established biofilm, targeting of biofilm matrix for degradation and interference with biofilm regulation. Several obstacles need to be overcome in the further development of these and other novel anti-biofilm agents. Most notably, although in vitro investigation has progressed over recent years, the need for biofilm models to closely mimic the in vivo situation is of paramount importance followed by controlled clinical trials. In this review we highlight the issues associated with staphylococcal colonisation of medical devices and potential new treatment options for the prevention and control of these significant infections.
Assuntos
Antibacterianos/farmacologia , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Modelos Biológicos , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/prevenção & controle , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificaçãoRESUMO
Protein adsorption is one of the key parameters influencing the biocompatibility of medical device materials. This study investigates serum protein adsorption and bacterial attachment on polymer coatings deposited using an atmospheric pressure plasma jet system. The adsorption of bovine serum albumin and bovine fibrinogen (Fg) onto siloxane and fluorinated siloxane elastomeric coatings that exhibit water contact angles (θ) ranging from superhydrophilic (θ < 5°) to superhydrophobic (θ > 150°) were investigated. Protein interactions were evaluated in situ under dynamic flow conditions by spectroscopic ellipsometry. Superhydrophilic coatings showed lower levels of protein adsorption when compared with hydrophobic siloxane coatings, where preferential adsorption was shown to occur. Reduced levels of protein adsorption were also observed on fluorinated siloxane copolymer coatings exhibiting hydrophobic wetting behaviour. The lower levels of protein adsorption observed on these surfaces indicated that the presence of fluorocarbon groups have the effect of reducing surface affinity for protein attachment. Analysis of superhydrophobic siloxane and fluorosiloxane surfaces showed minimal indication of protein adsorption. This was confirmed by bacterial attachment studies using a Staphylococcus aureus strain known to bind specifically to Fg, which showed almost no attachment to the superhydrophobic coating after protein adsorption experiments. These results showed the superhydrophobic surfaces to exhibit antimicrobial properties and significantly reduce protein adsorption.
Assuntos
Adsorção , Aderência Bacteriana , Proteínas Sanguíneas/química , Materiais Revestidos Biocompatíveis/química , Staphylococcus aureus/fisiologia , Animais , Bovinos , Fibrinogênio/química , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Soroalbumina Bovina/química , Siloxanas/químicaRESUMO
Hydrogen peroxide, Ecasol and Citrox aerosols were each tested for their ability to kill a range of nosocomial pathogens. Hydrogen peroxide had the broadest microbicidal activity but operational issues limit its use. Ecasol was effective against all micro-organisms, except Clostridium difficile, while Citrox aerosols were not effective against Gram-negative bacilli.
Assuntos
Aerossóis , Bactérias/efeitos dos fármacos , Descontaminação/métodos , Desinfetantes/farmacologia , Peróxido de Hidrogênio/farmacologia , Ácido Hipocloroso/farmacologia , Contagem de Colônia Microbiana , Concentração de Íons de Hidrogênio , Viabilidade Microbiana/efeitos dos fármacos , Projetos PilotoRESUMO
The effective disinfection of hospital surfaces is recognised as an important factor in preventing hospital-acquired infections. The purpose of this study was to quantify the disinfection rate of a novel gas plasma system on clinically relevant biofilms. Clinical isolates of Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus (MRSA) were grown as biofilms on glass surfaces and tested in a disinfection container remote from the plasma source. The strains used in this study were known to produce substantial quantities of biofilm and average log10 counts were 9.0 and 9.1 cfu/cm(2) for S. epidermidis and MRSA respectively. Counts were reduced by between 4 and 4.5 log10 after 1h of exposure for MRSA and S. epidermidis respectively. More prolonged treatment in the case of MRSA biofilms resulted in a 5.5 log10 reduction after 90 min. Biofilm samples were also placed in medical device packaging bags and similar rates of disinfection were observed.
