Development and Use of a Gene Deletion Strategy for … › content › jb › 193 › 10 › 2418.full.pdf · genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for

JOURNAL OF BACTERIOLOGY, May 2011, p. 2418–2428 Vol. 193, No. 100021-9193/11/$12.00 doi:10.1128/JB.00117-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Development and Use of a Gene Deletion Strategy forFlavobacterium johnsoniae To Identify the RedundantGliding Motility Genes remF, remG, remH, and remI�†

Ryan G. Rhodes, Halley G. Pucker, and Mark J. McBride*Department of Biological Sciences, University of Wisconsin—Milwaukee, P.O. Box 413, Milwaukee, Wisconsin 53201

Received 25 January 2011/Accepted 10 March 2011

Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought toinvolve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distalgenes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation ofspreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A genedeletion strategy was developed to determine whether these genes are involved in gliding. A spontaneousstreptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmidrestored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The genedeletion strategy employed a suicide vector carrying wild-type rpsL and used streptomycin for counterselection.This approach was used to delete the region spanning remF, remG, and fjoh_0982. The mutant cells formedspreading colonies, demonstrating that these genes are not required for normal motility. Analysis of thegenome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had noeffect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formednonspreading colonies. The motility defects resulting from the combination of mutations suggest that theparalogous proteins perform redundant functions in motility. The rpsL counterselection strategy allowsconstruction of unmarked mutations to determine the functions of individual motility proteins or to analyzeother aspects of F. johnsoniae physiology.

Cells of Flavobacterium johnsoniae crawl rapidly over sur-faces, a process referred to as gliding motility (20). Flavobac-terium gliding does not rely on flagella or pili, but rather in-volves the functioning of a novel motor that is thought topropel cell surface adhesins, such as SprB (15, 25). sprB is partof a seven-gene operon that spans 29.3 kbp of DNA (28).Mutations in any of the four distal genes of the operon (sprC,sprD, sprB, and sprF) cause motility defects that result in theformation of nonspreading colonies on agar. Analysis of acollection of polar and nonpolar mutations and complementa-tion with constructs expressing subsets of the genes demon-strated that sprC, sprD, sprB, and sprF are each required fornormal motility and for the formation of spreading colonies onagar. SprF appears to be required for secretion of SprB to thecell surface via a novel protein secretion system referred to asthe PorSS (28). PorSSs are only found in members of theBacteroidetes phylum, and they are not closely related to thetype I to type VII bacterial protein secretion systems (11, 32).Mutations in gldN and sprT, which are thought to encode twoof the core components of the PorSS, result in defects insecretion of SprB and of an extracellular chitinase (29, 32).Cells with mutations in sprF fail to secrete SprB, but they retainthe ability to secrete chitinase (28). SprF is thought to be an

adapter to the PorSS that is needed for secretion of SprB, butnot for secretion of other substrates, such as chitinase. Thereare multiple paralogs of sprF in the genome, and the productsof these genes may serve as adapters to the PorSS to allow thesecretion of other protein substrates. The exact functions ofSprC and SprD are not known, but they may also support SprBfunction.

The involvement of the three proximal genes of the sprBoperon (fjoh_0984, fjoh_0983, and fjoh_0982) in gliding motil-ity is less certain. Disruption of fjoh_0983 by plasmid-mediatedinsertion caused defects in gliding that resulted in the forma-tion of nonspreading colonies (28). However, the demon-strated polarity of this mutation on the expression of sprC,sprD, sprB, and sprF as well as the partial complementation ofthis mutant with plasmids carrying sprC, sprD, sprB, and sprFraised the possibility that the motility defects were the result ofpolarity rather than of involvement of Fjoh_0983 in motility.

F. johnsoniae has become the model organism for studies ofbacteroidete gliding motility, and a wide variety of genetictools have been developed for its manipulation. These includemethods of gene transfer by conjugation, electroporation andtransduction (23, 29), transposons for random mutagenesis (4,23), suicide vectors for construction of polar insertions in genesof interest (1), plasmids for complementation analyses (1, 16,23), and reporter plasmids to analyze gene expression (7).While multiple antibiotic resistance markers have been iden-tified that allow selection of plasmids or transposons in F.johnsoniae, the only counterselectable strategy that was suc-cessfully employed involved phage resistance and was thuslimited to construction of mutations in genes whose disruptionresulted in resistance to phages (29). Frequently used counter-

* Corresponding author. Mailing address: Department of BiologicalSciences, 181 Lapham Hall, University of Wisconsin—Milwaukee,3209 N. Maryland Ave., Milwaukee, WI 53211. Phone: (414) 229-5844.Fax: (414) 229-3926. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 18 March 2011.

