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Journal of Bacteriology, November 2008, p. 7219-7231, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.01003-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps{triangledown}

Evelyn Toh,1 Harry D. Kurtz Jr.,2 and Yves V. Brun1*

Department of Biology, Indiana University, Bloomington, Indiana 47405-3700,1 Department of Genetics and Biochemistry, 100 Jordan Hall, Clemson University, Clemson, South Carolina 296342

Received 21 July 2008/ Accepted 25 August 2008


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ABSTRACT
 
Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.


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INTRODUCTION
 
Bacterial adhesion plays a crucial role in the establishment of a community of microorganisms called a biofilm on host tissues and nonbiological surfaces. The strategies employed by different bacteria for mediating attachment to surfaces vary, but external appendages play important roles in adherence and colonization. In enteropathogenic Escherichia coli and enterohemorrhagic E. coli, the flagella can bind mucins on mucosal surfaces (13), while enterohemorrhagic E. coli produce adhesive type IV pili that form the physical bridges between bacteria adhering to human and bovine host cells (69). Polysaccharides also play important roles in adherence by both gram-positive and gram-negative bacteria. For example, E. coli produces an exopolysaccharide matrix composed of polymers of N-acetylglucosamine (GlcNAc) that influences the structural integrity of its biofilm, particularly the transition from temporary polar attachment of cells to permanent lateral attachment of cells to a substrate (1). Staphylococcus epidermidis produces a polysaccharide adhesin composed of a homopolymer of poly-β-1,6,GlcNAc (37) that is required for biofilm formation (65). A GlcNAc polysaccharide helps promote the transmission of Yersinia pestis from the flea to the mammalian host (21) and impacts bacterial colonization, virulence, and host immune evasion (56, 63).

Surface adhesion and biofilm formation by the gram-negative aquatic bacterium Caulobacter crescentus require the contribution of three polar structures: the flagellum, pili, and holdfast (6, 12, 34), whose biosynthesis is regulated in a cell cycle-dependent manner. C. crescentus produces two cell types at every cell division, a motile swarmer cell with a flagellum and pili at the same pole and a stalked cell with a cell envelope extension called the stalk tipped by the holdfast adhesin. The swarmer cell differentiates into a stalked cell by shedding the flagellum, retracting the pili, and synthesizing a holdfast and a stalk at the same pole (47). Although the C. crescentus pili and flagellum are important to optimize adhesion, the strong permanent attachment of C. crescentus to a substrate requires the holdfast. The holdfast is an elastic material with gel-like properties (35) and is strongly adhesive (61). Although the precise composition and structure of the holdfast material have not been determined, N-acetylglucosamine (GlcNAc) polymers are an important component of the holdfast and play a role in the elastic properties (35) and the force of adhesion of the holdfast (61). Previous studies of C. crescentus holdfasts indicated that the holdfast is sensitive to lysozyme and chitinase, glycolytic enzymes specific for cleavage of β-1,4 linkages in oligomers of GlcNAc, suggesting that the holdfasts might contain oligomers of β-1,4-linked GlcNAc (40). Lectin binding studies also revealed that wheat germ agglutinin (WGA), which recognizes GlcNAc polymers, binds specifically to the holdfast (40).

Adhesion-deficient mutants from a C. crescentus transposon library were previously grouped into three classes based on phenotypic characteristics, namely the holdfast biogenesis (hfs for holdfast synthesis) and holdfast anchor (hfa for holdfast anchor) classes and a class of some pleiotropic developmental mutants (podJ and pleC) (42, 46, 28, 29, 58). Mutations in the hfaABD operon result in various defects in surface adhesion, but most notably the holdfasts of these mutants have a reduced ability to attach to the tip of the stalk, causing the shedding of a fully adhesive holdfast polysaccharide (42, 46, 28, 29, 10). Insertional mutations in hfsDAB result in the complete abolishment of surface adhesion and holdfast production (58), indicating that these hfs genes are absolutely required for holdfast synthesis. Sequence and phenotypic analyses indicate that hfsDAB are required for holdfast polysaccharide export; however, the genes directly involved in holdfast polysaccharide biosynthesis were not identified in previous genetic screens.

Here, we characterized a class of adhesion-deficient mutants obtained in a previous transposon mutagenesis screen (42) and identified a cluster of four genes directly adjacent to hfsDABC that are involved in holdfast polysaccharide biosynthesis (hfsEFGH). We generated in-frame, nonpolar deletion mutants to assess the contribution of these hfsEFGH genes to C. crescentus surface adhesion and holdfast synthesis. We showed that hfsE has two paralogs that can substitute for hfsE in holdfast synthesis. Single-deletion hfsE, pssY, and pssZ mutants caused only a slight reduction in adhesion and holdfast synthesis, whereas a triple-deletion mutant was completely deficient in adhesion and holdfast synthesis. We identified a paralog to hfsC, which we call hfsI. An in-frame-deletion mutant with a deletion of the predicted polysaccharide polymerase gene hfsC had no surface adhesion phenotype (58). Just like an hfsC mutant, an hfsI deletion mutant does not display a holdfast synthesis defect. Deletion of both hfsC and hfsI results in a severe holdfast synthesis defect. We hypothesized that the paralogs of hfsE may serve to add various sugars in the holdfast polysaccharide repeat unit, while the paralog of hfsC may catalyze the formation of a different glycosidic linkage between these different repeat units.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. All bacterial strains and plasmids used in this study are listed in Table 1. All C. crescentus strains were cultured in peptone-yeast-extract (PYE) medium (48) at 30°C. Kanamycin (20 µg/ml [plate]; 5 µg/ml [broth]), tetracycline (2 µg/ml [plate]; 1 µg/ml [broth]), nalidixic acid (20 µg/ml [plate]), and 3% sucrose (plate) were used to supplement the C. crescentus media as necessary. E. coli strains were cultured at 37°C in Luria-Bertani (LB) medium. The LB medium was supplemented with kanamycin (50 µg/ml or 25 µg/ml [plate]; 30 µg/ml [broth]) and tetracycline (12 µg/ml [plate and broth]) when necessary.


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  		TABLE 1. Bacterial strains and plasmids used in this study

DNA manipulations and sequencing. All restriction enzymes used for standard molecular cloning in this study were purchased from New England Biolabs, Inc. (Ipswich, MA), and we followed standard molecular biology methods (3). All primers used in this study are listed in Table 2. Plasmid DNA was isolated by using a QIAprep miniprep kit, and PCR products were purified by using QIAquick spin columns (Qiagen, Valencia, CA) according to the procedures recommended by the manufacturer. Chromosomal DNA was isolated by using a Promega Magic miniprep DNA purification system (Promega, Madison, WI) according to the manufacturer's instructions. Sequencing reactions were performed in the Indiana Institute for Molecular Biology at Indiana University on an Applied Biosystems 3730 automated fluorescence sequencing system, using ABI Prism BigDye Terminator cycle sequencing version 3.1 (Applied Biosystems, Foster City, CA). Sequence data were analyzed using Sequencher 4.7 software (Gene Codes Corporation, Ann Arbor, MI) and the Codon Preference module of the GCG Wisconsin package.


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  		TABLE 2. Oligonucleotides used in this study

Analysis of hfs promoter by lacZ transcriptional fusions. Fragments were cloned upstream of the lacZ gene in pRKlac290 and analyzed for promoter activity in the wild-type C. crescentus strain CB15. The levels of promoter activity were determined by assaying for β-galactosidase activity as previously described (26), with the following modification: all measurements were done at 22°C. The levels of β-Galactosidase activity were expressed in Miller units and represent the averages of the results of five independent cultures done in triplicate, displayed with standard deviations. β-Galactosidase activity conferred by the plasmid alone was approximately 70 Miller units.

