Identiﬁcation of Genes Required for Synthesis of the ... · esis. These data suggest that the hfs...

JOURNAL OF BACTERIOLOGY, Feb. 2003, p. 1432–1442 Vol. 185, No. 40021-9193/03/$08.00�0 DOI: 10.1128/JB.185.4.1432–1442.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of Genes Required for Synthesis of the AdhesiveHoldfast in Caulobacter crescentus

Chris S. Smith, Aaron Hinz, Diane Bodenmiller, David E. Larson, and Yves V. Brun*Department of Biology, Indiana University, Bloomington, Indiana 47405

Received 11 July 2002/Accepted 1 November 2002

Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobactercrescentus is mediated by a polar organelle called the “holdfast,” which enables the bacterium to form stablemonolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizesto the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolationof adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfastbiogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfastattachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted ina severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeledlectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutantsfail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequencesimilarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequencesimilarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in theperiplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved insecretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogen-esis. These data suggest that the hfs genes play an important role in holdfast export.

The dimorphic bacterium Caulobacter crescentus undergoesprogrammed developmental changes during its cell divisioncycle, differentiating from a nonreplicative, motile stage(swarmer cell) to a sessile, replicative stage (stalked cell) (3).After an obligatory gap period, which is approximately one-third the length of one cell cycle, the swarmer cell sheds itsflagellum and forms a stalk at the pole that once held theflagellum. The stalked cell elongates and divides, giving rise tothe two different cell types. Primarily inhabiting freshwater andmarine environments dilute in nutrients, Caulobacter formsbiofilm monolayers on abiotic and biotic surfaces (29). Stableattachment to surfaces requires an adhesive holdfast. Attachedto the tip of the stalk, the holdfast can be used to maintainassociations with various surfaces, but never with the cell sur-face of another Caulobacter. Holdfasts can often be seen at thecenter of a group of cells (called a “rosette”) coordinated bytheir stalks (20).

The holdfast appears at the incipient stalked pole early dur-ing the differentiation of swarmer cells into stalked cells, re-sulting in its positioning at the tip of the stalk (9). Competitionexperiments with fluorescently labeled lectins, proteins with aspecific binding affinity for a polysaccharide moiety, demon-strated that the holdfast is composed in part of oligomers ofthe sugar residue, N-acetylglucosamine (GlcNac) (13). Thefluorescently labeled lectin fluorescein isothiocyanate-wheatgerm agglutinin (FITC-WGA) was shown to bind specificallyto the holdfast (13). The holdfast is resistant to both glycolytic

and proteolytic enzymes, except for lysozyme and chitinase,which are specific for oligomers of GlcNac (13).

A previous screen undertaken to identify adhesion-deficientmutants of C. crescentus CB2 identified four regions in thegenome potentially responsible for holdfast biogenesis (14).Mutants with insertions mapping to one of these regions, theholdfast attachment (hfa) operon (10), failed to bind to sur-faces but also did not attach their holdfasts to the stalk effi-ciently, a defect known as “holdfast shedding.” Studies of hfaexpression suggest that the holdfast attachment proteins arepreloaded in swarmer cells. The expression of the hfaA pro-moter occurs maximally in the swarmer compartment of thepredivisional cell, despite the fact that swarmer cells do nothave exposed holdfasts (9).

Genes involved in polar development are also thought toaffect holdfast biogenesis. Mutations in pleC and in podJ affectthe ordered development of C. crescentus, leading to the ab-sence or inactivation of the polar structures. Mutants withmutations in pleC and in podJ do not exhibit rosette formation,which can be an indicator of the presence of the holdfast (33).Nonetheless, the direct observation of the absence of a hold-fast in these mutants has not been made.

In order to identify genes required for holdfast biogenesis,we screened a C. crescentus CB15 transposon mutant libraryfor the absence of attachment to cellulose acetate. Twentymutant strains with severe adhesion deficiencies were furtherphenotypically characterized by cell morphology, lectin binding(FITC-WGA), bacteriophage sensitivity, and motility. Basedon the results of this analysis, the adhesion-deficient mutantswere divided into three classes: developmental mutants (podJand pleC), holdfast attachment mutants (hfa), and a novel classof holdfast biogenesis mutants (hfs, for holdfast synthesis). Thehfs genes are the first nonregulatory genes shown to be re-

* Corresponding author. Mailing address: Department of Biology,Jordan Hall 142, Indiana University, 1001 E. 3rd St., Bloomington, IN47405-3700. Phone: (812) 855-8860. Fax: (812) 855-6705. E-mail:[email protected].

1432

on Decem

ber 10, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

quired for holdfast synthesis or export. The proteins predictedto be encoded by these genes are an inner membrane-associ-ated periplasmic protein (HfsA), an outer membrane lipid-modified secretin (HfsD), and a novel protein (HfsB). Theseproteins bear significant sequence similarity to polysaccharideexport proteins of gram-negative bacteria, suggesting that theyare required for the export of the holdfast polysaccharide.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and mutagenesis. The bacterial strainsand plasmids used during the course of this study are listed in Table 1. AllCaulobacter strains were cultured with peptone-yeast extract (PYE) medium (20)or Hutner base, imidazole, buffered glucose-glutamate (HIGG) minimal medium(21) at 30°C. The antibiotics kanamycin and nalidixic acid (20 �g/ml each) wereused to supplement the Caulobacter media as necessary. Escherichia coli strainswere cultured with Luria-Bertani (LB) medium at 37°C. LB medium was sup-

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Description or construction Source or reference

StrainsE. coli

DH5�F� �80dlacZ�M15 �(lacZYA-argF)U169 endA1 recA1 hsdR17(r� m�) deoR thi-1supE44 �� gyrA96 relA1

12

MT607 pRK600 containing helper strain for conjugation 25S17-1 E. coli 294::RP4-2 (Tc::Mu)(Km::Tn7) 26YB1329 S17-1 �pir, pUT::mini-Tn5lacZ2 M. R. K. Alley, unpublished