2418

on July 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

http://jb.asm.org/

selectable strategies, such as those involving sucrose sensitivityconferred by sacB, failed to work in F. johnsoniae (M.McBride, unpublished results). Here we report the develop-ment of a system for making unmarked mutations that is basedon use of F. johnsoniae rpsL, which encodes the small subunitribosomal protein S12, as a counterselectable marker. Thisapproach relies on the dominance of the wild-type (streptomy-cin-sensitive) rpsL over a streptomycin-resistant rpsL allele andhas been used in other bacteria to select for loss of integratedplasmids that carry wild-type rpsL (8, 19, 31, 33). Using thisstrategy we constructed a strain lacking fjoh_0984 (remF),fjoh_0983 (remG), and fjoh_0982 and demonstrated that thisstrain exhibits normal motility. Further analysis revealed thatremF and remG are redundant motility genes. Cells lackingremF and its paralog (remH) and cells lacking remG and itsparalog (remI) exhibited motility defects that were not ob-served with any of the single mutations.

MATERIALS AND METHODS

Bacterial strains, bacteriophages, plasmids, and growth conditions. F. john-soniae ATCC 17061 (UW101) was the wild-type strain used in this study (21, 24).F. johnsoniae strains were grown in Casitone-yeast extract (CYE) medium at30°C, as previously described (23). To observe colony spreading, F. johnsoniaewas grown on PY2 agar medium (1) at 25°C. Motility medium (MM) was usedto observe movement of individual cells in wet mounts (18). The bacteriophagesactive against F. johnsoniae that were used in this study were �Cj1, �Cj13, �Cj23,�Cj28, �Cj29, �Cj42, �Cj48, and �Cj54 (6, 27, 34). Sensitivity to bacteriophageswas determined essentially as previously described, by spotting 5 �l of phagelysate (109 PFU/ml) onto lawns of cells in CYE overlay agar (14). The plateswere incubated for 24 h at 25°C to observe lysis. Strains and plasmids used in thisstudy are listed in Table 1. The plasmids used for complementation were allderived from pCP1 and have copy numbers of approximately 10 in F. johnsoniae(1, 16, 23). Primers used in this study are listed in Table S1 of the supplementalmaterial. Antibiotics were used at the following concentrations when needed:ampicillin, 100 �g/ml; cefoxitin, 100 �g/ml; erythromycin, 100 �g/ml; kanamycin,35 �g/ml; streptomycin, 100 �g/ml; tetracycline, 20 �g/ml.

Isolation of the streptomycin-resistant rpsL mutant CJ1827. Streptomycin-resistant F. johnsoniae cells were obtained by plating 109 wild-type cells on CYEagar containing streptomycin. Streptomycin-resistant clones were streaked forisolation on fresh medium, and the rpsL gene from each clone was PCR ampli-fied using primers 964 and 965 and sequenced. Point mutations in the rpsL geneconferring streptomycin resistance were identified by comparison to the wild-type rpsL gene sequence.

To determine if the wild-type rpsL gene was dominant to the mutant rpsLalleles, the wild-type rpsL gene was cloned into the shuttle vector pCP23. Spe-cifically, a 711-bp fragment spanning the wild-type rpsL gene and its presumedpromoter region was amplified using primers 979 and 980, which were designedwith engineered SphI and XbaI restriction sites, respectively. The fragment wasdigested with SphI and XbaI and cloned into the corresponding sites of pCP23to generate pRR50. pRR50 was introduced into streptomycin-resistant F. john-soniae by triparental conjugation as previously described (14), except that thehelper strain carried pRK2013 instead of R702.

Construction of the rpsL-containing suicide vector pRR51. To generate un-marked mutations using rpsL as a counterselectable marker, the wild-type rpsLgene was cloned into the suicide vector pLYL03. Primers 981 and 982 weredesigned with engineered EcoRI restriction sites and used to amplify a 681-bpfragment spanning rpsL and its putative promoter. The fragment was digestedwith EcoRI and cloned into the EcoRI site of pLYL03 to generate pRR51 (Fig.1). Orientation of the rpsL fragment in pRR51 was determined by sequencing.