Generation of in-frame-deletion mutants. In-frame markerless deletions of hfsE, hfsF, hfsG, hfsH, and hfsI were created by a two-step homologous recombination method, using upstream and downstream fragments of each gene cloned into nonreplicating plasmid pNPTS138, which carries a kanamycin resistance gene cassette (nptI), along with the sacB cassette that confers sucrose sensitivity, as previously described (19, 60). Primers NewFuphfsE, NewRuphfsE, FdwHindIIIhfsE, and RdwPstIhfsE were used in a PCR to amplify 500 bp directly upstream and downstream of the predicted start of the hfsE gene, leaving six codons at the start of the gene and seven codons at the end of the gene. Primers NewFuphfsF, NewRuphfsF, FdwHindIIIhfsF, and RdwSphIhfsF were used in PCR to amplify 500 bp directly upstream and downstream of the start of the hfsF gene, leaving 34 codons at the start of the gene and 7 codons at the end of the gene. Primers FupSphIhfsG, RupHindIIIhfsG, FdwHindIIIhfsG, and RdwBamHIhfsG were used in PCR to amplify 500 bp directly upstream and downstream of the predicted start of the hfsG gene, leaving 6 codons at the start of the gene and 10 codons at the end of the gene. Primers FupBamHIhfsH, RupEcoRIhfsH, FdwEcoRIhfsH, and RdwHindIIIhfsH were used in PCR to amplify 500 bp directly upstream and downstream of the predicted start of the hfsH gene, leaving three codons at the start of the gene and four codons at the end of the gene. Primers F499HindIII, R499XbaI, Fm499XbaI, and Rm499EcoRI were used in PCR to amplify 500 bp directly upstream and downstream of the predicted start of the hfsI gene, leaving three codons at the start of the gene and four codons at the end of the gene. The deletion mutants were confirmed by colony PCR, using the primers used to clone the upstream and downstream fragments, and were verified by sequencing.

Complementation of in-frame-deletion mutants. A series of pMR10-based plasmids was constructed to complement all the deletion mutants. The individual genes were PCR amplified separately from wild-type CB15 chromosomal DNA and introduced into pMR10, a medium-copy-number plasmid, together with its native promoter. Primers FupEcoRIhfsE and phfsEendNdeI were used to amplify the hfsE promoter. Primer pairs FupEcoRIhfsE and HfsEendPstI, HfsFstartNdeI and HfsFendHindIII, HfsGstartNdeI and HfsGendBamHI, and HfsHstartNdeI and HfsHendBamHI were used to amplify the entire hfsE, hfsF, hfsG, and hfsH genes, respectively. The appropriate plasmids carrying the different hfs genes under the control of the hfsE promoter were introduced to the individual in-frame-deletion mutants and assayed for the restoration of surface adhesion and lectin binding.

Surface binding assay. Polystyrene binding assays were performed as previously described (6), with the following modifications: the cells were allowed to adhere for 45 min, and 1.5 ml of a 1% crystal violet solution was used for staining.

Swarming motility assay. Exponential cultures (0.5 µl) grown in PYE medium and normalized to an optical density of 0.3 at 600 nm were stabbed into 0.3% agar semisolid agar PYE medium. The plates were incubated at room temperature for 5 days. A nonmotile {Delta}pleC mutant (31) was used as a negative control for swarming.

Phage sensitivity assay. Phage sensitivity assays were performed as previously described (53), using the caulophage {Phi}CbK.

Microscopy. Fluorescent lectin binding assays were performed as previously described (24), except that Alexa Fluor 488-conjugated WGA (AF488-WGA) was used to label the holdfast (Invitrogen Molecular Probes). A Nikon Eclipse E800 light microscope equipped with a 100x Plan Apo oil objective was used for phase-contrast microscopy, and a Nikon FITC-HyQ filter cube (Chroma Technology) was used for epifluorescence microscopy. Images were captured by using a Princeton Instruments cooled charge-coupled-device camera, model 1317, and MetaMorph imaging software, version 7.1.1 (Molecular Devices, Sunnyvale, CA). Transmission electron microscopy (TEM) was used to observe the appearance of the holdfast in the various mutants. Exponential-phase cells were mounted onto Formvar-coated, carbon film-stabilized copper grids (Electron Microscopy Sciences, Hatfield, PA) for 30 min. Each grid was washed in a drop of water, negatively stained with 7.5% uranyl magnesium acetate for 5 min, and washed five times with water after being stained with uranyl magnesium acetate. The grids were examined with a Jeol JEM-1010 transmission electron microscope set to 80 kV.


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RESULTS
 
Identification of a holdfast polysaccharide biosynthesis gene cluster adjacent to the hfsDABC genes. To identify the genes involved in the biosynthesis of the minimum-repeat units of the holdfast polysaccharide, we revisited the Tn5 mutagenesis screen for holdfast mutants of the freshwater C. crescentus strain CB2A and examined an uncharacterized gene cluster (cluster IV in the previous study of Mitchell and Smit [42]). All four Tn5 insertion mutants in cluster IV, with the original designations g3, g10, g4, and g6, were found to be adhesion deficient, based on a quantitative surface binding assay. CB2Ag3, CB2Ag10, CB2Ag4, and CB2Ag6 exhibited 9%, 8%, 9%, and 7% binding to a polystyrene surface, respectively, compared to 100% for wild-type C. crescentus CB2A.

A fragment of Tn5 with a flanking chromosomal DNA from mutants g3, g10, g4, and g6 was cloned into pUC18, and the DNA sequence of the chromosomal DNA portion was used to search the genome sequence of C. crescentus CB15 (45), since the genome sequence of C. crescentus CB2A is not known. Sequence analysis of the junction of the Tn5 fragment revealed that the g3, g10, g4, and g6 transposon insertion sites were found in a region containing four open reading frames (ORFs) downstream from and convergent to the hfsC gene (Fig. 1A). Sequence analysis of these four ORFs revealed that these genes (CC_2425 to CC_2428), hereinafter called hfsEFGH, are homologous to genes involved in polysaccharide biosynthesis (68). hfsE (CC_2425) encodes a predicted protein of 375 amino acids (aa) that is homologous to a family of integral membrane sugar transferases from the polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) family (32, 64), including specialized glycosyltransferases like WsaP in Geobacillus stearothermophilus NRS 2004/3a (59), WbaP in E. coli (11), and WbaP in Salmonella enterica (64). These sugar transferases catalyze the first step in polysaccharide biosynthesis by transferring hexose-1-phosphate residues from UDP-hexoses to the lipid carrier molecule undecaprenol phosphate in the inner membrane (4, 8). HfsE possesses the three characteristic blocks of highly conserved amino acids (KFRSM, DELPQ, and PGITG) that are typically found in the C-terminal halves of prototypes of this family of PHPT sugar transferases (Fig. 1B). hfsF (CC_2426) encodes a predicted integral membrane protein of 480 aa with 14 predicted transmembrane helices and a weakly conserved stretch of approximately 208 amino acids (polysacc_synt domain, Protein Families database of alignments; hidden Markov models [HMMs], http://cmr.tigr.org/cgi-bin/CMR/HmmReport.cgi?hmm_acc=PF01943) that is often present in Wzx flippases (formerly known as RfbX) (38). Some of the Wzx proteins have been shown to catalyze the translocation of undecaprenol diphosphate-linked K-repeating units formed at the cytoplasmic side of the inner membrane across this membrane (8, 51, 15). hfsG (CC_2427) encodes a predicted cytoplasmic protein of 309 aa homologous to family 2 glycosyltransferases that participate in a wide range of polysaccharide synthesis systems (5, 27, 33, 43, 52), including transferring sugar units from UDP-glucose to cellulose (56) and UDP-GlcNAc to oligosaccharide Nod factors, (17), and is thought to catalyze the polymerization of GlcNAc. hfsH (CC_2428) encodes a predicted cytoplasmic protein of 257 aa that belongs to carbohydrate esterase family 4 (CE4). All members of this family catalyze the hydrolysis of either N-linked acetyl groups from GlcNAc residues or O-linked acetyl groups from O-acetylxylose residues of their substrates (9). HfsH is similar to a NodB de-N-acetylase superfamily of polysaccharide deacetylases involved in the degradation and remodeling of GlcNAc substrates like chitin (20, 41), chito-oligosaccharide rhizobial Nod factors, and peptidoglycan (49). We hypothesize that hfsE, hfsF, hfsG, and hfsH catalyze the biosynthesis, remodeling, and flipping across the inner membrane of the holdfast polysaccharide repeat unit(s).