C. crescentusCB15 Wild type 20CB2A rsaA (S-layer) mutant of CB2 28NA1000 syn-1000, previously called NA1000; a derivative of CB15 that does not

synthesize holdfasts6

YB2861 CB15 pleC100::mini-Tn5lacZ2 This workYB2862 CB15 hfsB101::mini-Tn5lacZ2 This workYB2863 CB15 podJ102::mini-Tn5lacZ2 This workYB2864 CB15 podJ103::mini-Tn5lacZ2 This workYB2865 CB15 hfsA106::mini-Tn5lacZ2 This workYB2866 CB15 podJ107::mini-Tn5lacZ2 This workYB2867 CB15 podJ108::mini-Tn5lacZ2 This workYB2868 CB15 pleC109::mini-Tn5lacZ2 This workYB2869 CB15 hfsA111::mini-Tn5lacZ2 This workYB2870 CB15 hfsA118::mini-Tn5lacZ2 This workYB2877 CB15 hfsB176::mini-Tn5lacZ2 This workYB2878 CB15 hfsA125::mini-Tn5lacZ2 This workYB2879 CB2A hfsD172::mini-Tn5lacZ2 This workYB2779 CB15 hfaB113::mini-Tn5lacZ2 This workYB2780 CB15 hfaB115::mini-Tn5lacZ2 This workYB2781 CB15 hfa-120::mini-Tn5lacZ2 This workYB2782 CB15 hfa-121::mini-Tn5lacZ2 This workYB2783 CB15 hfa-122::mini-Tn5lacZ2 This workYB2784 CB15 hfaB123::mini-Tn5lacZ2 This workYB3738 CB15 hfa-105::mini-Tn5lacZ2 This workYB2833 CB15 hfsA::pNPTShfsA This workYB2837 CB15 hfsB::pNPTShfsB This workYB2841 CB15 hfsC::pNPTShfsC This workYB2845 CB15 hfsD::pNPTShfsD This workYB2832 CB15 pNPTS138 hfsA This workYB2836 CB15 pNPTS138 hfsB This workYB2840 CB15 pNPTS138 hfsC This workYB2845 CB15 pNPTS138 hfsD This workYB3136 CB15N::pLW56 (also CMS27) 34

PlasmidspRJ41 Cosmid 4-1, a pLaFR5-derived cosmid containing the hfaABDC region of CB15 9placHfa7 4.64-kb PstI-HindIII fragment containing the hfa promoter through the middle

of hfaC from pRJ41 was cloned into the PstI-HindIII sites of pRKlac290This work

pNPTS138 Litmus 38 derivative, oriT sacB Kanr Alley, unpublishedpNPTShfsA pNPTS138 parent vector containing 500-bp fragments upstream and

downstream of hfsAThis work

pNPTShfsB pNPTS138 parent vector containing 500-bp fragments upstream anddownstream of hfsB

This work

pNPTShfsC pNPTS138 parent vector containing 500-bp fragments upstream anddownstream of hfsC

This work

pNPTShfsD pNPTS138 parent vector containing 500-bp fragments upstream anddownstream of hfsD

This work

VOL. 185, 2003 CAULOBACTER HOLDFAST BIOGENESIS GENES 1433

on Decem


.org/D

ownloaded from

http://jb.asm.org/

plemented with kanamycin at 50 �g/ml when necessary. In order to create alibrary of adhesion mutants, a mini-Tn5lacZ2 transposon mutagenesis of C.crescentus CB15 and CB2A was performed as previously described (8). Aliquotsof mutagenized cells were stored at �80°C until plated. Transduction of kana-mycin resistance markers from the transposon mutants into C. crescentus CB15was performed as previously described (5).

Mutant identification. Genomic DNA isolated from the adhesion mutants wasused as a template for arbitrarily primed PCR (AP-PCR) to determine thelocation of each mini-Tn5lacZ2 insertion (8, 18). DNA sequencing was per-formed on an ABI 3700 automated DNA sequencer at the Institute for Molec-ular and Cellular Biology, Indiana University, Bloomington. C. crescentus ge-nome sequence data (16) were acquired from The Institute for GenomicResearch (TIGR). Sequence analysis was performed with the Genetics Com-puter Group GCG Wisconsin package, v.10 (Accelrys), and BLAST program(National Center for Biotechnology Information). Signal sequence and localiza-tion predictions were made with PSORT (15). Transmembrane prediction wasperformed with TMHMM (30a).

Southern blot analysis (2) was used to confirm the locations of the mini-Tn5lacZ2 insertions. Southern blot probes were prepared with the Roche DIGHigh Prime digoxigenin-UTP labeling kit. A 2.1-kb hfa probe, extending from230 bp upstream of the hfaA start codon to 612 bp downstream of the hfaD startcodon, was PCR amplified from the pRJ41 template with the oligonucleotideshfaA215Pst and hfaC2263HindIII. A 2.0-kb hfs probe, extending from 509 bpupstream of the hfsD stop codon to 661 bp downstream of the hfsA start codon,was PCR amplified from CB15 genomic DNA with the oligonucleotides5gumBC1367 and 3gumBC1568. A 3.0-kb pleC probe, extending from 188 bpupstream of the pleC start codon to 281 bp downstream of the stop codon, wasPCR amplified from CB15 genomic DNA with the oligonucleotides pleCfor andpleCrev. A 2.0-kb podJ probe, extending from 1,839 bp upstream to 175 bpdownstream of the start codon, was PCR amplified from CB15 genomic DNAwith the oligonucleotides 5podJ2508 and 3podJ4522. The oligonucleotide se-quences are available from the authors upon request. For Southern hybridiza-tion, genomic DNA prepared from the insertion mutants was digested withappropriate enzymes as follows. A 6-kb fragment containing the hfa operon wasreleased from the restriction enzyme digestion of genomic DNA with PstI. A4.93-kb fragment containing the hfs operon was released following the digestionof genomic DNA with HpaI and ClaI. A 3.13-kb fragment containing pleC wasreleased after digestion of genomic DNA with SalI. A 5.85-kb fragment contain-

ing podJ was released after the digestion of genomic DNA with SfiI. Transposoninsertions mapping to these fragments were identified by Southern hybridizationas shifts in their molecular weight compared to that of the wild type by QuickHyb(Stratagene) and anti-DIG-UTP alkaline phosphatase Fab fragment (Roche) asdescribed by the manufacturers.