Construction of the sprB deletion mutant CJ1922. A 2.1-kbp fragment span-ning the 3� end of sprD and including the first 168 bp of sprB was amplified usingprimers 1032 (introducing a BamHI site) and 1033 (introducing a SalI site). Thefragment was digested with BamHI and SalI and ligated into pRR51, which hadbeen digested with the same enzymes, to generate pRR66. A 2.8-kbp fragmentspanning sprF, pgk, and the final 222 bp of sprB was amplified with primers 720(introducing a SalI site) and 727 (introducing an SphI site). The fragment wasdigested with SalI and SphI and fused to the upstream region of sprB by ligation

with pRR66, which had been digested with the same enzymes, to generate thedeletion construct pRR67 (Fig. 2).

Plasmid pRR67 was introduced into the streptomycin-resistant wild-type F.johnsoniae strain CJ1827 by triparental conjugation followed by growth on CYEagar containing erythromycin to select for integration of the plasmid into thegenome by homologous recombination. An erythromycin-resistant (streptomy-cin-sensitive) clone was grown overnight in CYE in the absence of antibiotics,and loss of the plasmid by a second recombination event was selected by growthon PY2 agar medium containing streptomycin. Deletion of sprB in CJ1922 wasconfirmed by PCR amplification using primers 848 and 947, which flank the sprBcoding sequence, and sequencing the resulting 1.6-kbp product.

Construction of CJ1883, which has a deletion spanning remF, remG, andfjoh_0982. To generate a deletion in the three proximal genes of the sprB operon,a 2.5-kbp fragment spanning fjoh_0987, fjoh_0986, fjoh_0985, and the first 50 bpof fjoh_0984 (remF) was amplified using primers 983 (introducing a BamHI site)and 984 (introducing an XbaI site). The PCR fragment was digested with BamHIand XbaI and ligated into pRR51, which had been digested with the sameenzymes, to generate pRR52. The 2.4-kbp region spanning the final 191 bp offjoh_0982, sprC, and the 5� end of sprD was amplified with primers 985 (intro-ducing an XbaI site) and 986 (introducing a PstI site). The amplified product wasdigested with XbaI and PstI and ligated into pRR52, which had been digestedwith the same enzymes, to generate the deletion construct pRR53 (Fig. 2).

Plasmid pRR53 was introduced into CJ1827, and the double recombinationevent resulting in the deletion of remF, remG, and fjoh_0982 was selected for asdescribed above, except that CYE agar medium containing streptomycin wasused for the counterselection step. Streptomycin-resistant clones were tested forthe deletion by PCR using primers 831 and 842, which flanked the region ofinterest and resulted in either a 3.4-kbp PCR product for clones retaining remF,remG, and fjoh_0982 or a 1.2-kbp PCR product for clones in which these threegenes had been deleted. The deletion in strain CJ1883 was confirmed by se-quencing the 1.2-kbp PCR product.

Deletion of remH and remI. Separate constructs were generated to makedeletions of the remF paralog, remH (fjoh_3206), and the remG paralog, remI(fjoh_3194). For remH, a 2.7-kbp fragment spanning the 5� end of fjoh_3203,fjoh_3204, fjoh_3205, and the final 89 bp of remH was amplified with primers 999(introducing a BamHI site) and 1000 (introducing a SalI site). The PCR frag-ment was digested with BamHI and SalI and ligated into pRR51, which had beendigested with the same enzymes, to generate pRR55. The 2.6-kbp region up-stream of remH spanning the 5� end of fjoh_3209, fjoh_3208, fjoh_3207, and thefirst 60 bp of remH was amplified with primers 1001 (introducing a SalI site) and1002 (introducing an SphI site). The upstream fragment was digested with SalIand SphI and ligated into pRR55, which had been digested with the sameenzymes, to generate the remH deletion construct pRR61 (Fig. 3A). PlasmidpRR61 was used to generate remH deletions in various backgrounds as describedabove. Primers 1022 and 1023 flanking remH were used to identify streptomycin-resistant clones carrying the deletion. A 0.6-kbp PCR product was obtained forclones carrying the deletion, and a 0.9-kbp PCR product was obtained for clonesretaining the wild-type remH allele.

