Avibactam and class C ß-lactamases: mechanism of inhibition, conservation of the binding pocket, and implications for resistance.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that inhibits a wide range of ß-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of ß-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the ß-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first ß-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used ß-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C ß-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C ß-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.

Development of EUCAST zone diameter breakpoints and quality control range for Staphylococcus aureus with ceftaroline 5-µg disk.

This ceftaroline MIC/disk comparison study for Staphylococcus aureus was performed for the purpose of establishing EUCAST zone diameter breakpoints. Ceftaroline susceptibility for a challenge set of 70 methicillin resistant- and 30 methicillin susceptible-S. aureus was determined by 5-µg disk diffusion and broth microdilution methods. Seventeen isolates were retested by disk and MIC, and the remaining 83 isolates were retested by MIC. Molecular testing was performed on 19 isolates with borderline susceptible ceftaroline MIC results to assess any differences in mecA and epidemiological correlation. An additional set of 101 consecutive clinical S. aureus isolates were tested using the 5-µg disk. S. aureus ATCC 29213 was tested by multiple sites and media for QC range determination. Replicate MIC results were within ±1 doubling dilution, with tendency for slightly lower repeat MICs, and there was minimal variation in replicate zone results. Based on susceptible breakpoints for MIC of ≤1 mcg/mL and for disk of >20 mm, there was 100 % categorical agreement for 30 MSSA and 92 % categorical agreement for 70 MRSA. There were no common MLST or PBP changes for strains with MICs of 1 and 2 mcg/mL. All ceftaroline disk results for the consecutively collected isolates were >20 mm. EUCAST selected the ceftaroline 5-µg disk breakpoint of Susceptible ≥20, Resistant <20 mm because it correlated best with the MIC breakpoint of Susceptible ≤1, Resistant >1 mg/L. A ceftaroline 5-µg disk QC range for S. aureus ATCC 29213 of 24-30 mm was also established by EUCAST.

Helicobacter pylori: today's treatment, and possible future treatment.

Helicobacter pylori induces chronic superficial gastritis which in some patients may lead to peptic ulcer disease, while a subset of infected individuals develop gastric cancer or gastric lymphoma. Consensus guidelines recommend that patients with a known H. pylori infection receive eradication treatment. Successful treatment requires that antibiotics be used in combination with acid suppressants or bismuth, and although the list of effective antibacterials is short, regimens such as amoxicillin and clarithromycin or metronidazole and clarithromycin with the proton pump inhibitor omeprazole have achieved eradication rates of approximately 90% in trials. However lower eradication rates are probably more common, and strains resistant to clarithromycin or metronidazole, or both, are of concern. Stable amoxycillin resistance has also been reported. Efforts are underway to discover and develop novel therapeutics, both H. pylori specific antibacterial drugs and a therapeutic vaccine. Impetus to these efforts has been provided by the availability of the genome sequences of two different H. pylori isolates. In the case of drug discovery, a genome-based strategy facilitates the expeditious selection of novel lethal targets not used by today's antibiotics, providing the opportunity to identify novel classes of antibacterials. Vaccine discovery and development has largely focused on a small number of antigens selected by conventional means. Recent reports that mucosal and serum antibody titers do not appear to be essential for protection against H. pylori in murine models suggest that that a wider range of H. pylori proteins than those previously considered may be able to induce protective immunity. Progress towards development of new H. pylori therapeutics is discussed.

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

Involvement of a plasmid in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.

Comparative genomics of Helicobacter pylori: analysis of the outer membrane protein families.

The two complete genomic sequences of Helicobacter pylori J99 and 26695 were used to compare the paralogous families (related genes within one genome, likely to have related function) of genes predicted to encode outer membrane proteins which were present in each strain. We identified five paralogous gene families ranging in size from 3 to 33 members; two of these families contained members specific for either H. pylori J99 or H. pylori 26695. Most orthologous protein pairs (equivalent genes between two genomes, same function) shared considerable identity between the two strains. The unusual set of outer membrane proteins and the specialized outer membrane may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. One subfamily of proteins, which contains both channel-forming and adhesin molecules, is extremely highly related at the sequence level and has likely arisen due to ancestral gene duplication. In addition, the largest paralogous family contained two essentially identical pairs of genes in both strains. The presence and genomic organization of these two pairs of duplicated genes were analyzed in a panel of independent H. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates.

Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro.

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 microg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 microg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic beta-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, and hefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

Analysis of the genetic diversity of Helicobacter pylori: the tale of two genomes.