Figure 1
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  		FIG. 1. (A) Genetic organization of the hfsE, pssY, pssZ, and CC_1486 gene clusters. Each gene name and the gene's predicted functions and corresponding TIGR locus tags are listed below the arrows. GT stands for glycosyltransferase. Arrows indicate the direction of transcription for each gene relative to another. The dashed lines depict regions of the gene deleted in the various deletion mutants. (B) Alignment of the C-terminal region of HfsE and its paralogs, along with other PHPT family members. Highly conserved residues are shaded, while the asterisks indicate amino acids conserved across all paralogs. The conserved blocks I, II, and III are indicated by lines above them and are conserved across all PHPT homologs. The sugar transferases compared are as follows: C. crescentus CB15 HfsE (accession number AAK24396), C. crescentus PssY (accession number AAK22153), C. crescentus PssZ (accession number AAK24355), C. crescentus CC_1486 (accession number AAK23465), Burkholderia cenocepacia IST432 BceB (accession number ABC71344), E. coli WcaJ (accession number AP002647), Methylobacillus sp. strain 12S EpsB (accession number BAC41337), Xanthomonas campestris GumD (accession number AAA86372), Salmonella enterica serovar Typhimurium WbaP (formerly RfbP; accession number P26406), Erwinia amylovora AmsG (accession number Q46628), Sinorhizobium meliloti ExoY (accession number Q02731), Streptococcus pneumoniae serotype 8 WchA (accession number AAK20699), S. pneumoniae Cps14E (accession number CAA59777), Streptococcus salivarius CpsE (accession number CAC18355), Streptococcus thermophilus EpsE (accession number AAC44012), Lactococcus lactis B35 EpsD (accession number AAD22526), and Geobacillus stearothermophilus NRS 2004/3a (accession number AAR99615). All accession numbers are from GenBank.

Transcriptional and translational organization of the hfsEFGH genes. The organization of the hfsEFGH genes suggests that these genes might be transcribed as an operon. The ORFs of hfsF, hfsG, and hfsH overlap, with hfsF and hfsG sharing four nucleotides, whereas hfsG and hfsH share a single nucleotide; however, a 6-nucleotide space separates the end of hfsE and the start of hfsF. Two results of our initial deletion experiments, however, suggested that careful examination of the promoter organization in this region was required prior to phenotypic characterization of the deletion mutants. First, we were unable to complement an initial in-frame hfsE deletion mutant (data not shown), and second, the initial deletion of the hfsF gene from codon 5 to codon 475 suggested that a promoter was present in its coding sequence (data not shown). Examination of the hfsE annotation at The Institute for Genomic Research Comprehensive Microbial Resource used to design the gene deletion strongly suggested that the gene was misannotated. The annotation at the Comprehensive Microbial Resource indicated that the gene spanned nucleotide coordinates 2630635 to 2632173 in the C. crescentus genome, potentially encoding a protein of 512 aa. Amino acids 253 to 460 had good sequence similarity to the PHPT family members (Fig. 1B), whereas the first 180 aa had no similarity to other proteins. In addition, the third position GC bias and codon bias analyses suggested that the first 137 codons were unlikely to encode a protein. Based on the GC bias analysis and the region of similarity to PHPT family proteins, we hypothesized that the start codon of hfsE is an ATG starting at nucleotide coordinate 2631047; this coordinate was used in all our subsequent analyses. To determine the approximate location of the promoter(s) responsible for transcribing this gene cluster in order to design nonpolar deletions, we constructed a set of overlapping transcriptional fusions to a promoterless lacZ reporter gene in the plasmid pRKlac290 and assayed for β-galactosidase activity as a reporter of promoter activity (Fig. 2).


Figure 2
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  		FIG. 2. Promoter map of the hfsEFGH gene cluster. Transcriptional fusions to a promoterless lacZ were assayed for β-galactosidase activity. β-Galactosidase activities are expressed in Miller units with standard deviations and are the averages of five independent assays. The approximate locations of the hfs promoters are indicated by the bent arrows. The dotted lines under the bent arrows represent the positions of the promoters. All fusion fragments are drawn to scale and depicted by solid horizontal lines.

In order to verify that a promoter exists upstream of hfsE, we constructed a transcriptional fusion, plachfs12, which yielded 1,500 Miller units of β-galactosidase activity, indicating that there is a promoter residing in this 539-nucleotide fragment upstream of hfsE. To determine whether there were any promoters in the hfsE coding region, two additional transcriptional fusions were constructed: plachfs15 and plachfs9. Neither of these fusions had β-galactosidase activity, indicating that there are no promoters in the hfsE coding region. The plachfs3 fusion, which included 1,000 nucleotides upstream of the probable start codon of hfsF and 45 codons of hfsF, had 930 Miller units of β-galactosidase activity. Since the 5' end of plachfs3 overlaps with plachfs15 and plachfs9 and there is no promoter in the corresponding fragments, a promoter must lie between the 3' end of plachfs9 and that of plachfs3. To delineate this promoter further, we constructed plachfs13. The plachfs9 fusion had essentially no β-galactosidase activity, while the plachfs13 fusion had 500 Miller units of β-galactosidase activity, confirming the presence of a promoter in this region. These results indicate that there is a promoter in the coding region of hfsF, which resides in a 218-bp nucleotide fragment contained within the plachfs13 transcriptional fusion.

To determine if there are additional promoters driving hfsG and hfsH gene expression, we constructed two other transcriptional fusions: plachfs14 and plachfs5, neither of which had β-galactosidase activity (Fig. 2). These results indicate that there is no promoter between hfsF and hfsG or between hfsG and hfsH. Based on the results of this promoter fusion analysis, we concluded that there are at least two promoters present in the hfsEFGH gene cluster. Knowledge of the approximate location of these promoters was then used to construct nonpolar deletions of the hfsEFGH genes.

Contribution of hfsEFGH to holdfast synthesis. To determine the contribution of hfsEFGH to holdfast synthesis and to eliminate the possibility that the phenotypes of the CB2A transposon insertion mutants were due to polar effects on downstream genes, we generated in-frame-deletion mutants with deletions of each of the four genes, hfsE (CC_2425), hfsF (CC_2426), hfsG (CC_2427), and hfsH (CC_2428), in strain CB15 and assayed for their ability to adhere to a polystyrene surface and to bind a holdfast-specific lectin, WGA. Mutants with deletions in hfsE and hfsF adhered to polystyrene with only a small decrease in their binding abilities compared to the wild type, whereas the hfsG and hfsH mutants were severely impaired in their abilities to adhere to polystyrene (Fig. 3A). Lectin binding assays using AF488-WGA mimicked the adherence results: {Delta}hfsE and {Delta}hfsF predivisional cells had a slightly reduced ability to bind to AF488-WGA, whereas {Delta}hfsG and {Delta}hfsH predivisional cells were deficient in binding to AF488-WGA (Fig. 3B).


Figure 3
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  		FIG. 3. (A) Quantification of crystal violet-stained cells attached to polystyrene. CB15 is the wild type, and NA1000 is a nonadherent strain. A minus (–) indicates an hfsEFGH in-frame-deletion mutant, and a plus (+) indicates the respective complemented deletion mutant. The results represent the means of independent experiments; the means were calculated from a total of nine measurements derived from three samples per experiment, three independent times. Error bars represent standard deviations. (B) Quantitation of holdfast AF488-WGA labeling of predivisional (PD) cells. A total number of 500 predivisional cells were counted, and the percent values reflect the proportion of AF488-WGA-labeled predivisional cells versus the total of all predivisional cells counted. Error bars represent standard deviations.

