Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa.

Most strains of Pseudomonas aeruginosa can express two chemically and immunologically distinct types of lipopolysaccharide (LPS), an antigenically conserved form called A band and the serotype-specific form called B band. To study the molecular controls regulating expression of the A-band LPS antigen, we have cloned the genes involved with A-band LPS expression. Strain AK1401, a phage-resistant mutant of PAO1 which was shown previously to produce only A-band LPS and not the O-antigen-containing B-band LPS, was mutagenized by using ethyl methanesulfonate to generate an A-band-deficient mutant called rd7513. A cosmid clone bank of P. aeruginosa PAO1 whole genomic DNA was constructed in Escherichia coli. The gene bank was mobilized en masse into strain rd7513, and detection of complementation of synthesis of A band was done by screening transconjugants in a colony immunoblot assay with the A-band-specific monoclonal antibody N1F10. One recombinant cosmid, pFV3, complemented synthesis of A-band polysaccharide in rd7513. Silver-stained polyacrylamide gel and Western immunoblot analyses of LPS extracted from the transconjugant rd7513(pFV3) showed that the A band produced had a higher molecular weight than the A band of AK1401. Analysis of the plasmid pFV3 showed that it contained a chromosomal insert of 27 kb. Two subclones of pFV3, namely, pFV35 and pFV36, containing chromosomal inserts of 5.3 and 4.2 kb, respectively, also complemented A-band expression in rd7513. The LPS banding profile of rd7513(pFV35) was similar to that of AK1401, while the LPS profile of rd7513(pFV36) more closely resembled that of rd7513(pFV3). pFV3 complemented A-band expression in five of the six P. aeruginosa O serotypes which lack A band as well as in rough strain AK44 but failed to complement A-band expression in core mutants AK1012 and AK1282, suggesting that pFV3 contains genes for A-band expression and that synthesis of a complete core region in isogenic mutant strains is required for A-band synthesis.     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)

Previous work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb Xhol-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Synthesis of the A-band polysaccharide sugar D-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa

Pseudomonas aeruginosa is capable of producing various cell-surface polysaccharides including alginate, A-band and B-band lipopolysaccharides (LPS). The D -mannuronic acid residues of alginate and the D -rhamnose (D -Rha) residues of A-band polysaccharide are both derived from the common sugar nucleotide precursor GDP-D -mannose (D -Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP-D -Rha, have been localized to the 5′ end of the A-band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP-mannose pyrophosphorylases (GMPs) involved in GDP-D -Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP-D -Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A-band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP-D -Man, which is converted to GDP-D -Rha for A-band synthesis. Gmd, a previously reported A-band enzyme, and Rmd are predicted to perform the two-step conversion of GDP-D -Man to GDP-D -Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A-band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A-band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP-D -Man in the absence of WbpW and AlgA. Polymerase chain reaction (PCR) amplification and sequence analysis of this region reveals three open reading frames (ORFs), orf477, orf488 and orf303, arranged as an operon. ORF477 is homologous to initiating enzymes that transfer glucose 1-phosphate onto undecaprenol phosphate (Und-P), while ORF303 is homologous to L -rhamnosyltransferases involved in polysaccharide assembly. Chromosomal mapping using pulsed field gel electrophoresis (PFGE) and Southern hybridization places orf477, orf488 and orf303 between 0.3 and 0.9 min on the 75 min map of PAO1, giving it a map location distinct from that of previously described polysaccharide genes. This region may represent a unique locus within P. aeruginosa responsible for the synthesis of another polysaccharide molecule.     

Atomic force microscopy study of the effect of lipopolysaccharides and extracellular polymers on adhesion of Pseudomonas aeruginosa

