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.     

Verminephrobacter eiseniae type IV pili and flagella are required to colonize earthworm nephridia

The bacterial symbiont Verminephrobacter eiseniae colonizes nephridia, the excretory organs, of the lumbricid earthworm Eisenia fetida. E. fetida transfers V. eisenia into the egg capsule albumin during capsule formation and V. eiseniae cells migrate into the earthworm nephridia during embryogenesis, where they bind and persist. In order to characterize the mechanistic basis of selective tissue colonization, methods for site-directed mutagenesis and colonization competence were developed and used to evaluate the consequences of individual gene disruptions. Using these newly developed tools, two distinct modes of bacterial motility were shown to be required for V. eiseniae colonization of nascent earthworm nephridia. Flagella and type IV pili mutants lacked motility in culture and were not able to colonize embryonic earthworms, indicating that both twitching and flagellar motility are required for entrance into the nephridia.     

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.     

Swapping genes to survive - a new role for archaeal type IV pili

Type IV pili are filamentous structures that are found on the surface of many bacterial and archaeal cells, they are involved in cell motility and surface adhesion. In the crenarchaeon Sulfolobus solfataricus, type IV pili formation is strongly induced by UV irradiation and leads to cellular aggregation. The study by Ajon et al. (2011) published in this issue of Molecular Microbiology shows that UV-induced cellular aggregation greatly stimulates the exchange of chromosomal markers among irradiated cells, and that this strategy helps with cell survival. Sulfolobus knockout strains that are incapable of forming pili proved to be deficient in aggregation, and also showed decreased cellular survival after UV irradiation. The UV-induced pili of three different Sulfolobus species had distinct morphologies, and correspondingly these three species were able to aggregate only with their own kind. This work has defined a new role for type IV pili in both the transfer of genes within species and the recovery from UV-induced DNA damage.     

Denitrifying genes in bacterial and Archaeal genomes

Denitrification, the reduction of nitrate or nitrite to nitrous oxide or dinitrogen, is the major mechanism by which fixed nitrogen returns to the atmosphere from soil and water. Although the denitrifying ability has been found in microorganisms belonging to numerous groups of bacteria and Archaea, the genes encoding the denitrifying reductases have been studied in only few species. Recent investigations have led to the identification of new classes of denitrifying reductases, indicating a more complex genetic basis of this process than previously recognized. The increasing number of genome sequencing projects has opened a new way to study the genetics of the denitrifying process in bacteria and Archaea. In this review, we summarized the current knowledge on denitrifying genes and compared their genetic organizations by using new sequences resulting from the analysis of finished and unfinished microbial genomes with a special attention paid to the clustering of genes encoding different classes of reductases. In addition, some evolutionary relationships between the structural genes are presented.     

Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity

A large number of secretory proteins in the thermoacidophile Sulfolobus solfataricus are synthesized as a precursor with an unusual leader peptide that resembles bacterial type IV prepilin signal sequences. This set of proteins includes the flagellin subunit but also various solute binding proteins. Here we describe the identification of the S. solfataricus homolog of bacterial type IV prepilin peptidases, termed PibD. PibD is an integral membrane protein that is phylogenetically related to the bacterial enzymes. When heterologously expressed in Escherichia coli, PibD is capable of processing both the flagellin and glucose-binding protein (GlcS) precursors. Site-directed mutagenesis of the GlcS signal peptide shows that the substrate specificity of PibD is consistent with the variations found in proteins with type IV prepilin-like signal sequences of S. solfataricus. We conclude that PibD is responsible for the processing of these secretory proteins in S. solfataricus.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.     

Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient-dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One-half of these mutations was found to perturb normal flagellar function. Using defined fli , flh , mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose-specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.     

Mechanical removal of F pili, type I pili, and flagella from Hfr and RTF donor cells and the kinetics of their reappearance