Deletion analysis. Clean in-frame deletions of hfsA, hfsB, hfsC, and hfsD werecreated by homologous recombination between two fragments—one upstreamand one downstream of the gene—as previously described (8, 31). The upstreamand downstream fragments were cloned into the suicide vector pNPTS138, whichcarries a sacB cassette. hfsABamup, hfsAXhoup, hfsAXhoend, and hfsAHinendwere used to PCR amplify 500-bp DNA fragments directly upstream and down-stream of hfsA. hfsBBamu2, hfsBXhou2, hfsBXhoe2, and hfsBHine2 were usedto PCR amplify 500-bp DNA fragments directly upstream and downstream ofhfsB. hfsCBamup, hfsCXhoup, hfsCXhoend, and hfsCHinend were used to PCRamplify 500-bp DNA fragments directly upstream and downstream of hfsC.hfsDBamup, hfsDXhoup, hfsDXhoend, and hfsDHinend were used to PCRamplify the 500-bp downstream and upstream fragments of hfsD. The oligonu-cleotide sequences are available from the authors upon request. The upstreamfragments were cut with BamHI and XhoI, while the downstream fragments werecut with XhoI and HindIII. Each pair of fragments was ligated into the BamHI-and HindIII-digested vector pNPTS138. Deletions were confirmed by colonyPCR with primers used to clone fragments positioned upstream and downstreamof the gene. All deletions were transduced by �Cr30 (5) into a wild-type CB15background by using the closely linked kanamycin resistance marker from strainCMS27 (34). Deletions of the hfs genes were complemented by a low-copy-number plasmid carrying each gene. An 870-bp PCR fragment, extending 28 bpfrom the hfsD stop codon to 102 bp from the hfsD start codon, was amplified withhfsDxbadn2 and hfsDkpnup2. The PCR product was then cloned into the low-copy-number vector plac290 between the XbaI and KpnI restriction sites. A2.3-kb PCR fragment, extending 106 bp from the start codon of hfsA to 22 bpdownstream of hfsB, was amplified with hfsAbamup2 and hfsBhindend3. Theresulting PCR product was also cloned into plac290 between a BamHI site anda HindIII site. These plasmid constructs were transformed into E. coli S17-1 andmated into the various mutants. Rosette formation in liquid culture was deter-mined by phase-contrast light microscopy.

Microscopy. Lectin binding assays were performed as previously described(17). Two microliters of 5-mg/ml FITC-WGA (Molecular Probes) was added to200 �l of exponential-phase culture and incubated with gentle shaking at roomtemperature for 15 min. The culture was diluted fivefold with double-distilledwater (ddH2O), and the cells were harvested by centrifugation at 13,000 g for4 min (9). The cells were resuspended in 20 �l of SlowFade B (MolecularProbes) (9). Labeled cells were examined by epifluorescence on a Nikon E800light microscope equipped with a FITC-HYQ filter cube and 100 Plan Apo oilobjective. Image capture was carried out with a Princeton Instruments cooledcharge-coupled device (CCD) camera and Metamorph imaging software, v.4.5.

Electron microscopy was used to determine the nature of the adhesion defectof the mini-Tn5lacZ2 insertion mutants. Cells were mounted onto 0.25% Form-var-coated copper grids (Ted Pella) for 30 min. Each grid was washed three timesin a drop of water, followed by positive staining with 7.5% uranyl magnesiumacetate for 5 min. The grids were then washed again three times in a drop ofwater. Grids were examined with a JEOL JEM-1010 transmission electron mi-croscope (TEM) at 60 kV.

Surface binding assays. In order to screen for adhesion mutants, disks ofcellulose acetate (Apollo WO100C) were applied to patch plates of mini-Tn5lacZ2 mutants and screened as previously described (17).

As a semiquantitative method of measuring adhesion defects, approximately50 �l of exponential-phase culture concentrated to an optical density at 600 nm(OD600) of 4.8 was spotted onto a piece of cellulose acetate (or borosilicateglass). The cells were allowed to attach for 3 min, before each drop was removedwith a pipette tip. The cellulose acetate was washed thoroughly with a strong jetof cold water. The remaining adherent cells were visualized by staining the diskswith 0.1% Coomassie blue (R-250) in 10% acetic acid–10% isopropanol for 3min. Residual stain was removed by washing thoroughly in cold water.

The glass coverslip binding assay was adapted from an existing protocol (17).Overnight cultures were grown in PYE medium at 30°C. In the morning, cultureswere diluted in PYE medium to an OD600 of 0.15. Glass coverslips were dippedinto ethanol, exposed to flame, and placed into the well of a tissue culture dish(six-well, tissue culture, treated polystyrene dish, catalog no. 3506; Corning). A1.5-ml sample of cell culture was added to each well. The tissue culture plate wasplaced on a shaker (90 to 100 rpm) at 30°C and incubated for 4 to 5 h. Afterincubation, coverslips were rinsed with a steady stream of ddH2O from a waterbottle for 10 to 20 s. The coverslips were placed cell side up in a humid chamberand incubated with 50 �l of FITC-WGA at a concentration of 0.05 �g/�l for 30

TABLE 2. Phenotypic classification of the holdfast-deficientinsertional mutants

Strain (mutant gene)

Presence of:

�CbK/�Cr30resistant

Celluloseacetatebinding

Stalks Motility

YB2861 (pleC100) � � � Yes/noYB2868 (pleC109) � � � Yes/no

YB2863 (podJ102) � � � Yes/noYB2864 (podJ103) � � � Yes/noYB2866 (podJ107) � � � Yes/noYB2867 (podJ108) � � � Yes/no

YB2862 (hfsB101) � � � No/noYB2865 (hfsA106) � � � No/noYB2869 (hfsA111) � � � No/noYB2870 (hfsA118) � � � No/noYB2877 (hfsB176) � � � No/noYB2878 (hfsA125) � � � No/noYB2879 (hfsD172) � � � No/no

YB2779 (hfaB113) � � � No/noYB2780 (hfaB115) � � � No/noYB2781 (hfa-120) � � � No/noYB2782 (hfa-121) � � � No/noYB2783 (hfa-122) � � � No/noYB2784 (hfaB123) � � � No/noYB3738 (hfa-105) � � � NDa

a ND, not determined.

1434 SMITH ET AL. J. BACTERIOL.

on Decem


.org/D

ownloaded from

http://jb.asm.org/

min. The coverslips were rinsed with water as described above. The coverslipswere placed onto a slide with 2 �l of SlowFade A (Molecular Probes) solution(glycerol–phosphate-buffered saline), and examined by phase and epifluores-cence microscopy.

