The Multifaceted Proteins MvaT and MvaU,Members of the H-NS Family,Control Arginine Metabolism,Pyocyanin Synthesis,and Prophage Activation in Pseudomonas aeruginosa PAO1

The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the aotJQMOP-argR operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the aotJ regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in mvaT or mvaU, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the β-galactosidase activities from aotJ::lacZ fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of aotJ expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of cupA expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on cupA. Moreover, prophage Pf4 integrated into the chromosome of Pseudomonas aeruginosa PAO1 was activated in an mvaT mvaU double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the mvaT mvaU double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in P. aeruginosa.The MvaT and MvaU proteins of Pseudomonas aeruginosa belong to the H-NS family of small DNA-binding proteins. MvaT was initially identified in P. mevalonii as a positive regulator for mevalonate catabolism (25). Subsequently, MvaT homologues have been identified in other pseudomonads based on structural and functional similarities; five homologues have been identified in P. putida, three homologues have been identified in P. fluorescens, four homologues have been identified in P. syringae, and two homologues have been identified in P. aeruginosa. In P. putida, the TurA protein represses the Pu promoter of the TOL plasmid in a temperature-dependent manner (24). In P. fluorescens, the MvaT and MvaV proteins regulate the expression of two biocontrol exoproducts, 2,4-diacetyl phloroglucinol and pyoluteorin (1). In P. aeruginosa, MvaT is involved in quorum-sensing responses and biofilm formation. Inactivation of mvaT resulted in increased production of PA-IL lectin and the toxic exoproduct pyocyanin, reduced biofilm formation, increased drug resistance, and reduced swarming motility (5, 33). In addition, the MvaT protein is involved in the phase-variable expression of the fimbrial cup genes involved in biofilm formation. Moreover, DNA microarray analysis has shown that more than 150 genes are influenced by the MvaT protein (27).The MvaU protein shows high levels of similarity to MvaT and can perform some of the MvaT regulatory functions (28). Like other H-NS-related proteins, MvaT and MvaU possess two distinct domains: the N terminus for oligomerization and the C terminus for DNA-binding activity. The MvaT and MvaU proteins can interact through their N-terminal regions and form hetero- and homodimers (28). In general, mvaT mutation seems to have a much more profound effect than mvaU mutation. In a study using chromatin immunoprecipitation coupled with DNA microarrays, the potential MvaT and MvaU binding sites on the genome of P. aeruginosa were identified (3). In the same report, it was concluded that loss of both MvaT and MvaU from the cell cannot be tolerated.In this study, we identified MvaT and MvaU as components of a nucleoprotein complex that controls the aotJQMOP-argR operon for arginine uptake and regulation (21, 22). Using mvaT and mvaU single- and double-mutant constructs, we also demonstrated the consequences of MvaT and MvaU depletion for prophage activation, pyocyanin synthesis, and fimbrial cupA gene expression.     

Single-Molecule Study on Histone-Like Nucleoid-Structuring Protein (H-NS) Paralogue in Pseudomonas aeruginosa: MvaU Bears DNA Organization Mode Similarities to MvaT

Pseudomonas aeruginosa contains two distinct members of H-NS family of nucleoid-structuring proteins: MvaT and MvaU. Together, these proteins bind to the same regions of the chromosome and function coordinately in the regulation of hundreds of genes. Due to their structural similarity, they can associate to form heteromeric complexes. These findings left us wondering whether they bear similar DNA binding properties that underlie their gene-silencing functions. Using single-molecule stretching and imaging experiments, we found striking similarities in the DNA organization modes of MvaU compared to the previously studied MvaT. MvaU can form protective nucleoprotein filaments that are insensitive to environmental factors, consistent with its role as a repressor of gene expression. Similar to MvaT, MvaU filament can mediate DNA bridging while excessive MvaU can cause DNA aggregation. The almost identical DNA organization modes of MvaU and MvaT explain their functional redundancy, and raise an interesting question regarding the evolutionary benefits of having multiple H-NS paralogues in the Pseudomonas genus.     

Excisionase in Pf filamentous prophage controls lysis‐lysogeny decision‐making in Pseudomonas aeruginosa

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis‐lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5′‐untranslated regions overlap. XisF4 and Pf4r not only auto‐activate their own expression but also repress each other. Furthermore, two H‐NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella

Pseudomonas aeruginosa exhibits swarming motility on 0.5 to 1% agar plates in the presence of specific carbon and nitrogen sources. We have found that PAO1 double mutants expressing neither flagella nor type IV pili (fliC pilA) display sliding motility under the same conditions. Sliding motility was inhibited when type IV pilus expression was restored; like swarming motility, it also decreased in the absence of rhamnolipid surfactant production. Transposon insertions in gacA and gacS increased sliding motility and restored tendril formation to spreading colonies, while transposon insertions in retS abolished motility. These changes in motility were not accompanied by detectable changes in rhamnolipid surfactant production or by the appearance of bacterial surface structures that might power sliding motility. We propose that P. aeruginosa requires flagella during swarming to overcome adhesive interactions mediated by type IV pili. The apparent dependence of sliding motility on environmental cues and regulatory pathways that also affect swarming motility suggests that both forms of motility are influenced by similar cohesive factors that restrict translocation, as well as by dispersive factors that facilitate spreading. Studies of sliding motility may be particularly well-suited for identifying factors other than pili and flagella that affect community behaviors of P. aeruginosa.     