Assuntos
Biofilmes/efeitos dos fármacos , Desinfetantes/farmacologia , Desinfecção/métodos , Microbiologia Ambiental , Gases/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Contagem de Colônia Microbiana , Infecção Hospitalar/prevenção & controle , Radicais Livres/farmacologia , Humanos , Viabilidade Microbiana/efeitos dos fármacosRESUMO
AIMS: The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples. METHODS: The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gas-liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation. CONCLUSIONS: The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several micro-organisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm.
Assuntos
Técnicas Bacteriológicas/instrumentação , Biofilmes/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Meios de Cultura , Fermentação , Oxigênio/metabolismoRESUMO
Fifty-five Staphylococcus epidermidis isolates, classified as contaminants or causing device-related meningitis, from external ventricular drain (EVD) and non-EVD cerebrospinal fluid specimens were characterized. Thirty-three of 42 (78.6%) meningitis isolates were PCR-positive for ica and aap, known determinants of polysaccharide- and protein-mediated biofilm production, whereas five of 13 (38.5%) contaminants were ica- and aap-negative; 71.4% of meningitis isolates and 84.6% of contaminants produced biofilm. ica+aap+ meningitis isolates produced more biofilm than ica+aap- isolates (p 0.0020). ica+aap- isolates did not produce more biofilm than ica-aap+ isolates (p 0.4368). Apparently, ica and aap are associated with biofilm production in S. epidermidis device-related meningitis isolates.
Assuntos
Biofilmes , Derivações do Líquido Cefalorraquidiano/efeitos adversos , Meningites Bacterianas/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/fisiologia , Aderência Bacteriana , Técnicas de Tipagem Bacteriana , DNA Bacteriano/isolamento & purificação , Contaminação de Equipamentos , Genes Bacterianos , Genótipo , Humanos , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/etiologia , Óperon , Reação em Cadeia da Polimerase , Infecções Relacionadas à Prótese/líquido cefalorraquidiano , Infecções Relacionadas à Prótese/etiologia , Infecções Estafilocócicas/líquido cefalorraquidiano , Infecções Estafilocócicas/etiologia , Staphylococcus epidermidis/classificaçãoRESUMO
The production of polysaccharide intercellular adhesin (PIA) is an essential process in foreign body infections mediated by Staphylococcus epidermidis. Transcriptional regulation of the icaADBC operon, the genes responsible for production of enzymes that synthesize PIA, is multi-factorial and involves at least SarA and sigmaB. Transcriptional and promoter fusion studies revealed that the decreased transcription of the icaADBC operon observed in a S. epidermidis 1457 sigB mutant is not mediated through a direct interaction of sigmaB-RNA polymerase at the icaADBC promoter region but instead through the upregulation of IcaR, a known repressor of icaADBC transcription. Transcriptional analysis of a 1457 sigB-icaR double mutant confirmed that the decreased icaADBC transcript in 1457 sigB is IcaR dependent. Furthermore, primer extension studies suggest that the icaR promoter appears to be sigmaA dependent, suggesting that sigmaB indirectly controls icaR transcription through an unknown pathway. In addition, it was confirmed that the loss of SarA results in the loss of icaADBC transcription and PIA production in S. epidermidis. It was further demonstrated, through the over-production of SarA in 1457 sigB, that the loss of sarP1 promoter activity in 1457 sigB has little or no effect on the loss of PIA production in this mutant. Finally, it was demonstrated that PIA production could be restored in both 1457 sigB and 1457 sarA by complementing these mutants with a full-length icaADBC operon controlled by a cadmium-inducible noncognate promoter. It is concluded that sigmaB and SarA operate independently of each other to regulate PIA production and biofilm development in S. epidermidis.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Fator sigma/fisiologia , Staphylococcus epidermidis/fisiologia , Transativadores/fisiologia , Óperon/fisiologia , Staphylococcus epidermidis/patogenicidadeAssuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Resistência a Meticilina , Meticilina/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Glucose/metabolismo , Humanos , Unidades de Terapia Intensiva , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Staphylococcus epidermidis and Staphylococcus aureus are common causes of biofilm-mediated prosthetic device-related infection. The polysaccharide adhesion mechanism encoded by the ica operon is currently the best understood mediator of biofilm development, and represents an important virulence determinant. More recently, the contributions of other virulence regulators, including the global regulators agr, sarA and sigmaB, to the biofilm phenotype have also been investigated. Nevertheless, little has changed at the bedside; the clinical and laboratory diagnosis of device-related infection can be difficult, and biofilm resistance frequently results in failure of therapy. This review assesses the way in which advances in the understanding of biofilm genetics may impact on the clinical management of device-related infection.