The ability of cells to bind to various surfaces was measured by incubatingthose strains in 96-well microtiter plates. Each culture was grown in a test tubeat 30°C until exponential phase was reached, at an OD600 of 0.6. Two hundredmicroliters of each culture was pipetted into the wells of the microtiter plate,where it was incubated for 15 min at room temperature. After allowing time forthe cells to attach, a strong jet of cold water was used to displace any looselyattached cells. The microtiter plate was briefly air dried before 200 �l of 0.1%Coomassie blue (R-250) in a mixture of 10% acetic acid and 10% isopropanolwas pipetted into the wells. After incubation for 15 min, each well was emptiedby pipetting out the stain. The microtiter plate was washed with a strong jet ofcold water and air dried.

Swarming motility assay. Two microliters of overnight culture in PYE mediumwere stabbed into 0.3% semisolid agar PYE medium. The plates were incubatedat 30°C for 3 to 5 days.

Phage sensitivity assay. Phage sensitivity assays for determining resistance topolar caulophages, �Cbk and �Cr30, were performed as previously described(23).

RESULTS

Identification of adhesion mutants. While genes requiredfor holdfast attachment to the stalk have been identified pre-viously, no genes required for holdfast synthesis or export havebeen characterized. Therefore, we performed a screen in C.crescentus CB15 to identify genes required for holdfast biosyn-thesis and export. The screen used binding to cellulose acetateas its basis. Approximately 9,000 mini-Tn5lacZ2 mutants werescreened for absence of adhesion to cellulose acetate, resultingin the identification of 236 potential adhesion mutants. Inorder to decrease the number of potential false negatives,these adhesion mutants were put through a second screen thattested the adhesion of equal numbers of cells from exponen-tial-phase cultures to cellulose acetate. Twenty mutants wereidentified that did not adhere to cellulose acetate. These mu-tants were characterized by phase-contrast microscopy fortheir ability to form rosettes, overall cellular morphology, andmotility (Tables 2 and 3). Each insertional mutation was trans-duced into the wild-type background, and the resulting trans-ductants were examined for cellulose acetate and lectin bind-ing. All transductants displayed the same phenotype as theinsertional mutants, indicating that the observed phenotypewas the result of a single mini-Tn5lacZ2 insertion. Thirteen ofthe nonadherent mutants were deficient only in rosette forma-tion, suggesting specific defects in holdfast biogenesis. Six ofthe mutants had additional developmental defects, suggestingthat the mutations were in genes involved in the regulation ofdevelopment. The site of mini-Tn5lacZ2 insertion in variousmutants was determined by sequencing either AP-PCR prod-ucts or products of PCRs that used a transposon primer and aprimer from the suspected insertion region in cases in whichthe insertion had been roughly mapped by Southern hybrid-ization.

Developmental mutants. The two mutants YB2861 andYB2868 lacked discernible stalk structures and did not formrosettes (Fig. 1 and Table 2). However, upon growth in HIGGmedia limited for inorganic phosphate (30 �M), both strainsformed stalks (data not shown). These mutants were also un-able to swarm in 0.3% semisolid agar (Table 2). However, bothmutants were flagellated when examined by phase-contrastmicroscopy with a flagellar stain (data not shown). In addition,

YB2861 and YB2868 were resistant to the polar caulophage�CbK, but were sensitive to �Cr30 (Table 2). The caulophage�CbK binds to the polar pili of swarmer cells (27), suggestingthat YB2861 and YB2868 lack pili.

YB2861 and YB2868 were assayed for their binding of thefluorescently labeled lectin FITC-WGA, which specificallybinds oligomers of N-acetylglucosamine, a component of theholdfast. To ensure that counted cells had stalks, we scoredonly predivisional cells. Wild-type strain CB15 had 78% label-ing, whereas the holdfast-deficient strain NA1000 had less than1% labeling. Both YB2861 and YB2868 had less than 1% ofcells labeled with FITC-WGA, indicating a severe deficiency inholdfast synthesis (Table 3).

Mapping of the insertions in YB2861 and YB2868 indicatedthat the transposons were inserted in the pleC gene at approx-imately 500 bp from the end of the gene and exactly 1,523 bpfrom the start codon, respectively (Fig. 2). PleC is a dynami-cally localized histidine kinase required for polar development(7, 30, 33, 35). Our phenotypic characterization of these mu-tants matches previous findings.

Four other insertional mutants also displayed a pleiotropicphenotype. YB2863, YB2864, YB2866, and YB2867 failed toform rosettes, but otherwise their cellular morphology was wild

TABLE 3. Holdfast FITC-WGA labeling and rosette formation ofholdfast biogenesis mutants

Strain (mutant gene)% of predivisionalcells with FITC-WGA bindinga

Presence of:

Holdfastsheddingb

Rosetteformation

CB15 78 � �NA1000 0 � �YB2861 (pleC100) 1 � �YB2862 (hfsB101) 0 � �YB2863 (podJ102) 7 � �YB2864 (podJ103) 1 � �YB2865 (hfsA106) 2 � �YB2866 (podJ107) 2 � �YB2867 (podJ108) 0 � �YB2868 (pleC109) 0 � �YB2869 (hfsA111) 0 � �YB2870 (hfsA118) 0 � �YB2877 (hfsB176) 3 � �YB2878 (hfsA125) NDc ND NDYB2879 (hfsD172) 33 � �YB2779 (hfaB113) 2 � �YB2779 (hfaB113)/plac290hfa7 ND ND �YB2780 (hfaB115) 3 � �YB2780 (hfaB115)/plac290hfa7 ND ND �YB2781 (hfa-120) 15 � �YB2781 (hfa-120)/plac290hfa7 ND ND �YB2782 (hfa-121) 2 � �YB2782 (hfa-121)/plac290hfa7 ND ND �YB2783 (hfa-122) 0 � �YB2783 (hfa-122)/plac290hfa7 ND ND �YB2784 (hfaB123) 2 � �YB2784 (hfaB123)/plac290hfa7 ND ND �YB3738 (hfa-105) 31 � �YB3738 (hfa-105)/plac290hfa7 ND ND �

a FITC-WGA-labeled predivisional cells were counted for every 100 predivi-sional cells. Lectin binding experiments were performed three times.

b The number of shed holdfasts for every strain was compared to the numberof shed holdfasts per count of 100 predivisional cells for the holdfast-negativestrain NA1000.

c ND, not determined.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

type (Fig. 1 and Table 2). Phage sensitivity assays showed thatthese mutants were resistant to the polar caulophage �CbK(Table 2), suggesting that they lack pili (8a; this work). Swarm-ing motility in 0.3% semisolid agar was reduced relative to thatof the wild type, although cultures of the insertion mutantsclearly contained motile swarmer cells (Table 2). Three mu-tants (YB2864, YB2866, and YB2867) had no FITC-WGAlabeling of predivisional cells (Table 3). The other mutant,YB2863, displayed approximately 7% labeling (Table 3).