The effect of mechanical agitation (blending) on the removal of F pili, type I pili, and flagella from Hfr (high-frequency recombinant) and resistance transfer factor (RTF) fi(+)Escherichia coli cells was studied by electron microscopy. The reduction in number and length of appendages was measured as a function of blendor speed under standard conditions of temperature, medium, cell density, and blendor configuration. F pili and flagella were removed within the same narrow range of blendor speeds. Type I pili were removed within a higher and broader range of speeds. The speed which reduced the average length of type I pili to 50% was 3.5 times the speed which reduced the average length of F pili to 50%. None of the speeds employed inhibited cell growth, viability, or the ability to produce cell appendages. The kinetics of reappearance of F pili and type I pili after removal by blending were also different. F pili grew out very rapidly, reaching 50% of their full length in 30 sec and their full length in 4 to 5 min. The number of attached F pili per cell also increased rapidly, reaching a constant value in 4 to 5 min. After 5 min, F pilus lengths were distributed around a modal value of about 1.2 mum, and the numbers of F pili per cell were distributed according to a Poisson distribution, with an average of 1.0 per cell. These reappearance kinetics, length distributions, and number distributions are consistent with a model of F-pilus outgrowth in which new F pili appear at random locations on the cell surface at an average rate of about once every 4 min, grow to their characteristic length in about 4 min, and then separate from the cell. F pili which had separated could absorb to the cells, leading to the presence of two classes of F pili on cells: those in the process of natural out-growth and those attached by absorption. Type I pili increased in length much more slowly than did F pili, although the fraction of cells having visible type I pili increased very rapidly after blending because of the large number of type I pili per cell. The fraction of flagellated cells increased even more slowly, reaching only 30% of the unblended fraction in 30 min. The application of blending spectra and reappearance kinetics to the identification of cell functions with surface structures is discussed.     

Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ

Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.     

Cloning, expression, and functional analysis of molecular motor pilT and pilU genes of type IV pili in Acidithiobacillus ferrooxidans

PilT is a hexameric ATPase required for type IV pili (Tfp) retraction in gram-negative bacterium. Retraction of Tfp mediates intimate attachment and motility on inorganic solid surfaces. We investigated the cloning and expression of pilT and pilU genes of Acidithiobacillus ferrooxidans strains ATCC 23270, and the results indicate that PilT and PilU contain the canonical conserved AIRNLIRE and GMQTXXXXLXXL motifs that are the characteristic motifs of the PilT protein family; PilT and PilU also contain the canonical nucleotide-binding motifs, named with Walker A box (GxxGxGKT/S) and Walker B box (hhhhDE), respectively. The pilT and pilU genes were expressed to produce 37.1- and 42.0-kDa proteins, respectively, and co-transcribed induced by 10 % mineral powder. However, ATPase activity of PilT was distinctly higher than those of PilU. These results indicated that the PilT protein was the real molecular motor of Tfp, while PilU could play a key role in the assembly, modification, and twitching motility of Tfp in A. ferrooxidans. However, PilT and PilU were nonetheless interrelated in the forming and function of the molecular motor of Tfp.     

Ⅳ型菌毛可视化方法及其在菌毛功能研究中的应用

Ⅳ型菌毛(type Ⅳ pili, TFP)作为细菌表面的重要蛋白结构,是细菌的感知器官及运动器官,在细菌生理学、细胞黏附、宿主细胞入侵、DNA摄取、蛋白质分泌、生物被膜形成、细胞运动和电子传递等方面发挥着多种作用。近年来,随着研究方法的深入和技术设备的发展,尤其是随着多种菌毛可视化工具的开发,越来越多的研究揭示了它在生命活动中的各种功能,大大加快了微生物单细胞领域的研究步伐。本文重点讨论了TFP可视化方法及在菌毛功能研究中的应用,为更好地研究和利用TFP功能提供更多的思路,为其未来在生物学、医学以及生态学中的应用提供一定的理论基础。     

Relevance of posttranslational modifications for the arthritogenicity of type II collagen

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCII(pic)) and a baculovirus expression system (rCII(bac)). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCII(bac) was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCII(pic) was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCII(pic) induced a lower incidence of arthritis than tissue-derived CII, whereas rCII(bac) induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCII(pic) compared with mice immunized with tissue-derived CII, whereas that of rCII(bac) was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.     

Proteomics: posttranslational modifications, immune responses and current analytical tools

The publication of the human genome sequence enables most of the still unknown protein sequences to be added to the current databases. A sequence alone does not, however, give information about the possible expression level of the corresponding protein, neither does it inform about the possible posttranslational modifications, like phosphorylation, glycosylation or changes in individual amino acids. Thus, the human proteome project, a large scale analysis of the functions of gene products, will have an enormous impact on our understanding of the biochemistry of proteins, processes and pathways they are involved in. The diversity in proteins is considerably expanded by various posttranslational modifications. These also pose problems to the investigators, but their careful analysis often pays back because they can reveal important properties in proteins or peptides--like an increased antigenicity leading to (auto)immune responses or an active form of a signaling protein. Immune tolerance usually exists towards self-proteins, but in specific cases it may be broken by posttranslational modifications in the proteins. Novel mass spectrometric, affinity and display techniques offer valuable tools for the large-scale analysis of proteomes. In the present paper we discuss their use for the detection of posttranslational modifications, functional interactions and possible disease-associated abnormalities in proteins.     

Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria

Background

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.

Results

To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes.

Conclusions

We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins.     

Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases

The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.     

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.