Evidence that the algI/algJ gene cassette, required for O acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer

Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.     

Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation.

Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.     

Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation.

Mucoid strains of Pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of O-acetyl groups. To better understand the acetylation process, a gene involved in alginate acetylation called algF was identified in this study. We hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the P. aeruginosa chromosome. To isolate algF mutants, a procedure for localized mutagenesis was developed to introduce random chemical mutations into the P. aeruginosa alginate biosynthetic operon on the chromosome. For this, a DNA fragment containing the alginate biosynthetic operon and adjacent argF gene in a gene replacement cosmid vector was utilized. The plasmid was packaged in vivo into lambda phage particles, mutagenized in vitro with hydroxylamine, transduced into Escherichia coli, and mobilized to an argF auxotroph of P. aeruginosa FRD. Arg+ recombinants coinherited the mutagenized alginate gene cluster and were screened for defects in alginate acetylation by testing for increased sensitivity to an alginate lyase produced by Klebsiella aerogenes. Alginates from recombinants which showed increased sensitivity to alginate lyase were tested for acetylation by a colorimetric assay and infrared spectroscopy. Two algF mutants that produced alginates reduced more than sixfold in acetyl groups were obtained. The acetylation defect was complemented in trans by a 3.8-kb XbaI-BamHI fragment from the alginate gene cluster when placed in the correct orientation under a trc promoter. By a merodiploid analysis, the algF gene was further mapped to a region directly upstream of algA by examining the polar effect of Tn501 insertions. By gene replacement, DNA with a Tn501 insertion directly upstream of algA was recombined with the chromosome of mucoid strain FRD1. The resulting strain, FRD1003, was nonmucoid because of the polar effect of the transposon on the downstream algA gene. By providing algA in trans under the tac promoter, FRD1003 produced nonacetylated alginate, indicating that the transposon was within or just upstream of algF. These results demonstrated that algF, a gene involved in alginate acetylation, is located directly upstream of algA.     

Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm(-1). All strains produced biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 cm(-1) (amide II), 1,240 cm(-1) (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm(-1) (C---OH and P---O stretching) 1,450 cm(-1), and 1,400 cm(-1). The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm(-1) (C---OH stretching of alginate) and 1,250 cm(-1) (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 micrometer, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.     

Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.

Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.     

Microbial and algal alginate gelation characterized by magnetic resonance

Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca(2+) ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin-spin T(2) magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.     

Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of beta1-4-linked O-acetylated d-mannuronate (M) and l-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed beta-helix (RHbetaH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHbetaH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHbetaH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHbetaH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHbetaH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second d-mannuronate residue to l-guluronate along the epimerase catalytic face.     

In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa

An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Deltaalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. (1)H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.     

P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.     

The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa

Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide that confers a mucoid phenotype. Alginate is a linear polymer of d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate (G). AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate to the mixed M+G sequence of alginate. To understand better the role and mechanism of AlgG activity, a mutant was constructed in the mucoid strain FRD1 with a defined non-polar deletion of algG. Instead of producing poly mannuronate, the algG deletion mutant secreted dialysable uronic acids, as does a mutant lacking the periplasmic protein AlgK. High levels of unsaturated ends and the nuclear magnetic resonance spectroscopy pattern revealed that the small, secreted uronic acids were the products of extensive polymer digestion by AlgL, a periplasmic alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional with (i) epimerase activity and (ii) a role in protecting alginate from degradation by AlgL during transport through the periplasm. AlgK appears to share the second role. AlgG and AlgK may be part of a periplasmic protein complex, or scaffold, that guides alginate polymers to the outer membrane secretin (AlgE). To characterize the epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing FRD462 was shown to encode a protein lacking only the epimerase function. The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.     

The products of Rhizobium genes, psi and pss, which affect exopolysaccharide production, are associated with the bacterial cell surface

Translational fusions between a mutant phoA (lacking its promoter, ribosomal binding site and signal peptide sequence) and Rhizobium 'symbiotic' genes were isolated. Since these fusions expressed alkaline phosphatase (AP), the product of phoA, the genes into which phoA was inserted apparently specify proteins located in the bacterial periplasm or cell membrane, the compartment in which AP has activity. These genes were psiA and genes upstream of psiA (psiA is required for normal nodule development and strains with multicopy psiA fail to make exopolysaccharide (EPS) and to nodulate). Fusions between phoA and pss (exo) genes, which are required for EPS production, also resulted in the expression of AP indicating that products of these pss genes were located at the cell surface. Using gus fusions to psiA and pssA, we found that the former was expressed in N2-fixing bean root nodules but the latter was not.     

Putative membrane assembly of EtpM-colicin V chimeras

EtpM of the enterohemorrhagic E. coli O157:H7 is a bitopic membrane protein of the type II protein secretion apparatus. There is a twin-arginine (RR) motif in front of its signal anchor, suggesting a Tat-dependent membrane targeting of EtpM. By exploiting the periplasmic bactericidal activity of colicin V (ColV), we constructed EtpM-ColV fusions and studied the EtpM-mediated translocation of ColV. The wild type strain and the DeltatatC mutant were killed by the expressed fusions and were fully protected from the killing effect by the ColV-specific immunity protein. In contrast, cold-inactivation of YidC, which is generally required for integral membrane protein assembly, significantly attenuated the killing effect in the cold-sensitive yidC mutant. These results confirmed the predicted N(in)-C(out) EtpM topology, and suggests an EtpM-mediated, Tat-independent and YidC-dependent translocation of ColV.     

Bacterial alginates: from biosynthesis to applications

Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of β-d-mannuronic acid and its C5-epimer α- l-guluronic acid linked via β-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.     

Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium.

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane (C. A. Mulford and M. J. Osborn, Proc. Natl. Acad. Sci. USA 80:1159-1163, 1983). In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi] mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.     

Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Alginate, a co-polymer of O-acetylated beta-1,4-linked D-mannuronic acid and L-guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525. L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.     

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.