The insertions in YB2863, YB2864, YB2866, and YB2867were mapped to the polar organelle development gene, podJ(Fig. 2). The insertions interrupted the gene 277 (YB2863),800 (YB2864), 787 (YB2866), and 638 (YB2867) nucleotides(nt) after the start codon (Fig. 2). podJ mutants have beenshown to have defects in chemotaxis, rosette formation, andsensitivity to phage �CbK (33), and our results agree withthese findings.

Our analysis of pleC and podJ mutants extends previousresults by showing that these mutants are unable to bind sur-faces and to synthesize a holdfast. Since these mutants exhibitpleiotropic defects, pleC and podJ are clearly not specificallyinvolved in holdfast synthesis.

Holdfast attachment mutants. Seven mutants (YB2779,YB2780, YB2781, YB2782, YB2783, YB2784, and YB3738)were deficient in adhesion to surfaces, were wild type in termsof cellular morphology (Table 2), and formed rosettes at a lowfrequency or not at all compared to wild-type cells. Thesemutants frequently shed their holdfasts into the medium asmeasured by FITC-WGA assays (Table 3). The mutants ex-hibited various degrees of stalk-associated FITC-WGA label-ing (Table 3), indicating that the attachment of the holdfast tothe tip of the stalk was weak. This is similar to the phenotypeof previously identified holdfast attachment (hfa) mutants.

We used Southern hybridization to map the transposon in-sertions and found that all of them mapped to the previouslyidentified (10, 11) hfaABDC gene cluster (data not shown). Allthe mutants were fully complemented by plasmid placHfa7,which contains the hfaABD genes, indicating that the sheddingphenotype was due to mutations in the hfaABD region and thatnone of the mutations had dominant effects (Table 3). PCRamplification with primers in the hfaABDC region indicatedthat insertions 113, 115, and 123 were in hfaB (Fig. 2). The factthat all seven hfa adhesion mutants identified in this screen andthree hfa mutants identified previously (10) map to this genecluster strongly suggests that there are no other nonessentialgenes required for holdfast attachment to the stalk.

The hfa mutants were also analyzed by using a binding assayin which cultures of cells were allowed to adhere to a glasscoverslip (13, 17). Wild-type strain CB15 was able to formdense cellular biofilms. These biofilms were associated withspots of fluorescence, indicating the presence of holdfasts (Fig.3). Phase-contrast microscopy of coverslips exposed to theholdfast-deficient strain NA1000 indicated that no cells re-mained bound, and fluorescence microscopy showed thatFITC-WGA was unable to bind to the coverslips (Fig. 3). Thecells of all of the hfa mutants exhibited very poor binding toglass; however, spots of fluorescence densely stained the glass,indicating the presence of holdfasts (Fig. 3). These resultsindicate that the hfa mutants are able to synthesize holdfast

FIG. 1. Morphology of developmental mutants compared to that ofwild-type strain CB15. The cellular morphology of representative pop-ulations of C. crescentus (A) CB15, (B) YB2861, and (C) YB2863 wasimage captured with a Nikon E800 light microscope equipped with a100 Apo oil objective, Princeton Instruments cooled CCD camera,and Metamorph imaging software, v.4.5.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

material and that this holdfast material can bind to glass;however, the holdfasts do not remain attached to the stalk.

Holdfast biogenesis mutants. Seven insertion mutants(YB2862, YB2865, YB2869, YB2870, YB2877, YB2878, andYB2879) were adhesion deficient, yet wild type in other re-spects (Table 2). None of the mutants bound lectin to anyappreciable degree or shed holdfasts (Table 3).

All seven insertions were located in a previously uncharac-terized gene cluster (Fig. 2). The first two genes in the cluster,hfsD (CC2432) and hfsA (CC2431), are divergently transcribedbased on their orientation. The start codon of hfsB (CC2430)lies 78 bp downstream of the preceding gene, hfsA. The genedownstream of hfsB, hfsC (CC2429), overlaps the stop codonof the preceding gene (Fig. 2). This suggests that hfsB and hfsCare translationally coupled and form an operon; it is not clearif hfsA is cotranscribed with them. Three of the insertions inthis region were located in hfsA, interrupting the gene 614(YB2878), 680 (YB2865), and 1,085 (YB2869) bp after thestart codon (Fig. 2). Three insertions were located in hfsB,interrupting the gene 77 (YB2862), 115 (YB2870), and 279(YB2877) bp from the start codon (Fig. 2). Another insertionwas located in the N-terminal portion of hfsD (YB2879), whichinterrupts the gene 240 bp after the start codon (Fig. 2).

Sequence analysis indicated that hfsA, hfsC, and hfsD arehomologous to genes involved in exopolysaccharide (EPS) syn-thesis and export. hfsA encodes a predicted protein of 501amino acids (aa) (GCG). This protein is 21% identical and37% (1) similar to GumC from the plant pathogen Xanthamo-nas campestris. hfsB is predicted to encode a protein of 233 aa(GCG). HfsB has no homologues in the sequence databasesand possesses no obvious signal sequences with which to pre-dict its localization. hfsC is predicted to encode a protein of422 aa (GCG). HfsC is 29% identical and 39% (1) similar toExoQ from the plant symbiont Rhizobium melliloti. hfsD ispredicted to encode a protein of 246 aa (GCG). HfsD is 32%

identical and 50% (1) similar to Wza from E. coli. Wza is anouter membrane lipoprotein secretin that functions to exportpolysaccharides (4).