To generate a deletion in remI, a 2.7-kbp fragment spanning the 5� end offjoh_3191, fjoh_3192, fjoh3193, and the first 112 bp of remI was amplified withprimers 995 (introducing a BamHI site) and 996 (introducing a SalI site). ThePCR fragment was digested with BamHI and SalI and ligated into pRR51, whichhad been digested with the same enzymes, to generate pRR54. The 2.6-kbpregion downstream of remI spanning the 3� end of fjoh_3197, fjoh_3196,fjoh_3195, and the final 149 bp of remI was amplified with primers 997 (intro-ducing a SalI site) and 998 (introducing an SphI site). The downstream fragmentwas digested with SalI and SphI and ligated into pRR54, which had been digestedwith the same enzymes, to generate the remI deletion construct pRR60 (Fig. 3B).Plasmid pRR60 was used to generate remI deletions in various backgrounds asdescribed above. Primers 1020 and 1021 flanking remI were used to identifystreptomycin-resistant clones carrying the remI deletion. A 0.7-kbp product wasobtained for clones carrying the deletion, and a 1.8-kbp product was obtained forclones retaining the wild-type remI allele.

Construction of complementation plasmids. Plasmids carrying remF and remGwere constructed using shuttle vectors pCP23 and pCP29 and transferred tovarious F. johnsoniae strains by triparental conjugation for complementationstudies. We previously constructed pRR68, which carries remF, remG, andfjoh_0982 (28). To construct a plasmid containing only remF and remG, primers718 (introducing a BamHI site) and 1039 (introducing an SphI site) were used toamplify a 2.3-kbp fragment spanning remF and remG and the experimentallydetermined promoter region (28). The fragment was digested with BamHI andSphI and ligated into pCP23, which had been digested with the same enzymes, togenerate pRR72 (Fig. 2). Plasmids containing only remF or remG were also

VOL. 193, 2011 F. JOHNSONIAE GLIDING GENES remF, remG, remH, AND remI 2419


.org/D

ownloaded from

http://jb.asm.org/

constructed. An 881-bp fragment spanning remF and its promoter region wasamplified using primers 718 (introducing a BamHI site) and 1044 (introducing anSphI site). The fragment was digested with BamHI and SphI and ligated intopCP23, which had been digested with the same enzymes, to generate pRR73(Fig. 2). To construct a plasmid containing remG, a 1.9-kbp fragment spanningremG was amplified using primers 1038 and 1039, which were designed withengineered BamHI and SphI restriction sites, respectively. The fragment wasdigested with BamHI and SphI and ligated into pCP23 to generate pRR69 (Fig.2). The remG fragment in pRR69 did not carry its own promoter but wasoriented in the same direction as orf1 in pCP23 (2) and resulted in expressionfrom the vector promoter.

Plasmids containing the remF and remG paralogs, remH and remI, were alsoconstructed. To construct a plasmid containing remH, a 0.9-kbp fragment span-ning remH and its putative promoter was amplified with primers 1042 and 1043,

which were designed with engineered SalI and XmaI restriction sites, respec-tively. The fragment was digested with SalI and XmaI and ligated into pCP29,which had been digested with the same enzymes, to generate pRR75 (Fig. 3A).To generate a plasmid carrying remI, primers 1036 (introducing an SphI site) and1037 (introducing a BamHI site) were used to amplify a 2.0-kbp fragmentspanning remI and its putative promoter region. The fragment was digested withSphI and BamHI and inserted into pCP23, which had been digested with thesame enzymes, resulting in pRR70 (Fig. 3B). The 2.0-kbp KpnI-SphI fragment ofpRR70 spanning remI was ligated into pCP29, which had been digested with thesame enzymes, to generate pRR71.

Microscopic observations of cell movement. Wild-type and mutant cells of F.johnsoniae were examined for movement over glass by using phase-contrastmicroscopy. Cells in MM were spotted onto a glass microscope slide and werecovered with a glass coverslip, incubated for 1 min, and observed for motility

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Descriptiona Source orreference(s)

E. coli strainsDH5�mcr mcrA �(mrr-hsdRMS-mcrBC) �80dlacZ�M15 �(lacZYA-argF)U169 endA1 recA1 deoR thi-1

supE44 gyrA96 relA1 F� ��Bethesda Research

LaboratoriesHB101 recA13 proA2 leu lacY1 galK2 xyl-5 mtl-1 ara-14 hsdS20(rB

� mB�) supE44 rpsL20 F� �� 3

F. johnsoniae strainsUW101 (ATCC 17061) Wild type 21, 24CJ1708 Polar insertion mutation in remG; Emr 28CJ1827 rpsL2; Smr This studyCJ1883 rpsL2 �(remF remG fjoh_0982); Smr This studyCJ1898 rpsL2 �remI; Smr This studyCJ1899 rpsL2 �remH; Smr This studyCJ1912 rpsL2 �(remF remG fjoh_0982) �remI; Smr This studyCJ1913 rpsL2 �(remF remG fjoh_0982) �remH; Smr This studyCJ1922 rpsL2 �sprB; Smr This studyCJ1939 rpsL2 �(remF remG fjoh_0982) �remH �remI; Smr This studyCJ1988 rpsL2 �remH �remI; Smr This study