In order to observe the holdfasts present in the hfs deletion mutants, we performed TEM on negatively stained whole-cell mounts of these hfs deletion mutants. In a wild-type CB15 cell, the holdfast appeared as a darkly stained amorphous material localized at the tip of the stalk (Fig. 4, panel 1). In contrast, cells of NA1000, a spontaneous holdfast-deficient mutant of CB15, completely lacked a detectable holdfast (Fig. 4, panel 17). The hfsG and hfsH mutants were completely devoid of holdfast material (Fig. 4, panels 3 and 4), whereas the hfsE and hfsF mutants had holdfasts that were comparable to that of the wild-type cell (Fig. 4, panels 5 and 2).


Figure 4
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  		FIG. 4. TEM analysis of holdfasts of hfs deletion mutants and all the hfsE paralog combination mutants. Electron micrographs show holdfasts of representative stalked cells in the cell population at a magnification of x25,000. Labels: {Delta}E, {Delta}hfsE; {Delta}Y, {Delta}pssY; {Delta}Z, {Delta}pssZ; {Delta}1436, {Delta}CC_1486. Panels: 1, CB15; 2, {Delta}hfsF; 3, {Delta}hfsG; 4, {Delta}hfsH; 5, {Delta}hfsE; 6, {Delta}pssY; 7, {Delta}pssZ; 8, {Delta}CC_1486; 9, {Delta}hfsE {Delta}pssY {Delta}pssZ; 10, {Delta}pssY {Delta}pssZ; 11, {Delta}pssY {Delta}CC_1486; 12, {Delta}pssZ {Delta}CC_1486; 13, {Delta}hfsE {Delta}pssY {Delta}pssZ {Delta}CC_1486; 14, {Delta}hfsE {Delta}pssY; 15, {Delta}hfsE {Delta}pssZ; 16, {Delta}hfsE {Delta}CC_1486; 17, NA1000; 18, {Delta}hfsE {Delta}pssY {Delta}CC_1486; 19, {Delta}hfsE {Delta}pssZ {Delta}CC_1486; 20, {Delta}pssY {Delta}pssZ {Delta}CC_1486.

To confirm that the phenotype of each mutant was solely due to the deletions, we introduced a series of complementation plasmids carrying the native hfsE promoter fused to the hfsE, hfsF, hfsG, or hfsH gene into their respective deletion mutants and assayed for surface adhesion and lectin binding. All of the deletion mutants were complemented for adherence to polystyrene (Fig. 3A) and their ability to bind AF488-WGA (Fig. 3B), indicating that the phenotype of each mutant was caused by the individual gene deletions.

Although the holdfast is a critical player for the optimal attachment to surfaces, other polar structures such as pili and the flagellum have been shown to play a role in the attachment of C. crescentus to surfaces (6). To verify that the attachment deficiencies of the hfsEFGH deletion mutants were solely due to defects in holdfast synthesis and not the lack of pili or motility, we assayed for the presence of pili in these mutants, using a phage sensitivity assay with the caulophage {Phi}CbK, which requires pili for infection (55), and performed a swarm motility assay, which identifies swimming defects in semisolid agar. All of the hfsEFGH deletion mutants were sensitive to {Phi}CbK (data not shown), indicating that these deletion mutants possess pili and were motile in semisolid agar (data not shown), which indicates that flagellum function is not affected by these gene deletions.

We concluded that the hfsG and hfsH genes play a critical role in surface attachment and holdfast synthesis. Surprisingly, the deletion of hfsE and hfsF had only a minor effect on surface adhesion and holdfast synthesis.

hfsE has two paralogs that are redundant for holdfast synthesis. Based on amino acid sequence comparisons, HfsE should catalyze the first step in polysaccharide biosynthesis by transferring glucose-1-phosphate residues from UDP-GlcNAc to the lipid carrier molecule, undecaprenol phosphate, in the inner membrane. The loss of function of such an enzyme should render the cell incapable of manufacturing the holdfast polysaccharide. Since the hfsE mutant exhibited negligible defects in adhesion and holdfast synthesis, we considered the possibility that paralogs of hfsE might exist and provide redundant function. Analysis of the C. crescentus genome revealed the presence of three paralogs to hfsE: the exopolysaccharide production protein encoded by pssZ (CC_2384), the exopolysaccharide production protein encoded by pssY (CC_0166), and the protein encoded by the CC_1486 gene, which exhibit 51%, 41%, and 37% amino acid identity to the protein encoded by hfsE (CC_2425), respectively. All three predicted protein products share significant amino acid similarity with other known sugar transferases that function as initiating enzymes, including the three blocks of conserved amino acid residues found in other prototypes of these enzymes, suggesting that these proteins may have similar functions (Fig. 1B).

To assess whether any of the paralogs of hfsE can compensate for the function of hfsE, we constructed in-frame-deletion mutants with deletions of each of the individual paralogous genes and a comprehensive set of double-, triple-, and quadruple-combination mutants with hfsE and assayed for deficiencies in surface adhesion and the ability to synthesize a holdfast. Single-deletion mutants with deletions of hfsE, pssY, and pssZ had similar, slightly reduced abilities to bind to a polystyrene surface, whereas the CC_1486 mutant had a slightly increased surface adhesion phenotype (Fig. 5). The slight increase in surface adherence of the CC_1486 mutant is likely attributable to the overproduction of an exopolysaccharidelike material in the culture (Fig. 6), resulting in a massive cell aggregation phenotype exhibited by the single deletion of the CC_1486 mutant (Fig. 6). The large cell clumps made by {Delta}CC_1486 are often up to three times as wide as that depicted in the micrograph (Fig. 6).


Figure 5
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  		FIG. 5. Quantification of crystal violet-stained cells of various paralogous mutant combinations of hfsE attached to polystyrene. The results represent the means of measurements derived from three samples from each of three biological replicates. Error bars represent standard deviations.


Figure 6
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  		FIG. 6. Biofilm formation in culture tubes (top panel) and phase-contrast micrographs (bottom panel) of three C. crescentus strains: wild-type strain CB15, a nonadherent NA1000 strain, and the {Delta}CC_1486 strain, respectively.

Holdfast-specific lectin binding assays of the single-deletion mutants showed that they all bound lectin. In order to observe the holdfasts on these single-deletion mutants, TEM was performed on negatively stained whole-cell mounts. Each single mutant possessed holdfast material at the tip of its stalk (Fig. 4, panels 6 to 8). These results indicate that single mutations in hfsE, pssY, pssZ, and the CC_1486 gene alone do not prevent holdfast synthesis (Fig. 5).

In the set of double-deletion hfsE mutants and its paralogs, the {Delta}pssY {Delta}CC_1486 mutant exhibited essentially wild-type levels of surface adherence and the {Delta}pssY {Delta}pssZ and {Delta}pssZ {Delta}CC_1486 mutants had a similar, slightly reduced ability to bind to a polystyrene surface (Fig. 5). In contrast, the {Delta}hfsE {Delta}pssY and {Delta}hfsE {Delta}pssZ mutants exhibited a noticeable reduction in surface adherence compared to the wild type (Fig. 5). Interestingly, the {Delta}hfsE {Delta}CC_1486 mutant exhibited a significant increase in surface adherence compared to the wild type. All the double mutants bound the holdfast-specific lectin. TEM analyses on whole-cell mounts of these double mutants indicated that although all of them possessed some holdfast material at the tips of their stalks, there were some consistent differences in the holdfast appearance of a large proportion of some double mutants compared to that of the wild type (Fig. 4, panels 10 to 12 and 14 to 16). The holdfast of the {Delta}pssY {Delta}pssZ mutant appeared to be positioned off to one side of the stalk tip (Fig. 4, panel 10) compared to the holdfast of the wild-type CB15 cell (Fig. 4, panel 1). The {Delta}hfsE {Delta}pssY, {Delta}hfsE {Delta}pssZ, and {Delta}hfsE {Delta}CC_1486 mutants appeared to have less holdfast material at the stalk tip (Fig. 4, panels 14 to 16) than the wild-type cell (Fig. 4, panel 1). The holdfast of the {Delta}pssZ {Delta}CC_1486 mutant, on the other hand, appeared to be positioned only at the very tip of the stalk (Fig. 4, panel 12), instead of enveloping the entire stalk tip like that of the wild-type cell (Fig. 4, panel 1). These results revealed that double-combination mutations with hfsE, pssY, pssZ, or CC_1486 do not cause a dramatic reduction in surface adherence or in the inability to synthesize a holdfast but often cause holdfast misplacement at the tip of the stalk.