The roles of lipopolysaccharides (LPS) and extracellular polymers (ECP) on the adhesion of Pseudomonas aeruginosa PAO1 (expresses the A-band and B-band of O antigen) and AK1401 (expresses the A-band but not the B-band) to silicon were investigated with atomic force microscopy (AFM) and related to biopolymer physical properties. Measurement of macroscopic properties showed that strain AK1401 is more negatively charged and slightly more hydrophobic than strain PAO1 is. Microscopic AFM investigations of individual bacteria showed differences in how the biopolymers interacted with silicon. PAO1 showed larger decay lengths in AFM approach cycles, suggesting that the longer polymers on PAO1 caused greater steric repulsion with the AFM tip. For both bacterial strains, the long-range interactions we observed (hundreds of nanometers) were inconsistent with the small sizes of LPS, suggesting that they were also influenced by ECP, especially polysaccharides. The AFM retraction profiles provide information on the adhesion strength of the biopolymers to silicon (F_adh). For AK1401, the adhesion forces were only slightly lower (F_adh = 0.51 nN compared to 0.56 nN for PAO1), but the adhesion events were concentrated over shorter distances. More than 90% of adhesion events for AK1401 were at distances of <600 nm, while >50% of adhesion events for PAO1 were at distances of >600 nm. The sizes of the observed molecules suggest that the adhesion of P. aeruginosa to silicon was controlled by ECP, in addition to LPS. Steric and electrostatic forces each contributed to the interfacial interactions between P. aeruginosa and the silicon surface.     

Effect of O-Side-Chain-Lipopolysaccharide Chemistry on Metal Binding

Pseudomonas aeruginosa PAO1 produces two chemically distinct types of lipopolysaccharides (LPSs), termed A-band LPS and B-band LPS. The A-band O-side chain is electroneutral at physiological pH, while the B-band O-side chain contains numerous negatively charged sites due to the presence of uronic acid residues in the repeat unit structure. Strain PAO1 (A⁺ B⁺) and three isogenic LPS mutants (A⁺ B⁻, A⁻ B⁺, and A⁻ B⁻) were studied to determine the contribution of the O-side-chain portion of LPS to metal binding by the surfaces of gram-negative cells. Transmission electron microscopy with energy-dispersive X-ray spectroscopy was used to locate and analyze sites of metal deposition, while atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry were used to perform bulk quantitation of bound metal. The results indicated that cells of all of the strains caused the precipitation of gold as intracellular, elemental crystals with a d-spacing of 2.43 Å. This type of precipitation has not been reported previously for gram-negative cells and suggests that in the organisms studied gold binding is not a surface-mediated event. All four strains bound similar amounts of copper (0.213 to 0.222 μmol/mg [dry weight] of cells) at the cell surface, suggesting that the major surface metal-binding sites reside in portions of the LPS which are common to all strains (perhaps the phosphoryl groups in the core-lipid A region). However, significant differences were observed in the abilities of strains dps89 (A⁻ B⁺) and AK1401 (A⁺ B⁻) to bind iron and lanthanum, respectively. Strain dps89 caused the precipitation of iron (1.623 μmol/mg [dry weight] of cells) as an amorphous mineral phase (possibly iron hydroxide) on the cell surface, while strain AK1401 nucleated precipitation of lanthanum (0.229 μmol/mg [dry weight] of cells) as apiculate, surface-associated crystals. Neither iron nor lanthanum precipitates were observed on the cells of other strains, which suggests that the combination of A-band LPS and B-band LPS produced by a cell may result in a cell surface which promotes the formation of metal-rich precipitates. We therefore propose that the negatively charged sites located in the O-side chains are not directly responsible for the binding of metallic ions; however, the B-band LPS molecule as a whole may contribute to overall cell surface properties which favor the precipitation of distinct metal-rich mineral phases.     

Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C.

Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs. The relative expression levels of these two LPS types in P. aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide. A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody. Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) [A+B-]) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression. B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.     

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa.

Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.     

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a Truncated Lipopolysaccharide

The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL − Mg²⁺) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH₃⁺ groups in the core LPS. The zeta potentials of these cells were also measured in Na⁺, Ca²⁺, and La³⁺ electrolytes. Cells in the La³⁺ electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL − Mg²⁺) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca²⁺ was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg²⁺ concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.     

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz ( rol ), wzy ( rfc ), and wbpA – wbpN . A further 3 ORFs not thought to be involved with LPS synthesis were identified ( hisH , hisF , and uvrB ). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequence, IS 1209 , is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.     