PlasmidspCP23 E. coli-F. johnsoniae shuttle plasmid; Apr (Tc)r 1pCP29 E. coli-F. johnsoniae shuttle plasmid; Apr (Cfr Emr) 16pLYL03 Plasmid carrying ermF gene; Apr (Emr) 17pRK2013 Helper plasmid for triparental conjugation; IncP Tra� Kmr 9pRR50 rpsL gene cloned into SphI and XbaI sites of pCP23; Apr (Tcr) This studypRR51 rpsL-containing suicide vector; Apr (Emr) This studypRR53 Construct used to delete remF, remG, and fjoh_0982; 2.5-kbp region upstream of remF

fused to 2.4-kbp region downstream of fjoh_0982 and cloned into BamHI and PstI sitesof pRR51; Apr (Emr)

This study

pRR60 Construct used to delete remI; 2.7-kbp region upstream of remI fused to 2.6-kbp regiondownstream of remI and cloned into BamHI and SphI sites of pRR51; Apr (Emr)

This study

pRR61 Construct used to delete remH; 2.7-kbp region downstream of remH fused to 2.6-kbpregion upstream of remH and cloned into BamHI and SphI sites of pRR51; Apr (Emr)

This study

pRR67 Construct used to delete sprB; 2.1-kbp region upstream of sprB fused to 2.8-kbp regiondownstream of sprB and cloned into BamHI and SphI sites of pRR51; Apr (Emr)

This study

pRR68 2.65-kbp BamHI-SphI fragment of pSP1 spanning remF, remG, and fjoh_0982 inserted intoBamHI and SphI sites of pCP23; Apr (Tcr)

28

pRR69 1.9-kbp fragment amplified with primers 1038 and 1039 spanning remG and cloned intoBamHI and SphI sites of pCP23; Apr (Tcr)

This study

pRR70 2.0-kbp fragment amplified with primers 1036 and 1037 spanning remI and cloned intoSphI and BamHI sites of pCP23; Apr (Tcr)

This study

pRR71 2.0-kbp KpnI-SphI fragment from pRR70 spanning remI cloned into KpnI and SphI sitesof pCP29; Apr (Emr, Cfr)

This study

pRR72 2.3-kbp fragment amplified with primers 718 and 1039 spanning remF and remG andcloned into BamHI and SphI sites of pCP23; Apr (Tcr)

This study

pRR73 881-bp fragment amplified with primers 718 and 1044 spanning remF and cloned intoBamHI and SphI sites of pCP23; Apr (Tcr)

This study

pRR75 0.9-kbp fragment amplified with primers 1042 and 1043 spanning remH and cloned intoSalI and XmaI sites of pCP29; Apr (Emr, Cfr)

This study

pSN60 pCP29 carrying sprB; Apr (Cfr Emr) 25

a Antibiotic resistance phenotypes: ampicillin, Apr; cefoxitin, Cfr; erythromycin, Emr; kanamycin, Kmr; streptomycin, Smr, tetracycline; Tcr. Unless indicatedotherwise, the plasmid-encoded antibiotic resistance phenotypes are those expressed in E. coli. The plasmid-encoded antibiotic resistance phenotypes given inparentheses are those expressed in F. johnsoniae but not in E. coli.

2420 RHODES ET AL. J. BACTERIOL.


.org/D

ownloaded from

http://jb.asm.org/

using an Olympus BH-2 phase-contrast microscope with a heated stage set at25°C. For analyses of cell tracks, cell movements were observed using Palmercounting cells (Wildlife Supply Company, Saginaw, MI) as previously described(25), except that 22-mm2 glass coverslips were used. Images were recorded usinga Photometrics CoolSNAPcf

2 camera and were analyzed using MetaMorph soft-ware (Molecular Devices, Downingtown, PA). Tracks illustrating the movementsof cells were obtained by superimposing individual digital video frames using thelogical AND operation of MetaMorph.