Of all the triple-combination mutants, only the {Delta}hfsE {Delta}pssY {Delta}pssZ mutant exhibited a severe deficiency in surface adherence (Fig. 5) and a complete loss of binding to AF488-WGA and lacked any holdfast material at the tip of the stalk, based on TEM (Fig. 4, panel 9). In contrast, the {Delta}pssY {Delta}pssZ {Delta}CC_1486, {Delta}hfsE {Delta}pssY {Delta}CC_1486, and {Delta}hfsE {Delta}pssZ {Delta}CC_1486 mutants were all competent in surface adherence, and the {Delta}hfsE {Delta}pssY {Delta}CC_1486 and {Delta}hfsE {Delta}pssZ {Delta}CC_1486 mutants exhibited an increase in surface adherence compared to the wild-type (Fig. 5). The {Delta}hfsE {Delta}pssY {Delta}CC_1486, {Delta}hfsE {Delta}pssZ {Delta}CC_1486, and {Delta}pssY {Delta}pssZ {Delta}CC_1486 mutants all bound the holdfast-specific lectin and possessed holdfast material at the tips of their stalks (Fig. 4, panels 18 to 20), although the holdfast of the {Delta}hfsE {Delta}pssY {Delta}CC_1486 mutant also appeared to be positioned off to one side of the stalk tip, while there appeared to be less holdfast material at the stalk tips of the {Delta}pssY {Delta}pssZ {Delta}CC_1486 and {Delta}hfsE {Delta}pssZ {Delta}CC_1486 mutants. The phenotype of the {Delta}hfsE {Delta}pssY {Delta}pssZ {Delta}CC_1486 quadruple mutant closely mimicked that of the {Delta}hfsE {Delta}pssY {Delta}pssZ triple mutant. The {Delta}hfsE {Delta}pssY {Delta}pssZ mutant did not bind the holdfast-specific lectin, but when complemented by the introduction of a plasmid harboring hfsE fused to its native promoter, 70% of the predivisional cells bound the holdfast-specific lectin, compared to 78% of wild-type predivisional cells. These results indicate that pssY, pssZ, or hfsE is redundant for holdfast synthesis.

hfsI is a paralog of hfsC that is redundant for holdfast synthesis. Unraveling the existence of compensatory paralogs for hfsE led us to reinvestigate the role of hfsC. HfsC is a predicted integral membrane protein with 11 transmembrane helices with sequence similarity to Wzy-like proteins. Wzy proteins are O-antigen polymerases that are involved in the polymerization of high-molecular-weight exopolysaccharide (44) and succinoglycan (50). Previous work has shown that an hfsC mutant could still bind to surfaces and to AF488-WGA lectin, suggesting that hfsC does not play a role in holdfast synthesis, despite the fact that it is located in the holdfast biosynthesis gene cluster or that there was a redundant gene (58). Sequence analysis revealed a possible redundant protein encoded by CC_0499 (hereinafter called hfsI) exhibiting 60% sequence identity to HfsC. We constructed an in-frame hfsI deletion mutant and assayed for any surface adherence and AF488-WGA lectin binding deficiencies. The {Delta}hfsI mutant had close to wild-type levels of surface adherence (Fig. 7A), while the {Delta}hfsC mutant exhibited 78% surface adherence compared to that of the wild type. The levels of AF488 lectin binding of the {Delta}hfsC mutant and the {Delta}hfsI mutant were similar to each other but were slightly reduced compared to that of the wild type (Fig. 7B). We constructed an hfsC and hfsI double mutant and assayed for surface adherence and holdfast-specific lectin binding. The hfsC hfsI double mutant was completely incapable of binding to a polystyrene surface (Fig. 7A) and failed to bind the holdfast-specific lectin AF488-WGA (Fig. 7B). In addition, the holdfast phenotype of the hfsC hfsI double mutant could be complemented by the introduction of a plasmid harboring hfsC fused to the hfsA promoter. We concluded that hfsC and hfsI are redundant for holdfast synthesis.


Figure 7
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  		FIG. 7. (A) Quantification of crystal violet-stained cells of {Delta}hfsC, {Delta}hfsI, and {Delta}hfsC {Delta}hfsI mutants attached to polystyrene. The results represent the means of independent experiments; the means were calculated from a total of nine measurements each derived from three samples per experiment, three independent times. Error bars represent standard deviations. (B) Quantitation of holdfast AF488-WGA labeling of predivisional (PD) cells. A total number of 500 predivisional cells were counted, and the percent values reflect the proportion of AF488-WGA-labeled predivisional cells versus the total of all predivisional cells counted. Error bars represent standard deviations.


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DISCUSSION
 
In this study, we identified a set of polysaccharide biosynthesis genes that are involved in the biosynthesis (hfsEFGH, pssY, and pssZ) and polymerization (hfsC and hfsI) of the repeat unit of the holdfast polysaccharide. Together with the previously identified hfsDAB (58) and hfaABD genes, involved in the export and anchoring of the holdfast polysaccharide to the stalk (28, 29, 10), we defined for the first time a pathway that unifies the distinct processes for holdfast polysaccharide synthesis, export, and anchoring to the stalk.

Since the hfsEFGH and hfsCBAD gene clusters encode the functions required for polysaccharide synthesis, we expected that mutations in any of these genes would abolish surface adhesion and holdfast production; however, our results showed that although hfsDAB, hfsG, and hfsH are essential for surface adhesion and holdfast synthesis, hfsE and hfsF are not. It is not surprising to find that mutations in polysaccharide deacetylase archetypes like HfsH result in a severe deficiency in surface adhesion and holdfast production, because the conversion of an acetylated to a deacetylated form of the sugar may serve as a signal for polysaccharide secretion. For example, there is evidence that the N-deacetylation modification of the E. coli polysaccharide adhesin facilitates its secretion (23). In contrast, hfsE and hfsF are dispensable for surface adhesion and holdfast production, and the dispensability of hfsE is explained by the existence of two paralogs of hfsE, pssY and pssZ. Our results revealed that either hfsE, pssY, or pssZ, but not their paralog the CC_1486 gene, is sufficient for holdfast synthesis and surface adhesion. Intriguingly, strains in which only one of these three redundant paralogs is present exhibit a slight reduction of surface adhesion, which suggests that each gene contributes to the biosynthesis of the holdfast polysaccharide. PssZ is 51% identical to HfsE, and pssZ (CC_2384) is found in a gene cluster on the C. crescentus chromosome with a putative gene for a protein tyrosine phosphatase (CC_2385), a UDP-glucose-4 epimerase (CC_2383), and a UDP-glucose-6 dehydrogenase (CC_2382), suggesting that these gene products may synthesize an activated sugar precursor that could be used in polysaccharide synthesis by the initiating glycosyltransferase, PssZ. PssY exhibits 41% amino acid identity to HfsE, and pssY (CC_0166) is found in a gene cluster on the C. crescentus chromosome with a putative transglutaminase like cysteine peptidase (CC_0167), an O-antigen polymerase gene (CC_0165), and a chain-length-determinant gene (CC_0164). This genetic organization of the pssY gene cluster, comprising a chain-length-determinant protein adjacent to a polymerase protein, is typical of gene clusters involved in the biosynthesis of the O antigen of lipopolysaccharide, suggesting that the primary function of PssY is in lipopolysaccharide synthesis but that pssY can substitute for hfsE and pssZ because of sequence similarity. CC_1486 is unable to compensate for the loss of hfsE and its paralogs, indicating that this gene does not play a role in holdfast polysaccharide synthesis. Compared to the other paralogs, CC_1486 exhibits the lowest amino acid sequence identity to HfsE (37%). CC_1486 is found in a gene cluster with tipN (CC_1485), a gene involved in establishing polarity and the correct placement of polar organelles (22, 30) instead of being clustered with other polysaccharide biosynthetic genes, suggesting that CC_1486 may have other functions in the cell.