Infection with Helicobacter pylori has been linked to numerous severe gastroduodenal diseases including peptic ulcer and gastric cancer. Several techniques have been used to measure the genetic heterogeneity of H. pylori at several different levels and to determine whether there is any correlation with severity of disease. The availability of two completed genome sequences from unrelated strains (J99 and 26,695) has allowed an analysis of the level of diversity from a large-scale yet detailed perspective. Although the two chromosomes are organized differently in a limited number of discrete regions, the genome size and gene order of these two "high-virulence" (cagA+ and vacA+) H. pylori isolates was found to be highly similar. The regions of organizational difference are associated with insertion sequences, DNA restriction/modification genes, repeat sequences, or a combination of the above. A significant level of variation at the nucleotide level is seen across the genome, providing an explanation for why the nucleotide-based typing techniques have such high discriminatory power among independent H. pylori isolates. This nucleotide variation together with the organizational rearrangements appears to have provided an over-estimation of the gene order diversity of H. pylori as assessed by pulse-field gel electrophoresis. Functional assignments are assigned to approximately only 60% of the gene products in each strain, with one-half of the remaining gene products of unknown function having homologues in other bacteria, while the remainder appear to be H. pylori-specific. Between 6% and 7% of the coding capacity of each strain are genes that are absent from the other strain, with almost one-half of these strain-specific genes located in a single hypervariable region called the plasticity zone. The majority of the strain-specific genes in each strain are also H. pylori-specific, with no homologues being identified in the public databases. Significantly, over one-half of the functionally assigned strain-specific genes in both H. pylori J99 and 26695 encode DNA restriction/modification enzymes. Analysis of the level of conservation between orthologues from the two strains indicates that the H. pylori specific genes have a lower level of conservation than those orthologues to which a putative function can be assigned. The plasticity zone represents one of several regions across each genome that is comprised of lower (G+C)% content DNA, some of which has been detected in self-replicating plasmids, suggesting that both horizontal transfer from other species and plasmid integration are responsible for the strain-specific diversity at this locus. These analyses have yielded results with important implications for understanding the genetic diversity of H. pylori and its associated diseases, and imply a need to reassess the respective roles of bacterial and host factors in H. pylori associated diseases.

Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes.

The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.

Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa.

Type-4 fimbriae are filamentous polar organelles which are found in a wide variety of pathogenic bacteria. Their biogenesis and function is proving to be extremely complex, involving the expression and coordinate regulation of a large number of genes. Type-4 fimbriae mediate attachment to host epithelial tissues and a form of surface translocation called twitching motility. In Pseudomonas aeruginosa they also appear to function as receptors for fimbrial-dependent bacteriophages. Analysis of mutants defective in fimbrial function has allowed the identification of many of the genes involved in the biogenesis of these organelles. Thus far over 30 genes have been characterized, which fall into two broad categories: those encoding regulatory networks that control the production and function of these fimbriae (and other virulence determinants such as alginate) in response to alterations in environmental conditions; and those encoding proteins involved in export and assembly of these organelles, many of which are similar to proteins involved in protein secretion and DNA uptake. These systems all appear to be closely related and to function in the assembly of surface-associated protein complexes that have been adapted to different biological functions.

The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa--a review.

Type-4 fimbriae (or pili) are filaments found at the poles of a wide range of bacterial pathogens, including Neisseria gonorrhoeae, Moraxella bovis, Dichelobacter nodosus and Pseudomonas aeruginosa. They are composed of a small subunit which is highly conserved among different species and appear to mediate adhesion and translocation across epithelial surfaces via a phenomenon termed "twitching motility'. These fimbriae are key host colonisation factors and important protective antigens. We have analysed the genetics and biosynthesis of type-4 fimbriae in P. aeruginosa, which is an opportunistic pathogen of compromised individuals, including those suffering cystic fibrosis, AIDS or burns. A library of P. aeruginosa transposon mutants was constructed which exhibited loss of twitching motility, as determined by altered colony morphology. Analysis of these mutants, and of similar collections by other groups, have revealed that there are at least 22 genes involved in type-4 fimbrial assembly and function. A large number (pilA, B, C, D, E, M, N, O, P, Q, T, U, V and Z) appear to be involved in the biogenesis of the fimbriae and to represent a subset of a supersystem involved in the assembly of surface-associated protein complexes. Homologs of at least some of these genes have subsequently been identified in other type-4 fimbriate bacteria. In P. aeruginosa, the system is also regulated via two signal transduction pathways-a classic sensor-regulator system (encoded by pilS, pilR and rpoN) which controls transcription of the fimbrial subunit, presumably in response to host cues, and a chemotactic system (encoded by pilG, H, I, J, K and L) which may be involved in the directional or rate control of twitching motility in response to local environmental variables.

Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa.