hfsA, hfsB, and hfsD, but not hfsC, are required for holdfastsynthesis. In order to rule out possible polar effects of inser-tions on the observed phenotype of the hfs mutants, cleanin-frame deletions of each gene were created. The deletions ofhfsA, hfsB, and hfsD (YB2833, YB2837, and YB2845) resultedin the same cellulose acetate adhesion defect observed with theinsertion mutants. In order to determine if the adhesion defectobserved with the deletion mutants and the insertion mutantswas global for different types of surfaces, we performed asurface binding assay with polypropylene, polyvinylchloride,polystyrene, and borosilicate glass. We found that the hfsA,hfsB, and hfsD deletion mutants were defective in attaching toall of these surfaces (data not shown). C. crescentus CB15,however, bound noticeably to each of the surfaces, whereas thenegative control NA1000 did not bind. The cellular morphol-ogy of the hfs deletion mutants was indistinguishable from thatof the wild type, as determined by phase-contrast microscopy(Table 2). When examining whole-cell mounts of hfsA, hfsB,and hfsD deletion mutants by TEM, however, the stalked cellsall lacked dark staining material where the holdfast normallyresides (Fig. 4). No other morphological differences betweenthe wild type and the deletion mutants were apparent. The factthat hfsA, hfsB, and hfsD deletion mutants lack holdfasts wasbolstered by the lectin binding results. The deletion mutantsdid not bind FITC-WGA (Fig. 5). The hfsC deletion mutant(YB2841) exhibited no phenotypic differences from the wildtype for binding to surfaces or to FITC-WGA (Fig. 5), indi-cating that hfsC does not have a role in holdfast synthesis andin binding to the surfaces tested. The hfs deletion mutants weretransduced into a clean background in order to further rule outunlinked mutations as the cause of the holdfast biogenesisphenotype. In each case, we recovered transductants that did

FIG. 2. Location of transposon insertions in holdfast-deficient mutants. Genes are delineated by boxes with corresponding gene name andGenBank accession number. Arrows indicate the direction of transcription for each gene relative to one another. Wedges indicate the mappedlocations of mini-Tn5lacZ2 insertions.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

not form rosettes at the predicted frequency, which suggeststhat the deletions are the sole cause of the phenotype (data notshown). Introduction of a low-copy-number plasmid carryinghfsD fully restored rosette formation in the hfsD deletion mu-tant, and introduction of a plasmid carrying hfsAB fully re-stored rosette formation to the hfsA and hfsB deletion mutants(data not shown).

DISCUSSION

In this paper, we report the results of a saturating mini-Tn5screen for Caulobacter mutants with adhesion defects. Thesemutants can be grouped into three classes: developmental mu-tants (pleC and podJ), holdfast synthesis or export mutants

(hfs), and mutants that disrupt the attachment of the holdfastto the tip of the stalk (hfa).

The developmental mutants displayed additional polar de-fects, in addition to a lack of adhesion and an absence of aholdfast. The pleC mutants were deficient in stalk synthesis andflagellar rotation and were resistant to caulophage �CbK asshown previously (33). PleC is a histidine kinase that localizesto the flagellated pole of swarmer cells and of late predivisionalcells and is involved in coordinating many aspects of polarmorphogenesis (35). The podJ mutants had defects in chemo-taxis and were resistant to �CbK, as shown previously (33), inaddition to their deficiency in surface adhesion and holdfastsynthesis. We have recently shown that PodJ is localized to the

FIG. 3. Glass coverslip binding assay of holdfast-shedding transposon mutants. Phase-contrast (left panels) and fluorescence (right panels)images of representative areas of glass slides submerged in cultures of various strains and assayed for lectin binding with FITC-WGA are shownfor the following strains: CB15 (A and B), NA1000 (C and D), YB2779 (E and F), YB2782 (G and H), YB2780 (I and J), YB2783 (K and L),YB2781 (M and N), and YB2784 (O and P).


on Decem


.org/D

ownloaded from

http://jb.asm.org/

flagellated pole of swarmer cells, disappears from that pole instalked cells, and localizes to the opposite pole, where it re-mains for the rest of the cell cycle (8a). PodJ is required forPleC localization (8a), suggesting that the holdfast synthesisdeficiency of podJ mutants may be due to PleC delocalization(8a).

The hfa genes were identified previously (11) and are tran-scribed from a promoter upstream of hfaA (9, 10; J. Cole, D.Bodenmiller, and Y. V. Brun, unpublished observations). Non-polar mutants of hfaA, hfaB, and hfaD produce holdfasts, asshown by their binding to fluorescently labeled lectins, but donot attach them to the stalk at wild-type levels (Cole et al.,unpublished). This leads to an overall defect in adhesion. HfaB

shares some similarity with the curli attachment gene, csgG,from E. coli and has been experimentally proven to be a li-poprotein (Cole et al., unpublished). Curli are proteinaceousfibers that mediate attachment to surfaces. hfaA and hfaDshare no significant sequence similarity with any characterizedproteins. Both HfaB and HfaD have been shown to localize tothe stalk (Cole et al., unpublished), suggesting that they aredirectly involved in attaching the holdfast to the stalk. The hfagenes appear to be the only nonessential genes required for theattachment of the holdfast to the tip of the stalk based on thefact that 10 independent insertions have been isolated at thislocus (10; this work).

The hfs genes are likely to be involved in holdfast export.hfsA, hfsB, and hfsD mutants all show a dramatic defect incellular adhesion to a variety of surfaces. Lectin binding studiesalso show that hfs mutants do not shed holdfasts in the me-dium, further separating this new class of mutants from the hfamutants. Lectin binding experiments and electron microscopyshow the absence of holdfast material at the tip of the stalk.The gene products of the hfs operon have a high degree ofsequence similarity to polysaccharide export components.HfsD resembles an oligomeric secretin, Wza, from E. coli. Anouter membrane lipoprotein, Wza functions as a hexamericchannel for export of capsular polysaccharide to the surface(4). Wza belongs to the outer membrane auxiliary, or OMA,family of proteins (19). HfsD also carries a potential lipopro-tein sorting signal sequence at its N-terminal portion (15). Inaddition, based upon the presence of noncharged residues atthe �2 and �3 positions of the predicted mature lipoprotein,HfsD is predicted to reside in the outer leaflet of the outermembrane (15, 24).