Similarly, we have identified hfsI (CC_0499), a paralog of hfsC that is able to compensate for its loss. We observed dramatic defects in surface adhesion and holdfast synthesis caused by an hfsC and hfsI double-deletion mutant. There is precedence for redundancy in Wzy-like proteins. Two versions of Wzy proteins exist in Pseudomonas aeruginosa and Salmonella enterica serovar Anatum (25, 39). Each of the P. aeruginosa Wzy proteins catalyzes a different glycosidic linkage ({alpha} or β) of the O antigen, and mutations of one of the wzy genes result in the production of an O antigen missing the specific {alpha} or β linkage. hfsI is not part of a gene cluster of polysaccharide biosynthesis genes but is instead adjacent to genes encoding a putative RDD (arginine-aspartate-aspartate) family of transporter proteins (CC0498) and a phage tail fiber adhesin (CC_0500). We therefore hypothesized that hfsI primarily functions in holdfast synthesis.

Although it is possible that there may be other flippases that can compensate for the loss of HfsF function, the identification of flippases is made difficult because these proteins are composed mostly of transmembrane helices (12-14) and have no amino acid sequence similarity or an obvious signature sequence except a weakly conserved stretch of approximately 208 amino acids (polysacc_synt domain, Protein Families database of alignments; HMMs, http://cmr.tigr.org/cgi-bin/CMR/HmmReport.cgi?hmm_acc=PF01943) (38). Alternatively, we hypothesized that there may be another polysaccharide efflux system that can translocate the repeat units across the inner membrane, such as the ABC transporter superfamily of proteins that constitutes the second pathway of polysaccharide biosynthesis (2), which is distinct and independent from the Wzy-dependent pathway. A hallmark of the ABC transporter-dependent polysaccharide translocation system is the absence of flippases (66).

Our current understanding of polysaccharide biosynthesis stems from years of research in capsular polysaccharide biosynthesis (62, 66). Capsular polysaccharide biosynthesis and assembly occur by two main mechanisms. The first mechanism governs group 1 capsular polysaccharide synthesis and involves two integral inner membrane proteins in polymer translocation across the inner membrane, namely the polymerase protein Wzy and the flippase protein Wzx (67). The presence of these two proteins defines this pathway. Wzy-dependent pathways typically manufacture heteropolymeric polysaccharides (62). In the second mechanism, which governs group 2 capsular polysaccharide synthesis, polymer translocation is facilitated by an ATP binding cassette (ABC) transporter. The presence of the ABC transporter defines this pathway. Homopolymeric polysaccharides are usually synthesized in an ABC transporter-dependent fashion (7). Based on the presence of the Wzy(s) and Wzx homologs in C. crescentus, HfsC or HfsI, and HfsF, respectively, we propose a working model for the synthesis, export, and anchoring of the holdfast polysaccharide to the stalk that is analogous to group I capsular polysaccharide synthesis in E. coli (Fig. 8). An initiating glycosyltransferase (HfsE, PssY, or PssZ) adds the first sugar to the lipid carrier undecaprenol phosphate on the cytoplasmic side of the inner membrane. Subsequently, the glycosyltransferase HfsG elongates the growing polysaccharide repeat unit. Upon the de-O- or de-N-acetylation of substituted saccharides by the carbohydrate esterase HfsH, the polysaccharide repeat unit is flipped across the inner membrane by the action of the flippase, HfsF, and polymerization of the polysaccharide repeat units is accomplished by the polymerases HfsC and HfsI. The polysaccharide chain undergoes continued polymerization, and the polymerization of the polysaccharide is controlled by the combined action of HfsA and HfsB, representing the periplasmic N-terminal and cytoplasmic C-terminal domains, respectively, of a polysaccharide export protein. The secretin HfsD then provides the channel for export of the polysaccharide across the peptidoglycan and outer membrane. Subsequently, the holdfast polysaccharide is anchored to the cell surface by the concerted action of the holdfast attachment proteins, HfaABD (G. G. Hardy et al., submitted for publication).


Figure 8
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  		FIG. 8. Model of holdfast biosynthesis, export, and attachment in C. crescentus. IM, inner membrane; PG, peptidoglycan; OM, outer membrane, LPS, lipopolysaccharide. The protein names in parentheses are the E. coli homologs. (1) Activated precursors (nucleotide monophospho and diphospho sugars [denoted by gray diamonds]) in the cytoplasm are assembled on a lipid carrier, undecaprenyl phosphate (PP~), by the glycosyltransferase HfsE, PssY, or PssZ. (2) The glycosyltransferase HfsG participates in polysaccharide elongation by transferring sugar subunits to the growing repeat unit. (3) The carbohydrate esterase HfsH deacetylates one or more sugar residues (denoted by black diamonds). The PP~-linked repeats are then flipped across the inner membrane by the flippase HfsF. (4) Repeat units are polymerized by the polymerases HfsC and HfsI. (5) The polysaccharide is exported by the concerted actions of membrane periplasmic auxiliary protein 1, HfsA; a tyrosine autokinase, HfsB; and an outer membrane channel formed by HfsD (6). The polysaccharide is anchored to the cell by the secreted Hfa proteins.

In summary, we showed that there are redundancies for two key enzymes involved in the initiating step and polymerization of the holdfast polysaccharide. To our knowledge, this level of gene redundancy for the membrane glycosyltransferase that initiates the first step in polysaccharide biosynthesis is unprecedented. Interestingly, we did not notice any redundancies in the enzymes involved in translocation through the outer membrane. This finding supports the current paradigm that enzymes involved in polysaccharide synthesis and polymerization are substitutable but the polysaccharide translocation machineries are specialized (66). While hfsEFGH and hfsCBAD encode the proteins required for the biosynthesis and export of the holdfast polysaccharide, the presence of redundant genes involved in the initiating and polymerization steps may provide the ability to add additional sugars and linkages, or both, to the holdfast polysaccharide. Future work will be needed to dissect the precise roles that each of these redundant enzymes plays in polysaccharide synthesis.


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ACKNOWLEDGMENTS
 
We thank the members of the Brun lab and the Kurtz lab for critical readings of the manuscript. We also express special gratitude to Pamela Bonner-Brown, Patrick Curtis, Clay Fuqua, and Ellen M. Quardokus for helpful scientific discussions.

