Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. (1/116)

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.  (+info)

Characterization of Pseudomonas aeruginosa bacteriophage UNL-1, a bacterial virus with a novel UV-A-inducible DNA damage reactivation phenotype. (2/116)

UNL-1, a lytic virus of Pseudomonas aeruginosa, was observed to express a novel inducible DNA damage reactivation activity in UV-A-irradiated P. aeruginosa host cells. The expression of bacteriophage reactivation was quantified in hosts exposed to either UV-C or UV-A radiation. While reactivation of UV-C-damaged UNL-1 was not inducible in UV-C-irradiated host cells, an approximately 13-fold induction was observed in UV-A-irradiated host cells. When host cells were exposed to sunlight, reactivation of damaged UNL-1 virus increased eightfold. The UV-A induction of UNL-1 DNA damage reactivation was supported in hosts lacking recA gene function. This report is the first description of a recA-independent, UV-inducible virus DNA damage repair system. Our findings suggest that a combination of both host and virus DNA repair processes contribute to the persistence and sustained replication of some bacterial viruses in aquatic environments.  (+info)

Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. (3/116)

Bacterial integral inner membrane proteins are either translocated across the lipid bilayer using an energy-driven enzyme, such as the Sec translocase, or they might interact directly with the membrane due to hydrophobic forces. We report that the single-spanning Pf3 coat protein is spontaneously inserted into the membrane of Escherichia coli and requires the electrical component of the membrane potential (DeltaPsi) to translocate its N-terminal region. This results in a final N(out)C(in) orientation of the protein in the cytoplasmic membrane, due the potential-driven translocation of the aspartyl residue at position 18 in the hydrophilic N-terminal tail. Uncharged protein tails are only translocated when the hydrophobic transmembrane region of the protein has been extended. An extended transmembrane anchor allows membrane insertion in the absence of an electrochemical membrane potential, but also causes the loss of a strict determination of the topology.  (+info)

Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control. (4/116)

Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida.  (+info)

Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112. (5/116)

Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.  (+info)

Characterization of phi8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to Phi6. (6/116)

The three double-stranded RNA genomic segments of bacteriophage Phi8 were copied as cDNA, and their nucleotide sequences were determined. Although the organization of the genome is similar to that of Phi6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence. Several features of the viral proteins differ markedly from those of Phi6. Although both phages are covered by a lipid-containing membrane, the protein compositions are very different. The most striking difference is that protein P8, which constitutes a shell around the procapsid in Phi6, is part of the membrane in Phi8. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus. The host range of Phi8 includes rough strains of Salmonella typhimurium and of pseudomonads  (+info)

The three-dimensional structure of bacteriophage PP7 from Pseudomonas aeruginosa at 3.7-A resolution. (7/116)

The three-dimensional structure of phage PP7 from Pseudomonas aeruginosa has been determined to 3.7-A resolution. A comparison with distantly related small RNA phages showed that the biggest differences were found in the FG loops, forming the contacts around the fivefold and threefold axes. In contrast to the situation in other phages, the FG loops of phage PP7 are very similar in all three subunits. This supports the hypothesis that no switches are needed for the assembly control in these viruses. Some of the most conserved residues lie within the region involved in RNA binding in the related phages MS2 and seem to have the same function.  (+info)

Sequence of the genome of the temperate, serotype-converting, Pseudomonas aeruginosa bacteriophage D3. (8/116)

Temperate bacteriophage D3, a member of the virus family Siphoviridae, is responsible for serotype conversion in its host, Pseudomonas aeruginosa. The complete sequence of the double-stranded DNA genome has been determined. The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs. The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome. D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG. The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin. The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97. Among the ORFs was discovered the gene encoding the fucosamine O-acetylase, which is in part responsible for the serotype conversion events.  (+info)