HfsA has sequence similarity in its 400-aa periplasmic loopto the polysaccharide transport protein, GumC, from X.campestris. GumC has been shown to be involved in the exportof the EPS xanthan gum from the cytoplasm to the cellularexterior (32). Export of high-molecular-weight EPS is requiredfor the invasion of plant hosts by X. campestris. GumC is amember of membrane periplasmic auxiliary (MPA-1) family ofpolysaccharide export proteins (19). One of the defining char-acteristics of the genes encoding the MPA-1 is the nearbypresence of an OMA gene, which participates in EPS export(19). Normally, members of the MPA-1 protein family share aninner membrane topology and possess a large cytoplasmic do-main containing a Walker A (GXXXXGKT/S) ATP bindingmotif, which is responsible for providing the energy necessaryfor driving the export process in the form of ATP hydrolysis(19). Although HfsA possesses the required two-transmem-brane-helix topology, with a large periplasmic loop, it lacks asizeable cytoplasmic domain (30a) and the necessary Walker Amotif (1). However, two other members of the MPA-1 familyalso lack the relatively well-conserved C-terminal cytoplasmicdomain: GumC and OtnB (19). In these cases, energy may beprovided by an ABC cassette transport system. Members of theMPA-2 family, such as KpsE and CtrB, work in concert withthese cytoplasmic membrane transporters (19). It is unknownhow the 400-aa periplasmic loop of the MPA-1 proteins func-tions to facilitate transport of polysaccharide residues from thecytoplasmic membrane face of the periplasm to the outermembrane face.

HfsC has sequence similarity to ExoQ from R. melliloti.

FIG. 4. Analysis of the holdfasts of hfs deletion mutants as com-pared to CB15 and NA1000 by TEM. Electron micrographs of repre-sentative members of stalked cell population (positive stained with7.5% uranyl magnesium acetate) at 50,000 magnification. (A) CB15.(B) NA1000. (C) CB15 �hfsA. (D) CB15 �hfsB. (E) CB15 �hfsC.(F) CB15 �hfsD. Bars, 100 nm.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

HfsC adopts a similar membrane topology to ExoQ, with 11(rather than 12) predicted transmembrane helices spanningthe inner membrane (30a). The region of highest similaritybetween the two proteins occurs in an 75-aa periplasmic loop

(30a) in which they are located (33% identical and 46% simi-lar). ExoQ has been shown to be directly involved in the po-lymerization of a high-molecular-weight EPS, succinoglycan,which is required for nodule formation (22). An hfsC deletion

FIG. 5. Detection of N-acetylglucosamine in the holdfast of hfs deletion mutants. FITC-WGA was used to label the holdfasts of CB15 (A),NA1000 (B), CB15 �hfsA (C), CB15 �hfsB (D), CB15 �hfsC (E), and CB15 �hfsD (F).


on Decem


.org/D

ownloaded from

http://jb.asm.org/

mutant does not have an adhesion-deficient phenotype to sur-faces such as borosilicate glass, polyvinylchloride, polypro-pylene, and polystyrene and binds lectin at wild-type levels.The data do not rule out an effect on binding to differentsurfaces, but clearly hfsC is not required for holdfast synthesis.

Finally, HfsB has no significant similarity to available pro-tein sequences. Combined with the absence of a signal se-quence or hydrophobic regions (15, 30a), it is difficult to pro-pose a role for HfsB in holdfast biogenesis.

No biosynthetic genes were isolated in the cellulose acetatescreen; however, the possibility remains that hfsB functions inthis capacity. One explanation for the absence of biosyntheticmutants is that the screen is not saturated. However, we haveidentified multiple insertions at every locus (seven at hfa, sevenat hfs, two at pleC, and four at podJ). On the basis of thisevidence, we believe the screen has been saturated for knock-out mutations that abolish adhesion. Another possibility is thatmutations in some genes required for optimal adhesion havebeen missed in this screen. For example, mutations in pilus andflagellar structural genes reduce, but do not completely abolishadhesion (D. Bodenmiller and Y. V. Brun, unpublished data;Cole et al., unpublished). Similarly, it is possible that mutationsthat eliminate some yet unknown component of the holdfastreduce but do not abolish adhesion to the cellulose acetateused in the screen. Isolation of these potential mutants wouldrequire a screen for binding to materials with different surfacechemistries. Finally, since N-acetylglucosamine is a componentof both the holdfast and peptidoglycan, mutations that abolishits synthesis would be lethal and would not be representedamong our collection of insertional knockouts. EPS subunitsare synthesized in the cytoplasm from precursors. The EPSsubunits are then attached to a lipid carrier residing in theinner membrane, which is energized to flip across the mem-brane to the periplasm. In a poorly understood process, theEPS subunits are oligomerized into their final form, modifiedif necessary, and exported to the outer membrane, where theyare transported to the exterior matrix via a secretin. Such asystem would take advantage of the fact N-acetylglucosaminesubunits are present in the periplasm for peptidoglycan bio-synthesis, where the hfs gene products could utilize N-acetyl-glucosamine for holdfast biogenesis.

In conclusion, we have identified the first nonregulatorygenes known to be required for holdfast synthesis. Based ontheir sequence, we hypothesize that the gene products of thehfs cluster are involved in EPS export from the periplasmicspace to the cellular exterior. This particular area of the EPSbiosynthesis pathway has not been fully elucidated. HfsA mightprovide periplasmic export functions for holdfast polysaccha-ride subunits with the energy provided by an ABC transportcomplex. It is also possible that HfsA plays a role in the pro-cessing of holdfast subunits, which could be required for ex-port. HfsD probably terminates the export branch of holdfastbiogenesis by serving as the site of secretion to the eventual siteof holdfast localization, the tip of the stalk.

We thank members of our laboratory for critical reading of themanuscript and for helpful discussions.

This work was supported by a National Institutes of Health grant(GM51986) and a National Science Foundation CAREER Award(MCB-9733958) to Y.V.B., a Beckman Scholarship to A.H., an Amer-ican Society for Microbiology Undergraduate Research Fellowship to

D.L., and a National Institutes of Health Predoctoral Fellowship(GM07757) to C.S.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

2. 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. JohnWiley/Greene, New York, N.Y.

3. Brun, Y., G. Marczynski, and L. Shapiro. 1994. The expression of asymmetryduring cell differentiation. Annu. Rev. Biochem. 63:419–450.

4. Drummelsmith, J., and C. Whitfield. 2000. Translocation of group 1 capsularpolysaccharide to the surface of Escherichia coli requires a multimeric com-plex in the outer membrane. EMBO J. 19:57–66.

5. Ely, B., and R. C. Johnson. 1977. Generalized transduction in Caulobactercrescentus. Genetics 87:391–399.

6. Evinger, M., and N. Agabian. 1977. Envelope-associated nucleoid from Cau-lobacter crescentus stalked and swarmer cells. J. Bacteriol. 132:294–301.