Early stages of this work were supported by grant GM51986 from the National Institutes of Health to Y.V.B and by funds from the College of Agriculture, Forestry and Life Sciences of Clemson University to H.D.K. Most of this work was supported by grant GM077648 from the National Institutes of Health to Y.V.B. and by the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Indiana University, Bloomington, Indiana 47405-3700. Phone: (812) 855-8860. Fax: (812) 855-6705. E-mail: ybrun{at}indiana.edu Back

{triangledown} Published ahead of print on 29 August 2008. Back


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REFERENCES
 
    1
  1. Agladze, K., X. Wang, and T. Romeo. 2005. Spatial periodicity of Escherichia coli K-12 biofilm microstructure initiates during a reversible, polar attachment phase of development and requires the polysaccharide adhesin PGA. J. Bacteriol. 187:8237-8246.[Abstract/Free Full Text]
    2. 2
  2. Alaimo, C., I. Catrein, L. Morf, C. L. Marolda, N. Callewaert, M. A. Valvano, M. F. Feldman, and M. Aebi. 2006. Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO J. 25:967-976.[CrossRef][Medline]
    3. 3
  3. Ausubel, F. M., R. Brent, R. E. Kingston, D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley/Greene, New York, NY.
    4. 4
  4. Becker, A., A. Kleickmann, H. Kuster, M. Keller, W. Arnold, and A. Puhler. 1993. Analysis of the Rhizobium meliloti genes exoU, exoV, exoW, exoT, and exoI involved in exopolysaccharide biosynthesis and nodule invasion: exoU and exoW probably encode glucosyltransferases. Mol. Plant-Microbe Interact. 6:735-744.[Medline]
    5. 5
  5. Becker, A., S. Ruberg, H. Kuster, A. A. Roxlau, M. Keller, T. Ivashina, H. P. Cheng, G. C. Walker, and A. Puhler. 1997. The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J. Bacteriol. 179:1375-1384.[Abstract/Free Full Text]
    6. 6
  6. Bodenmiller, D., E. Toh, and Y. V. Brun. 2004. Development of surface adhesion in Caulobacter crescentus. J. Bacteriol. 186:1438-1447.[Abstract/Free Full Text]
    7. 7
  7. Bronner, D., B. R. Clarke, and C. Whitfield. 1994. Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. Mol. Microbiol. 14:505-519.[CrossRef][Medline]
    8. 8
  8. Bugert, P., and K. Geider. 1995. Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol. Microbiol. 15:917-933.[Medline]
    9. 9
  9. Caufrier, F., A. Martinou, C. Dupont, and V. Bouriotis. 2003. Carbohydrate esterase family 4 enzymes: substrate specificity. Carbohydr. Res. 338:687-692.[CrossRef][Medline]
    10. 10
  10. Cole, J. L., G. G. Hardy, D. Bodenmiller, E. Toh, A. Hinz, and Y. V. Brun. 2003. The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk. Mol. Microbiol. 49:1671-1683.[CrossRef][Medline]
    11. 11
  11. Drummelsmith, J., and C. Whitfield. 1999. Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30). Mol. Microbiol. 31:1321-1332.[CrossRef][Medline]
    12. 12
  12. Entcheva-Dimitrov, P., and A. Spormann. 2004. Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus. J. Bacteriol. 186:8254-8266.[Abstract/Free Full Text]
    13. 13
  13. Erdem, A. L., F. Avelino, J. Xicohtencatl-Cortes, and J. A. Giron. 2007. Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli. J. Bacteriol. 189:7426-7435.[Abstract/Free Full Text]
    14. 14
  14. Evinger, M., and N. Agabian. 1977. Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J. Bacteriol. 132:294-301.[Abstract/Free Full Text]
    15. 15
  15. Feldman, M. F., C. L. Marolda, M. A. Monteiro, M. B. Perry, A. J. Parodi, and M. A. Valvano. 1999. The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J. Biol. Chem. 274:35129-35138.[Abstract/Free Full Text]
    16. 16
  16. Freiberg, C., R. Fellay, A. Bairoch, W. J. Broughton, A. Rosenthal, and X. Perret. 1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature 387:394-401.[CrossRef][Medline]
    17. 17
  17. Geremia, R. A., P. Mergaert, D. Geelen, M. Van Montagu, and M. Holsters. 1994. The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase. Proc. Natl. Acad. Sci. USA 91:2669-2673.[Abstract/Free Full Text]
    18. 18
  18. Gober, J. W., and L. Shapiro. 1992. A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements. Mol. Biol. Cell 3:913-926.[Abstract]
    19. 19
  19. Gonin, M., E. M. Quardokus, D. O'Donnol, J. Maddock, and Y. V. Brun. 2000. Regulation of stalk elongation by phosphate in Caulobacter crescentus. J. Bacteriol. 182:337-347.[Abstract/Free Full Text]
    20. 20
  20. Hekmat, O., K. Tokuyasu, and S. G. Withers. 2003. Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS. Biochem. J. 374:369-380.
    21. 21
  21. Hinnebusch, B. J., and D. L. Erickson. 2008. Yersinia pestis biofilm in the flea vector and its role in the transmission of plague. Curr. Top. Microbiol. Immunol. 322:229-248.[CrossRef][Medline]
    22. 22
  22. Huitema, E., S. Pritchard, D. Matteson, S. K. Radhakrishnan, and P. H. Viollier. 2006. Bacterial birth scar proteins mark future flagellum assembly site. Cell 124:1025-1037.[CrossRef][Medline]
    23. 23
  23. Itoh, Y., J. D. Rice, C. Goller, A. Pannuri, J. Taylor, J. Meisner, T. J. Beveridge, J. F. Preston III, and T. Romeo. 2008. Roles of pgaABCD genes in synthesis, modification, and export of the Escherichia coli biofilm adhesin poly-β-1,6-N-acetyl-D-glucosamine. J. Bacteriol. 190:3670-3680.[Abstract/Free Full Text]
    24. 24
  24. Janakiraman, R. S., and Y. V. Brun. 1999. Cell cycle control of a holdfast attachment gene in Caulobacter crescentus. J. Bacteriol. 181:1118-1125.[Abstract/Free Full Text]
    25. 25
  25. Kaluzny, K., P. D. Abeyrathne, and J. S. Lam. 2007. Coexistence of two distinct versions of O-antigen polymerase, Wzy-{alpha} and Wzy-β, in Pseudomonas aeruginosa serogroup O2 and their contributions to cell surface diversity. J. Bacteriol. 189:4141-4152.[Abstract/Free Full Text]
    26. 26
  26. Kelly, A. J., M. J. Sackett, N. Din, E. M. Quardokus, and Y. V. Brun. 1998. Cell cycle-dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter. Genes Dev. 12:880-893.[Abstract/Free Full Text]
    27. 27
  27. Król, J., and A. Skorupska. 1997. Identification of genes in a Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins. Microbiology 143:1389-1394.[Abstract/Free Full Text]
    28. 28
  28. Kurtz, H. D., Jr., and J. Smit. 1992. Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell. J. Bacteriol. 174:687-694.[Abstract/Free Full Text]
    29. 29
  29. Kurtz, H. D., Jr., and J. Smit. 1994. The Caulobacter crescentus holdfast: identification of holdfast attachment complex genes. FEMS Microbiol. Lett. 116:175-182.[CrossRef][Medline]
    30. 30
  30. Lam, H., W. B. Schofield, and C. Jacobs-Wagner. 2006. A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell. Cell 124:1011-1023.[CrossRef][Medline]
    31. 31
  31. Lawler, M. L., D. E. Larson, A. J. Hinz, D. Klein, and Y. V. Brun. 2006. Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus. Mol. Microbiol. 59:301-316.[CrossRef][Medline]
    32. 32
  32. Leipold, M. D., N. A. Kaniuk, and C. Whitfield. 2007. The C-terminal domain of the Escherichia coli WaaJ glycosyltransferase is important for catalytic activity and membrane association. J. Biol. Chem. 282:1257-1264.[Abstract/Free Full Text]
    33. 33
  33. Lellouch, A. C., and R. A. Geremia. 1999. Expression and study of recombinant ExoM, a β1-4 glucosyltransferase involved in succinoglycan biosynthesis in Sinorhizobium meliloti. J. Bacteriol. 181:1141-1148.[Abstract/Free Full Text]
    34. 34
  34. Levi, A., and U. Jenal. 2006. Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development. J. Bacteriol. 188:5315-5318.[Abstract/Free Full Text]
    35. 35