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções Relacionadas à Prótese/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética , Staphylococcus/patogenicidade , Adesinas Bacterianas/fisiologia , Antibacterianos/uso terapêutico , Regulação Bacteriana da Expressão Gênica , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Óperon/genética , Polissacarídeos Bacterianos/biossíntese , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus/enzimologia , Ácidos Teicoicos/metabolismoAssuntos
Infecção Hospitalar/microbiologia , Variação Genética , Unidades de Terapia Intensiva , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/genética , Biofilmes/crescimento & desenvolvimento , Impressões Digitais de DNA , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Epidemiologia Molecular , Óperon , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologiaRESUMO
Production of biofilm in Staphylococcus epidermidis is mediated through enzymes produced by the four-gene operon ica and is subject to phenotypic variation. The purpose of these experiments was to investigate the regulation of ica and icaR transcription in phenotypic variants produced by multiple unrelated isolates of S. epidermidis. Ten isolates were chosen for the study, four of which contained IS256. IS256 mediates a reversible inactivation of ica in approximately 30 % of phenotypic variants. All ten strains produced at least two types of phenotypic variant (intermediate and smooth) in which biofilm formation was significantly impaired. Reversion studies indicated that all phenotypic variants were stable after overnight growth, but began to revert to other phenotypic forms after 5 days of incubation at 37 degrees C. ica transcriptional analysis was performed on phenotypic variants from three IS256-negative isolates; 1457, SE5 and 14765. This analysis demonstrated that ica transcription was significantly reduced in the majority of phenotypic variants, although two variants from SE5 and 1457 produced wild-type quantities of ica transcript. Analysis of seven additional phenotypic variants from SE5 revealed that ica expression was only reduced in three. Expression of icaR transcript was unaffected in all smooth phenotypic variants. Mutations within ica were identified in two SE5 variants with wild-type levels of ica transcription. It is concluded that mutation and transcriptional regulation of ica are the primary mechanisms that govern phenotypic variation of biofilm formation within IS256-negative S. epidermidis.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Mutação , Staphylococcus epidermidis/classificação , Proteínas de Bactérias/genética , Genótipo , Humanos , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/crescimento & desenvolvimento , Transcrição GênicaRESUMO
Staphylococcus epidermidis is a common cause of prosthetic device-related infection in the intensive care unit (ICU). The environmentally regulated ica operon encodes a polysaccharide adhesin which is a key virulence determinant in the development of S. epidermidis biofilms. To evaluate the capacity of ICU S. epidermidis isolates to form biofilm, we measured biofilm production by 18 isolates associated with device-related infection and 20 contaminating isolates that were not associated with clinically diagnosed infection. Biofilm assays were performed in brain-heart infusion (BHI) medium and in BHI supplemented with salt, ethanol or subinhibitory tetracycline, all of which have the potential to promote biofilm formation. Polymerase chain reaction (PCR) was used to screen for the presence of the ica genes. A significant proportion of S. epidermidis strains associated with device-related infections (89%) were found to contain the ica locus compared with 50% of contaminating isolates (P = 0.01). However only four of 26 (15.3%) of all ica-positive isolates were biofilm-positive when grown in BHI medium, indicating that no significant association existed between the presence of the ica locus and biofilm-forming capacity, under standard growth conditions. In contrast the number of ica-positive isolates that were biofilm-positive under stress-inducing growth conditions or in the presence of