7. Fukuda, A., K. Miyakawa, H. Iida, and Y. Okada. 1976. Regulation of polarsurface structures in Caulobacter crescentus: pleiotropic mutations affect thecoordinate morphogenesis of flagella, pili, and phage receptors. Mol. Gen.Genet. 149:167–173.

8. 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.

8a.Hinz, A., D. E. Larson, C. S. Smith, and Y. V. Brun. The Caulobactercrescentus polar organelle development protein PodJ is differentially local-ized and is required for polar targeting of the PleC development regulator.Mol. Microbiol., in press.

9. Janakiraman, R. S., and Y. V. Brun. 1999. Cell cycle control of a holdfastattachment gene in Caulobacter. J. Bacteriol. 181:1118–1125.

10. Kurtz, H. D., Jr., and J. Smit. 1992. Analysis of a Caulobacter crescentus genecluster involved in attachment of the holdfast to the cell. J. Bacteriol. 174:687–694.

11. Kurtz, H. D., Jr., and J. Smit. 1994. The Caulobacter crescentus holdfast:identification of holdfast attachment complex genes. FEMS Microbiol. Lett.116:175–182.

12. Liss, L. R. 1987. New M13 host: DH5�F� competent cells. Focus 9:13.13. Merker, R. I., and J. Smit. 1988. Characterization of the adhesive holdfast of

marine and freshwater caulobacters. Appl. Environ. Microbiol. 54:2078–2085.

14. Mitchell, D., and J. Smit. 1990. Identification of genes affecting productionof the adhesion organelle of Caulobacter crescentus CB2. J. Bacteriol. 172:5425–5431.

15. Nakai, K., and M. Kanehisa. 1991. Expert system for predicting proteinlocalization sites in gram-negative bacteria. Proteins 11:95–110.

16. Nierman, W. C., T. V. Feldblyum, M. T. Laub, I. T. Paulsen, K. E. Nelson,J. 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 Caulobactercrescentus. Proc. Natl. Acad. Sci. USA 98:4136–4141.

17. Ong, C. J., M. L. Y. Wong, and J. Smit. 1990. Attachment of the adhesiveholdfast organelle to the cellular stalk of Caulobacter crescentus. J. Bacteriol.172:1448–1456.

18. O’Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation inPseudomonas fluorescens WCS365 proceeds via multiple, convergent signal-ling pathways: a genetic analysis. Mol. Microbiol. 28:449–461.

19. Paulsen, I. T., A. M. Beness, and M. H. Saier, Jr. 1997. Computer-basedanalyses of the protein constituents of transport systems catalysing export ofcomplex carbohydrates in bacteria. Microbiology 143:2685–2699.

20. Poindexter, J. S. 1964. Biological properties and classification of the Cau-lobacter group. Bacteriol. Rev. 28:231–295.

21. Poindexter, J. S. 1978. Selection for nonbuoyant morphological mutants ofCaulobacter crescentus. J. Bacteriol. 135:1141–1145.

22. Reuber, T. L., and G. C. Walker. 1993. Biosynthesis of succinoglycan, asymbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74:269–280.

23. Schoenlein, P. V., L. S. Gallman, and B. Ely. 1989. Organization of the flaFGgene clusters and identification of two additional genes involved in flagellumbiogenesis in Caulobacter crescentus. J. Bacteriol. 171:1544–1553.

24. Seydel, A., P. Gounon, and A. P. Pugsley. 1999. Testing the “�2 rule” forlipoprotein sorting in the Escherichia coli cell envelope with a new geneticselection. Mol. Microbiol. 34:810–821.

25. Sharma, S. B., and E. R. Signer. 1990. Temporal and spatial regulation of thesymbiotic genes of Rhizobium meliloti in planta revealed by transposon Tn5-gusA. Genes Dev. 4:344–356.

26. Simon, R., U. Prieffer, and A. Puhler. 1983. A broad host range mobilization


on Decem


.org/D

ownloaded from

http://jb.asm.org/

system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1:784–790.

27. Skerker, J. M., and L. Shapiro. 2000. Identification and cell cycle control ofa novel pilus system in Caulobacter crescentus. EMBO J. 19:3223–3234.

28. Smit, J., and N. Agabian. 1984. Cloning of the major protein of the Cau-lobacter crescentus periodic surface layer: detection and characterization ofthe cloned peptide by protein expression assays. J. Bacteriol. 160:1137–1145.

29. Smit, J., C. S. Sherwood, and R. F. Turner. 2000. Characterization of highdensity monolayers of the biofilm bacterium Caulobacter crescentus: evalu-ating prospects for developing immobilized cell bioreactors. Can. J. Micro-biol. 46:339–349.

30. Sommer, J. M., and A. Newton. 1989. Turning off flagellum rotation requiresthe pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenicpathways in Caulobacter crescentus. J. Bacteriol. 171:392–401.

30a.Sonnhammer, E. L., G. V. Heijne, and A. Krogh. 1998. A hidden Markov,model for predicting transmembrane helices in protein sequences, p. 175–182. In J. I. Glasgow, T. G. Littlejohn, F. Major, R. H. Lathrop, D. Sankoff,

C. Sensen (ed.) Proceedings of the 6th International Conference on Intelli-gent Systems for Molecular Biology. AAAI Press, Menlo Park, Calif.

31. 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.

32. Vojnov, A. A., A. Zorreguieta, J. M. Dow, M. J. Daniels, and M. A. Dankert.1998. Evidence for a role for the gumB and gumC gene products in theformation of xanthan from its pentasaccharide repeating unit by Xanthomo-nas campestris. Microbiology 144:1487–1493.

33. Wang, S. P., P. L. Sharma, P. V. Schoenlein, and B. Ely. 1993. A histidineprotein kinase is involved in polar organelle development in Caulobactercrescentus. Proc. Natl. Acad. Sci. USA 90:630–634.

34. West, L., D. Yang, and C. Stephens. 2002. Use of the Caulobacter crescentusgenome sequence to develop a method for systematic genetic mapping. J.Bacteriol. 184:2155–2166.

35. Wheeler, R. T., and L. Shapiro. 1999. Differential localization of two histi-dine kinases controlling bacterial cell differentiation. Mol. Cell 4:683–694.


on Decem


.org/D

ownloaded from

http://jb.asm.org/

Identiﬁcation of Genes Required for Synthesis of the ... · esis. These data suggest that the hfs...

Documents

Transcript of Identiﬁcation of Genes Required for Synthesis of the ... · esis. These data suggest that the hfs...