Mutational analysis of the TonB1 energy coupler of Pseudomonas aeruginosa

Siderophore-mediated iron transport in Pseudomonas aeruginosa is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the tonB1 gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both P. aeruginosa and Escherichia coli. Appending it to the N terminus of the nonfunctional (in P. aeruginosa) Escherichia coli TonB protein (TonB(Ec)) rendered TonB(Ec) weakly active in P. aeruginosa and did not compromise the activity of this protein in E. coli. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in P. aeruginosa and E. coli. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB(Ec) function in E. coli, was shown here to be essential for TonB1 activity in E. coli but not in P. aeruginosa. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both P. aeruginosa and E. coli, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in E. coli but not in P. aeruginosa. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in P. aeruginosa. Several TonB1-TonB(Ec) chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in P. aeruginosa, although chimeras possessing an E. coli C terminus were active in E. coli.     

A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa

This study revealed that a Pseudomonas aeruginosa tonB1 mutant was unable to produce a mature biofilm and showed reduced swarming and twitching motilities compared with the parent strain. The tonB1 mutant was also found to produce significantly lower cell-free and cell-associated levels of the quorum sensing (QS) signal molecule 3-oxo-C12-AHL. Altered biofilm and motility phenotypes were restored to wildtype with the addition of exogenous N-acylhomoserine lactones. These functions were independent of the role of TonB1 in iron uptake. This is the first time that a link has been established between TonB1 activity and QS.     

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.     

The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

Summary: Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Role of polysaccharides in Pseudomonas aeruginosa biofilm development

During the past decade, there has been a renewed interest in using Pseudomonas aeruginosa as a model system for biofilm development and pathogenesis. Since the biofilm matrix represents a crucial interface between the bacterium and the host or its environment, considerable effort has been expended to acquire a more complete understanding of the matrix composition. Here, we focus on recent developments regarding the roles of alginate, Psl, and Pel polysaccharides in the biofilm matrix.     

Acquisition and Role of Molybdate in Pseudomonas aeruginosa

In microaerophilic or anaerobic environments, Pseudomonas aeruginosa utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO₄²⁻). Here, we show that molybdate acquisition in P. aeruginosa occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the modA gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of P. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth of P. aeruginosa and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.     

左氧氟沙星浸涂导管对铜绿假单胞菌生物膜的影响

目的评估左氧氟沙星（levofloxacin,LFX）浸涂导管抑制铜绿假单胞菌粘附、定植,防止生物膜形成的能力。方法体外部分：制备LFX浸涂导管。LFX浸涂导管、PVC导管分别浸没在5 mL 50%LB培养液中（含PAO1 108CFU/mL）,37℃孵育6、12、24和48 h,在各时间点,予导管表面和导管培养液进行细菌计数。体内部分：小鼠皮下植入LFX浸涂导管或PVC导管,沿着导管注射PAO1菌液50μL（107CFU）。第1、5天,对植入导管及导管周围组织进行细菌计数及扫描电镜（SEM）观察。结果 （1）LFX浸涂导管显示药物的快速释放。（2）在各孵育时间点,LFX浸涂导管及导管培养液的细菌数较PVC导管均明显减少（P〈0.05）。（3）小鼠感染第1、5天,LFX浸涂植入导管表面没有或很少细菌;LFX浸涂导管较PVC导管能明显减少植入导管周围组织的细菌量（P〈0.05）。（4）SEM观察：感染第1、5天,LFX浸涂导管表面散在单个细菌或者没有细菌;而第1天,PVC导管表面大量细菌分散存在。第5天,导管表面＂珊瑚状＂生物膜形成。结论 LFX浸涂导管能抑制铜绿假单胞菌粘附、定植,防止生物膜形成,从而有效降低导管生物膜相关感染的发生。     

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

Abstract A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.     

Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida

A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5 g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4.7H2O and 0.025% CaCl2.2H2O, with shaking at 200 rpm for an incubation period of 100 h at 30 degrees C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes.     

The Role of ExoS in Dissemination of Pseudomonas aeruginosa during Pneumonia

Hospital-acquired pneumonia is associated with high rates of morbidity and mortality, and dissemination to the bloodstream is a recognized risk factor for particularly poor outcomes. Yet the mechanism by which bacteria in the lungs gain access to the bloodstream remains poorly understood. In this study, we used a mouse model of Pseudomonas aeruginosa pneumonia to examine this mechanism. P. aeruginosa uses a type III secretion system to deliver effector proteins such as ExoS directly into the cytosol of eukaryotic cells. ExoS, a bi-functional GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT), inhibits phagocytosis during pneumonia but has also been linked to a higher incidence of dissemination to the bloodstream. We used a novel imaging methodology to identify ExoS intoxicated cells during pneumonia and found that ExoS is injected into not only leukocytes but also epithelial cells. Phagocytic cells, primarily neutrophils, were targeted for injection with ExoS early during infection, but type I pneumocytes became increasingly injected at later time points. Interestingly, injection of these pneumocytes did not occur randomly but rather in discrete regions, which we designate ““fields of cell injection” (FOCI). These FOCI increased in size as the infection progressed and contained dead type I pneumocytes. Both of these phenotypes were attenuated in infections caused by bacteria secreting ADPRT-deficient ExoS, indicating that FOCI growth and type I pneumocyte death were dependent on the ADPRT activity of ExoS. During the course of infection, increased FOCI size was associated with enhanced disruption of the pulmonary-vascular barrier and increased bacterial dissemination into the blood, both of which were also dependent on the ADPRT activity of ExoS. We conclude that the ADPRT activity of ExoS acts upon type I pneumocytes to disrupt the pulmonary-vascular barrier during P. aeruginosa pneumonia, leading to bacterial dissemination.