Binding and movement of protein G-coated polystyrene spheres carryingantibodies against SprB. Movement of surface-localized SprB was detected aspreviously described (25). Cells were grown overnight at 25°C in MM withoutshaking. Purified anti-SprB (1 �l of a 1:10 dilution of a 300-mg/liter stock),0.5-�m-diameter protein G-coated polystyrene spheres (1 �l of a 0.1% stockpreparation; Spherotech Inc., Libertyville, IL), and bovine serum albumin (1 �lof a 1% solution) were added to 7 �l of cells (approximately 5 108 cells per ml)in MM. The cells were spotted on a glass slide, covered with a glass coverslip, andexamined by phase-contrast microscopy at 25°C. Samples were examined 1 minafter spotting, and images were captured for 30 s.

Western blot analyses. Wild-type and mutant F. johnsoniae cells were grown tomid-log phase in MM at 25°C without shaking (100-ml cultures in 500-ml Er-lenmeyer flasks). Cells were harvested by centrifugation at 4,000 g, and pelletswere concentrated in 1 ml of lysis buffer (20 mM sodium phosphate [pH 7.4], 10mM EDTA). Ten microliters of 100 HALT protease inhibitor (Thermo FisherScientific, Waltham, MA) was added, and cells were lysed with a French pressurecell. Cell lysates were solubilized in SDS-PAGE loading buffer by boiling for 5min, and proteins were separated by SDS-PAGE on 3-to-8% Criterion XTTris-acetate-acrylamide gradient gels (Bio-Rad, Hercules, CA). Detection ofSprB was carried out as previously described (29).

Measurement of chitin digestion. Chitin utilization on agar plates was detectedas previously described (29). Briefly, an MYA agar (0.5 mM MgSO4, 0.05 mMFeSO4, 0.04 mM EDTA, 20 mM potassium phosphate, [pH 7.25], 0.1 g of yeast

FIG. 1. Map of the rpsL-containing suicide vector pRR51. pRR51was constructed by cloning the wild-type F. johnsoniae rpsL gene intothe EcoRI site of the bacteroidete suicide vector pLYL03. Integrationof pRR51 into the genome of the streptomycin-resistant F. johnsoniaestrain CJ1827 results in streptomycin sensitivity, and loss of the plas-mid from the cell results in streptomycin resistance. Numbers imme-diately inside the ring refer to kilobase pairs of the sequence. ori refersto the origin of replication that functions in E. coli but not in F.johnsoniae. oriT refers to the conjugative origin of transfer. bla confersampicillin resistance to E. coli but not F. johnsoniae. ermF conferserythromycin resistance to F. johnsoniae but not E. coli.

FIG. 2. Map of the sprB operon. Numbers below the map refer to kilobase pairs of sequence. The regions of DNA carried by plasmids usedin this study are indicated beneath the map. pRR53 and pRR67 are derivatives of the suicide vector pRR51 and were used in the construction ofCJ1883 [�(remF remG fjoh_0982)] and CJ1922 (�sprB), respectively. pSN60, pRR68, pRR69, pRR72, and pRR73 were used for complementationexperiments. The promoter sequence (underlined) and transcription start site (�1) for the sprB operon are indicated upstream of remF. The remFstart codon is indicated in bold. An inverted repeat (underlined) that may function in transcription termination is indicated downstream of the sprFstop codon (shown in bold).



.org/D

ownloaded from

http://jb.asm.org/

extract per liter, 15 g of agar per liter) plate was overlaid with 3 ml of a 2% chitinslurry and allowed to dry overnight at room temperature. Cells were grownovernight in MM, and 3 �l (approximately 106 cells) was spotted onto the plate,allowed to dry, and incubated at 25°C for 4 days.

RESULTS

Development of rpsL as a counterselectable marker for F.johnsoniae and construction of an sprB deletion mutant. Fivespontaneous streptomycin-resistant mutants of wild-type F.johnsoniae were isolated, and the rpsL genes were amplifiedand sequenced. Four of the mutants had an adenine-to-gua-nine point mutation at bp 263 of the rpsL coding sequence(rpsL1) that resulted in a K88R substitution in RpsL. This isthe same lysine residue altered in streptomycin-resistant Esch-erichia coli (10) and Borrelia burgdorferi (8). The fifth mutant,CJ1827, had an adenine-to-guanine point mutation at bp 128(rpsL2), resulting in a K43R substitution in RpsL. The mutantsproducing K88R RpsL formed colonies that spread slightly lesswell than the wild type, whereas CJ1827 (rpsL2) was indistin-guishable from the wild type except for its resistance to strep-tomycin. In E. coli, wild-type rpsL is dominant to the mutantallele, and merodiploids are sensitive to streptomycin (30). Wetested whether this was also true for F. johnsoniae. pRR50,which carries wild-type F. johnsoniae rpsL inserted into theshuttle vector pCP23, was introduced into CJ1827. CJ1827without the plasmid grew well in the presence of 100 �g per ml

streptomycin, whereas CJ1827 carrying pRR50 failed to growunder these conditions.