Many bacterial pathogens produce a class of surface structures called type 4 fimbriae. In Pseudomonas aeruginosa these fimbriae are responsible for adhesion and translocation across host epithelial surfaces. We have identified a novel gene involved in the complex process of type 4 fimbrial biogenesis. This gene, termed pilF, is located on SpeI fragment S at 30 min on the P. aeruginosa genomic map, which is the sixth region on the chromosome shown to contain a fimbrial-associated gene. The PilF protein has a predicted M(r) of 22402, and together with a highly homologous upstream ORF shares a chromosomal arrangement similar to that found in Haemophilus influenzae. A pilF mutant is blocked in the export/assembly of the fimbrial subunit PilA, and accumulates this protein in the membrane fraction. Complementation studies indicate that the cloned pilF gene is able to restore the expression of surface fimbriae, twitching motility and susceptibility to fimbrial-specific bacteriophage.

Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are surface organelles produced by a wide range of bacterial pathogens. In Pseudomonas aeruginosa they are associated with a form of surface translocation known as twitching motility and have also been implicated as the receptor for a number of fimbrial-specific bacteriophages. The infrastructural machinery required for type 4 fimbrial biogenesis appears to be conserved as heterologous subunits from other species can be expressed in P. aeruginosa. All of these studies have, until now, been performed in non-functional Pseudomonas host strains which lack twitching motility. We have constructed isogenic mutants of two commonly studied wild-type P. aeruginosa strains, PAK and PAO1, by replacing the entire pilA gene which encodes the fimbrial subunit. Fimbrial expression and twitching motility were restored by complementation in trans with either the homologous or heterologous subunits from these strains, as well as that from another type 4 fimbriate species, Dichelobacter nodosus. The expression of different subunits allowed us to investigate the precise role that the individual subunit proteins contribute to bacteriophage infection by several fimbrial-specific bacteriophages. Sensitivity to bacteriophages B3cts and D3112cts was restored by the expression of any fimbrial subunit in both PAO1 and PAK cells, indicating that infection by these bacteriophages is fimbrial dependent but not fimbrial specific. In contrast, while sensitivity to the PAK-specific bacteriophage PO4 was restored by the expression of any fimbrial subunit in PAK cells, this did not occur in PAO1 cells except when expressing the PAK subunit. In all cases, the presence of fimbriae was absolutely required to allow a productive bacteriophage infection to occur.

Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue.

Type 4 fimbriae of Pseudomonas aeruginosa are surface filaments involved in host colonization. They mediate both attachment to host epithelial cells and flagelia-independent twitching motility. Four additional genes, pilW, pilX, pilY1 and pilY2, are located on Spel fragment E in the 5 kb intergenic region between the previously characterized genes pilV and pilE, which encode prepilin-like proteins involved in type 4 fimbrial biogenesis. The phenotypes of a transposon insertion and other mutations constructed by allelic exchange show that these genes are involved in the assembly of type 4 fimbriae. The PilW and PilX proteins are membrane located, possess the hydrophobic N-terminus characteristic of prepilin-like proteins, and appear to belong to the GspJ and GspK group of proteins that are required for protein secretion in a wide range of Gram-negative bacteria. These findings increase the similarities between the fimbrial biogenesis and the Gsp-based protein-secretion supersystems. PilY1 is a large protein with C-terminal homology to the PilC2 protein of Neisseria gonorrhoeae, thought to be a fimbrial tip-associated adhesin, and which, like PilY1, is involved in fimbrial assembly. PilY1 appears to be located in both the membrane and the external fimbrial fractions. PilY2 is a small protein that appears to play a subtle role in fimbrial biogenesis and represents a new class of protein.

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces. In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage. Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P. aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA*. The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli. Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes. However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis. The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.

Construction of improved vectors for protein production in Pseudomonas aeruginosa.

We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.

Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. We have used transposon mutagenesis to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. A subset of these mutants is resistant to fimbria-specific phage. One of these mutants (R270) was found to contain a transposon insertion in a new gene, termed pilZ, which is located on chromosomal SpeI fragment I at about 40 min on the P. aeruginosa map, a position remote from other loci involved in fimbrial biogenesis. pilZ appears to be linked to and possibly forms an operon with a gene, holB*, which is homologous to the gene encoding the delta' subunit of Escherichia coli DNA polymerase III. The product of the pilZ gene is a protein of 118 amino acids (predicted molecular weight, 12,895) which probably has a cytoplasmic location. PilZ appears to be a new class of protein which has not hitherto been represented in the sequence databases, and its function is unknown. Complementation studies indicate that pilZ is able to restore the expression of fimbriae on the surface of P. aeruginosa, as well as twitching motility and sensitivity to fimbria-specific phage when provided in trans to the R270 mutant.