Higher order oligomerization is required for H-NS family member MvaT to form gene-silencing nucleoprotein filament

MvaT from Pseudomonas aeruginosa is a member of the histone-like nucleoid structuring protein (H-NS) family of nucleoid-associated proteins widely spread among Gram-negative bacteria that functions to repress the expression of many genes. Recently, it was reported that H-NS from Escherichia coli can form rigid nucleoproteins filaments on DNA, which are important for their gene-silencing function. This raises a question whether the gene-silencing function of MvaT, which has only ∼18% sequence similarity to H-NS, is also based on the formation of nucleoprotein filaments. Here, using magnetic tweezers and atomic force microscopy imaging, we demonstrate that MvaT binds to DNA through cooperative polymerization to form a nucleoprotein filament that can further organize DNA into hairpins or higher-order compact structures. Furthermore, we studied DNA binding by MvaT mutants that fail to repress gene expression in P. aeruginosa because they are specifically defective for higher-order oligomer formation. We found that, although the mutants can organize DNA into compact structures, they fail to form rigid nucleoprotein filaments. Our findings suggest that higher-order oligomerization of MvaT is required for the formation of rigid nucleoprotein filaments that silence at least some target genes in P. aeruginosa. Further, our findings suggest that formation of nucleoprotein filaments provide a general structural basis for the gene-silencing H-NS family members.     

The adsorption of Pseudomonas aeruginosa bacteriophage φKMV is dependent on expression regulation of type IV pili genes

Pseudomonas aeruginosa bacteriophage φKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host ( P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore φKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage φKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1Δ pilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage φKMV infectivity.     

Novel anti-repression mechanism of H-NS proteins by a phage protein

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT–DNA complex. Structural investigations suggest that gp4 acts as an ‘electrostatic zipper’ between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their ‘half-open’ conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.     

Biofilm formation by Pseudomonas aeruginosa wild type,flagella and type IV pili mutants

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.     

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa -induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt⁻). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.     

Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.     

Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.     

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical. Of five resistant mutants examined, all were defective in the production of pili and did not adsorb either phage. P. aeruginosa PAK strains altered in pilus expression, such as hyperpiliated or nonpiliated mutants, adsorbed the phage but were not productively infected, implying that an additional host function was required for infection. The cell-associated lipopolysaccharide was not required for D3112 or B3 infection, since mutants deficient in O side-chain and core biosynthesis were still capable of adsorption and productive infection. This is in contrast to Escherichia coli mutator phages Mu and D108, which are dependent on lipopolysaccharide for adsorption. The P. aeruginosa phages adsorbed only to cells grown on solid media or in liquid media supplemented with agents that increase the macroviscosity, such as polyvinylpyrrolidone. Adsorption time course studies of D3112 and B3 using cells grown in solid media revealed similar but not identical adsorption patterns. These studies suggested that expression of the D3112 and B3 cell receptor is induced by growth on solid media.     

铜绿假单胞菌蹭行运动相关基因的研究

应用Mu转座重组技术研究铜绿假单胞菌 (Pseudomonasaeruginosa)蹭行运动 (Twitchingmotility)的相关基因。通过转座突变、表型筛选 ,得到 8个Twitchingmotility缺陷或减弱的突变子。经过基因克隆、核苷酸测序研究 ,鉴定转座子插入到基因组中的位置。结果表明 ,在其中 4个突变子中 ,转座子分别插入到与IV型菌毛生物合成和功能相关的 3个已知基因中 (其中有两个突变子转座子插入到同一基因的不同位置 ) ,它们是pilV ,pilQ ,algR。另外 4个突变子中 ,有 3个是转座子分别插入到基因pilL基因的前端 ,中部和后端 ,均引起Twitchingmotility功能缺失。另一个突变子中 ,转座子插入到基因PA1 82 1中 ,引起Twitchingmotility功能减弱。PilL和PA1 82 1的编码产物均属于 3 类蛋白质 ,它们的功能是根据其保守的氨基酸基序或基因序列与已知功能基因的相似性推测得出的。但缺乏详细的试验证据。研究结果为pilL控制Twichingmotility提供了有力的证据。并证实基因PA1 82 1与Twitchingmotility有关。将Mu转座重组技术应用到假单胞菌的研究中 ,国内外均未见报道。由于该技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点。保证了表型的改变与转座子插入位点的基因突变的一一对应关系。为进一步研究铜绿假     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.     

Upstream migration of Xylella fastidiosa via pilus-driven twitching motility

Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.