Wild-type rpsL was introduced into the suicide vectorpLYL03 to generate pRR51 (Fig. 1), which was designed tofacilitate construction of unmarked mutations. As a test case,an sprB deletion mutant was constructed. Regions upstreamand downstream of sprB were cloned in pRR51, and the plas-mid was introduced into CJ1827 by conjugation. Erythromycin-resistant colonies arose as a result of integration of the plasmidinto the chromosome. Cells were plated on a medium contain-ing streptomycin to select for loss of the plasmid by a secondrecombination event, and thousands of streptomycin-resistantcolonies were obtained. Depending on the site of the secondrecombination event, the resulting colonies were expected tohave either a wild-type sprB locus or an in-frame deletionspanning most of sprB, and colonies of each type were identi-fied. Deletion of sprB in CJ1922 was verified by PCR andsequencing as described in Materials and Methods. CJ1922formed nonspreading colonies (Fig. 4B), as previously re-ported for other sprB mutants (25, 28). CJ1922 also exhibitedresistance to the same bacteriophages (see Fig. S1 in the sup-plemental material), as do other sprB mutants (25, 28). Com-plementation with pSN60 (which carries sprB) restored theability to form spreading colonies (Fig. 4C), verifying the lackof polarity of the unmarked in-frame deletion. In contrast,polar mutations in sprB are not complemented by pSN60 butrather require plasmids expressing both sprB and the down-stream gene, sprF, for complementation (28).

Deletion of the region spanning remF, remG, and fjoh_0982does not disrupt motility. sprB is part of a seven-gene operon,

FIG. 3. Maps of the regions surrounding remH (A) and remI (B).Numbers below the maps refer to kilobase pairs of the sequences. Theregions of DNA carried by plasmids used in this study are indicatedbeneath the maps. pRR61 and pRR60 are derivatives of the suicidevector pRR51 and were used in the construction of CJ1899 (�remH)and CJ1898 (�remI), respectively. Plasmid pRR75 (remH) and plas-mids pRR70 and pRR71 (remI) were used for complementation ex-periments.

FIG. 4. remF, remG, and fjoh_0982 are not required for formationof spreading colonies. Colonies were incubated at 25°C on PY2 agarmedium for 48 h, and photomicrographs were taken with a Photomet-rics CoolSNAPcf

2 camera mounted on an Olympus IMT-2 phase-con-trast microscope. (A) Streptomycin-resistant “wild-type” strainCJ1827. (B) sprB deletion mutant CJ1922. (C) CJ1922 complementedwith pSN60. (D) remG polar insertion mutant CJ1708. (E) CJ1883,which has a deletion spanning remF, remG, and fjoh_0982. Bar, 1 mm.



.org/D

ownloaded from

http://jb.asm.org/

and each of the distal genes (sprC, sprD, sprB, and sprF) isrequired for normal motility and for the formation of spread-ing colonies. The situation was less clear for the proximalgenes, remF, remG, and fjoh_0982. CJ1708, which has an in-sertion in remG, formed nonspreading colonies (Fig. 4D), butthe insertion resulted in polar effects on the downstream genesthat could have been responsible for this phenotype (28). Weused pRR53 to construct CJ1883, which has a deletion span-ning remF, remG, and fjoh_0982. Western blot analysis re-vealed that the deletion in CJ1883 did not exhibit polar effectson expression of the downstream gene, sprB (Fig. 5). Cells ofCJ1883 formed spreading colonies on agar that were similar tothose formed by wild-type cells (Fig. 4E), indicating that remF,remG, and fjoh_0982 are not required for the formation ofspreading colonies.