Regulation of Caenorhabditis elegans and Pseudomonas aeruginosa machinery during interactions

The amenability of Caenorhabditis elegans against pathogen provides a valuable tool for studying host–pathogen interactions. Physiological experiments revealed that the P. aeruginosa was able to kill C. elegans efficiently. The effects of P. aeruginosa PA14, PAO1 and their isolated lipopolysaccharide (LPS) on the host system were analyzed. The LPS at higher concentrations (≥2 mg/ml) was toxic to the host animals. Kinetic studies using qPCR revealed the regulation of host-specific candidate antimicrobial genes during pathogen-mediated infections. In addition, the pathogen-specific virulent gene, exoT expression, was anlyzed and found to be varied during the interactions with the host system. Ability of the pathogens to modify their internal machinery in the presence of the host was analyzed by XRD, FTIR and PCA. LPS isolated from pathogens upon exposure to C. elegans showed modifications at their functional regions. LPS from PAO1 showed difference in d-spacing angle (Å) and °2Th position. FTIR spectra revealed alterations in polysaccharide (1,200–900 cm⁻¹) and fatty acid (3,000–2,800 cm⁻¹) regions of LPS from P. aeruginosa PAO1 exposed to the host system. These data provide additional insights on how the pathogens subvert its own and host machinery during interactions.     

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.     

The Occurrence of 4-Amino-4-Deoxy-Arabinose in LPS of Supersusceptible Strain Pseudomonas aeruginosa Z61: Noncorrelation with Polymyxin Resistance

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. ³¹P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance. Received: 31 August 1998 / Accepted: 5 November 1998     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

Isolation of genes required for hydrogenase synthesis in Escherichia coli

A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa

Type 4 fimbriae are produced by a variety of pathogens, in which they appear to function in adhesion to epithelial cells, and in a form of surface translocation called twitching motility. Using transposon mutagenesis of Pseudomonas aeruginosa, we have identified a new locus required for fimbrial assembly. This locus contains the gene pilQ which encodes a 77 kDa protein with an N-terminal hydro-phobic signal sequence characteristic of secretory proteins, pilQ mutants lack the spreading colony morphology characteristic of twitching motility, are devoid of fimbriae, and are resistant to the fimbrial-specific bacteriophage PO4. The pilQ gene was mapped to Spel fragment 2, which is located at 0–5 minutes on the P. aeruginosa PAO1 chromosome, and thus it is not closely linked to the previously characterized pilA-D, pilS,R or pilT genes. The pilQ region also contains ponA, aroK and aroB-like genes in an organization very similar to that of corresponding genes in Escherichia coli and Haemophilus influenzae. The predicted amino acid sequence of PilQ shows homology to the PulD protein of Kleb-siella oxytoca and related outer membrane proteins which have been found in association with diverse functions in other species including protein secretion, DNA uptake and assembly of filamentous phage. PilQ had the highest overall homology to an outer membrane antigen from Neisseria gonorrhoeae, encoded by omc, that may fulfil the same role in type 4 fimbrial assembly in this species.     

Heterotrophic bacteria of the freshwater neuston and their ability to act as plasmid recipients under nutrient deprived conditions

Significantly higher numbers of Gram-negative heterotrophic bacteria were present at the air-water interface (neston) of freshwater lakes than in the bulk water. Neuston bacteria were distinguished as a population distinct from bacteria in the bulk water by a higher incidence of pigmented colony types and significantly greater levels of multiple resistance to antibiotics and heavy metals. The incidence of plasmids in 236 neuston and 229 bulk water strains were similar (14 and 16.2%, respectively). Nine of 168 plasmid-free strains and 2 of 14 plasmid carrying strains, isolated from both bulk water and neuston, acted as recipients of plasmid R68.45 in plate matings with aPseudomonas aeruginosa donor strain PAO4032 at 21°C, but at frequencies below that of matings with a restriction-minus recipient strain ofP. aeruginosa, strain PAO1168. In a model system composed of nutrient-free synthetic lake water, plasmid R68.45 was shown to transfer betweenP. aeruginosa strains at frequencies between 10⁻³ and 10⁻⁵. Transconjugants were detected about 100 times more frequently at the interface than in the bulk water, which in part reflected a greater enrichment of the donor at this site. None of the aquatic isolates were able to act as recipients of plasmid R68.45 in this model system with strain PAO4032 as donor. The results suggest that under nutrient deprived conditions, the spread of plasmid R68.45 and similar plasmids by lateral transfer into this particular aquatic population would be a rare event.