  35. Li, G., C. S. Smith, Y. V. Brun, and J. X. Tang. 2005. The elastic properties of the Caulobacter crescentus adhesive holdfast are dependent on oligomers of N-acetylglucosamine. J. Bacteriol. 187:257-265.[Abstract/Free Full Text]
    36. 36
  36. Liss, L. R. 1987. New M13 host: DH5{alpha}F' competent cells. Focus 9 3:13.[Medline]
    37. 37
  37. Mack, D., W. Fischer, A. Krokotsch, K. Leopold, R. Hartmann, H. Egge, and R. Laufs. 1996. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear β-1,6-linked glucosaminoglycan: purification and structural analysis. J. Bacteriol. 178:175-183.[Abstract/Free Full Text]
    38. 38
  38. Marolda, C. L., J. Vicarioli, and M. A. Valvano. 2004. Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit. Microbiology 150:4095-4105.[Abstract/Free Full Text]
    39. 39
  39. McConnell, M. R., K. R. Oakes, A. N. Patrick, and D. M. Mills. 2001. Two functional O-polysaccharide polymerase wzy (rfc) genes are present in the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum. FEMS Microbiol. Lett. 199:235-240.[CrossRef][Medline]
    40. 40
  40. Merker, R. I., and J. Smit. 1988. Characterization of the adhesive holdfast of marine and freshwater caulobacters. Appl. Environ. Microbiol. 54:2078-2085.[Abstract/Free Full Text]
    41. 41
  41. Mishra, C., C. E. Semino, K. J. McCreath, H. de la Vega, B. J. Jones, C. A. Jones, and P. W. Robbins. 1997. Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae. Yeast 13:327-336.[CrossRef][Medline]
    42. 42
  42. Mitchell, D., and J. Smit. 1990. Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2. J. Bacteriol. 172:5425-5431.[Abstract/Free Full Text]
    43. 43
  43. Mukhopadhyay, P., J. Williams, and D. Millis. 1988. Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae. J. Bacteriol. 170:5479-5488.[Abstract/Free Full Text]
    44. 44
  44. Nakhamchik, A., C. Wilde, and D. A. Rowe-Magnus. 2007. Identification of a Wzy polymerase required for group IV capsular polysaccharide and lipopolysaccharide biosynthesis in Vibrio vulnificus. Infect. Immun. 75:5550-5558.[Abstract/Free Full Text]
    45. 45
  45. Nierman, W. C., T. V. Feldblyum, M. T. Laub, I. T. Paulsen, K. E. Nelson, J. A. Eisen, J. F. Heidelberg, M. R. Alley, N. Ohta, J. R. Maddock, I. Potocka, W. C. Nelson, A. Newton, C. Stephens, N. D. Phadke, B. Ely, R. T. DeBoy, R. J. Dodson, A. S. Durkin, M. L. Gwinn, D. H. Haft, J. F. Kolonay, J. Smit, M. B. Craven, H. Khouri, J. Shetty, K. Berry, T. Utterback, K. Tran, A. Wolf, J. Vamathevan, M. Ermolaeva, O. White, S. L. Salzberg, J. C. Venter, L. Shapiro, and C. M. Fraser. 2001. Complete genome sequence of Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 98:4136-4141.[Abstract/Free Full Text]
    46. 46
  46. Ong, C. J., M. L. Y. Wong, and J. Smit. 1990. Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus. J. Bacteriol. 172:1448-1456.[Abstract/Free Full Text]
    47. 47
  47. Pierce, D. L., and Y. V. Brun. 2008. Developmental control in Caulobacter crescentus: strategies for survival in oligotrophic environments, p. 10. In D. E. Whitworth (ed.), Myxobacteria multicellularity and differentiation. ASM Press, Washington, DC.
    48. 48
  48. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295.[Free Full Text]
    49. 49
  49. Psylinakis, E., I. G. Boneca, K. Mavromatis, A. Deli, E. Hayhurst, S. J. Foster, K. M. Varum, and V. Bouriotis. 2005. Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis. J. Biol. Chem. 280:30856-30863.[Abstract/Free Full Text]
    50. 50
  50. Reuber, T. L., and G. C. Walker. 1993. Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74:269-280.[CrossRef][Medline]
    51. 51
  51. Rick, P. D., K. Barr, K. Sankaran, J. Kajimura, J. S. Rush, and C. J. Waechter. 2003. Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. J. Biol. Chem. 278:16534-16542.[Abstract/Free Full Text]
    52. 52
  52. Saxena, I. M., R. M. Brown, Jr., M. Fevre, R. A. Geremia, and B. Henrissat. 1995. Multidomain architecture of β-glycosyl transferases: implications for mechanism of action. J. Bacteriol. 177:1419-1424.[Free Full Text]
    53. 53
  53. Schoenlein, P. V., L. S. Gallman, and B. Ely. 1989. Organization of the flaFG gene cluster and identification of two additional genes involved in flagellum biogenesis in Caulobacter crescentus. J. Bacteriol. 171:1544-1553.[Abstract/Free Full Text]
    54. 54
  54. Simon, R., U. Prieffer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1:784-790.[CrossRef]
    55. 55
  55. Skerker, J. M., and L. Shapiro. 2000. Identification and cell cycle control of a novel pilus system in Caulobacter crescentus. EMBO J. 19:3223-3234.[CrossRef][Medline]
    56. 56
  56. Sloan, G. P., C. F. Love, N. Sukumar, M. Mishra, and R. Deora. 2007. The Bordetella Bps polysaccharide is critical for biofilm development in the mouse respiratory tract. J. Bacteriol. 189:8270-8276.[Abstract/Free Full Text]
    57. 57
  57. Smit, J., and N. Agabian. 1984. Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays. J. Bacteriol. 160:1137-1145.[Abstract/Free Full Text]
    58. 58
  58. Smith, C. S., A. Hinz, D. Bodenmiller, D. E. Larson, and Y. V. Brun. 2003. Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus. J. Bacteriol. 185:1432-1442.[Abstract/Free Full Text]
    59. 59
  59. Steiner, K., R. Novotny, K. Patel, E. Vinogradov, C. Whitfield, M. A. Valvano, P. Messner, and C. Schaffer. 2007. Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a. J. Bacteriol. 189:2590-2598.[Abstract/Free Full Text]
    60. 60
  60. Stephens, C., A. Reisenauer, R. Wright, and L. Shapiro. 1996. A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. Proc. Natl. Acad. Sci. USA 93:1210-1214.[Abstract/Free Full Text]
    61. 61
  61. Tsang, P. H., G. Li, Y. V. Brun, L. B. Freund, and J. X. Tang. 2006. Adhesion of single bacterial cells in the micronewton range. Proc. Natl. Acad. Sci. USA 103:5764-5768.[Abstract/Free Full Text]
    62. 62
  62. Valvano, M. A. 2003. Export of O-specific lipopolysaccharide. Front. Biosci. 8:s452-s471.[Medline]
    63. 63
  63. Vuong, C., J. M. Voyich, E. R. Fischer, K. R. Braughton, A. R. Whitney, F. R. DeLeo, and M. Otto. 2004. Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell. Microbiol. 6:269-275.[CrossRef][Medline]
    64. 64
  64. Wang, L., D. Liu, and P. R. Reeves. 1996. C-terminal half of Salmonella enterica WbaP (RfbP) is the galactosyl-1-phosphate transferase domain catalyzing the first step of O-antigen synthesis. J. Bacteriol. 178:2598-2604.[Abstract/Free Full Text]
    65. 65
  65. Wang, X., J. F. Preston III, and T. Romeo. 2004. The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation. J. Bacteriol. 186:2724-2734.[Abstract/Free Full Text]
    66. 66
  66. Whitfield, C. 2006. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75:39-68.[CrossRef][Medline]
    67. 67
  67. Whitfield, C., and A. Paiment. 2003. Biosynthesis and assembly of Group 1 capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria. Carbohydr. Res. 338:2491-2502.[CrossRef][Medline]
    68. 68
  68. Wilbur, W. J., and D. J. Lipman. 1983. Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA 80:726-730.[Abstract/Free Full Text]
    69. 69
  69. Xicohtencatl-Cortes, J., V. Monteiro-Neto, M. A. Ledesma, D. M. Jordan, O. Francetic, J. B. Kaper, J. L. Puente, and J. A. Giron. 2007. Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7. J. Clin. Investig. 117:3519-3529.[CrossRef][Medline]

Journal of Bacteriology, November 2008, p. 7219-7231, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.01003-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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