subinhibitory tetracycline increased significantly to 73% (P = 0.02). These findings suggest that the presence of the ica locus alone is not sufficient for biofilm formation and that regulation of biofilm formation under altered growth conditions, which may exist in the in vivo environment, also plays a possible role in the pathogenesis of biomaterial-related S. epidermidis infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecção Hospitalar/etiologia , Contaminação de Equipamentos/estatística & dados numéricos , Unidades de Terapia Intensiva , Infecções Estafilocócicas/etiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Staphylococcus epidermidis/genética , Infecção Hospitalar/prevenção & controle , DNA Bacteriano/análise , DNA Bacteriano/genética , Contaminação de Equipamentos/prevenção & controle , Hospitais de Ensino , Humanos , Controle de Infecções , Irlanda , Resistência a Meticilina/genética , Óperon/genética , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/genética , Estudos Prospectivos , Infecções Estafilocócicas/prevenção & controleRESUMO
The fim switch of Escherichia coli is responsible for phase-variable expression of type 1 fimbriae. Switching in the ON-to-OFF and OFF-to-ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON-to-OFF direction. The effects of local promoter activity and the H-NS nucleoid-associated protein on inversion of the switch were assessed. In contrast to FimB-mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H-NS status of the cell. Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+-like phenotype in the absence of the FimE protein. This biasing was H-NS dependent and was also contingent on transcription from within the switch. These data show that local transcription and a nucleoid-associated protein both contribute to the modulation of a site-specific recombination event on the bacterial chromosome.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Recombinação Genética , Proteínas Repressoras , Transcrição Gênica , Alelos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Integrases/genética , Integrases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
To further understand the proposed signal transduction pathway involving the presumed redox proteins RdxBH and cbb3 cytochrome oxidase in Rhodobacter sphaeroides 2.4.1, a series of mutants lacking components of both the Prr two-component activation system and the cbb3-type cytochrome oxidase or RdxBH were constructed. We report that under highly aerobic conditions, aberrant photosynthesis gene expression and spectral complex formation typical of cbb3- or RdxBH-deficient mutants were no longer observed when either prrA (encoding the response regulator of the Prr system) or prrB (encoding the presumed sensor kinase) was also deleted. These double-mutant strains are phenotypically identical to single-mutant PrrA and PrrB strains, suggesting that the signal(s) originating from the cbb3 terminal oxidase affects downstream puc and puf operon expression by acting exclusively through the Prr system. When the same double-mutant strains were examined under anaerobic dark dimethyl sulfoxide growth conditions, photosynthesis gene expression was obligatorily linked to the two-component activation system. However, photosynthesis gene expression under the same growth conditions was significantly higher in the cbb3 mutant strain when compared to that in the wild type. Similarly, under anaerobic photosynthetic conditions the high levels of the oxidized carotenoid, spheroidenone, which accumulate in cbb3-deficient mutants were nearly restored to normal in a PrrB- CcoP- double mutant. This observation, together with previously published results, suggests that the regulation of the CrtA-catalyzed reaction possesses both transcriptional and posttranscriptional regulatory effectors. We propose that the cbb3 cytochrome oxidase, which by definition can interact with external oxygen, serves to control the activity of the Prr two-component activation system under both aerobic and anaerobic conditions. Although independent from the cbb3 oxidase, the RdxBH proteins are also required for normal functioning of the Prr two-component activation system and are therefore believed to lie between the cbb3 oxidase in this oxygen-sensing, redox signaling pathway and the Prr activation system.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Fotossíntese/genética , Rhodobacter sphaeroides/genética , Transativadores , Aerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Teste de Complementação Genética , Histidina Quinase , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas/fisiologia , Mutagênese , Oxirredução , Oxirredutases/metabolismo , Oxigênio/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.