Deletion of remH and remI cause motility defects in CJ1883[�(remF remG fjoh_0982)]. Analysis of the F. johnsoniae ge-nome (24) revealed a paralog of remF (fjoh_3206, which wenamed remH) and a paralog of remG (fjoh_3194, which wenamed remI) (Fig. 3). remF and remG are cotranscribed withsprB (28), but remH and remI appear to be transcribed as singlegenes, and they are not located near sprB paralogs or nearknown motility genes. RemF and RemH exhibit 92% identityover their entire length (150 amino acids each), and RemG(430 amino acids in length) and RemI (429 amino acids inlength) exhibit 80% identity with each other over 428 aminoacids. Several additional remG paralogs (fjoh_0546, fjoh_3856,and fjoh_4889) were also identified, but the proteins encodedby these genes were less similar to RemG (31% to 47% aminoacid identity), and they were not considered further in thisstudy. RemF, RemG, RemH, and RemI are each predicted tohave N-terminal signal peptides targeting them for exportacross the cytoplasmic membrane by the sec system. PSORTand CELLO analyses predict that RemF and RemH areperiplasmic proteins and that RemG and RemI are outermembrane or extracellular proteins. RemF and RemH aresimilar in sequence to predicted proteins of unknown functionfrom several members of the phylum Bacteroidetes, but they donot share significant sequence similarity with predicted pro-

teins from other phyla of bacteria or from members of thedomains Archaea and Eukarya. RemG and RemI exhibit se-quence similarity to many bacteroidete proteins of unknownfunction, and they exhibit more limited sequence similarity topredicted proteins of unknown function from members ofother bacterial phyla.

The ability to make unmarked deletions allowed us to re-peatedly use the same selectable marker (erythromycin resis-tance) and counterselectable marker (streptomycin resistance)to construct strains with multiple deletions. A collection ofstrains with multiple deletions was constructed to determinethe effect of elimination of either set of paralogs (remF andremH or remG and remI). Strains with single deletions of remHor remI or strains missing both remH and remI formed spread-ing colonies that were similar to those of the wild type or ofCJ1883 [�(remF remG fjoh_0982)] (Fig. 6A to E). However,introduction of either a remH deletion or a remI deletion intoCJ1883 resulted in motility defects and the formation of non-spreading colonies (Fig. 6F and K). The ability to form spread-ing colonies was restored by introduction of remF and remG onpRR68 or pRR72, or by the introduction of remH and remI onpRR75 and pRR70, verifying that the mutations in remF,remG, remH, and remI were responsible for the nonspreadingphenotypes. fjoh_0982 is not required for formation of spread-ing colonies, since CJ1913 [�(remF remG fjoh_0982) �remH]and CJ1912 [�(remF remG fjoh_0982) �remI] were comple-mented by either pRR68 (which carries remF, remG, andfjoh_0982) or by pRR72 (which carries just remF and remG).The phenotypes associated with mutations in the paralogousgenes suggest that the encoded proteins perform redundant orsemiredundant roles associated with motility. Surprisingly, in-troduction of pRR75, which carries remH, into CJ1913[�(remF remG fjoh_0982) �remH] resulted in the formation ofcolonies that had a few tiny flares (Fig. 6I) but spread muchless well than wild-type colonies or than colonies of CJ1883[�(remF remG fjoh_0982)]. Further analysis revealed the likelyreason for the poor complementation, since expression ofremH from pRR75 in wild-type cells also resulted in a dramaticreduction of colony spreading (Fig. 7). The motility defectcaused by pRR75 could be corrected by introducing remI onpRR70 (Fig. 6J and 7), suggesting the possibility that overex-pression of remH causes a motility defect and overexpressionof remI in the same cells counteracts the deleterious effects ofhigh levels of RemH.

To determine which combination of genes allows formationof spreading colonies, we constructed CJ1939, which is missingremF, remG, fjoh_0982, remH, and remI, and introduced com-binations of genes on plasmids (Fig. 8). As expected, introduc-tion of the individual genes (remF, remG, remH, and remI)failed to restore colony spreading. Introduction of remF andremG, remH and remI, or remF and remI resulted in formationof spreading colonies, whereas introduction of remG and remHdid not. Apparently RemF can function with either RemG orRemI, whereas RemH (the paralog of RemF) can functionwith RemI (paralog of RemG) but not with RemG. Furtherevidence that the presence of RemH and RemG is not suffi-cient for formation of spreading colonies was obtained fromcomplementation experiments performed with CJ1912[�(remF remG fjoh_0982) �remI]. Introduction of remI on

FIG. 5. Western immunoblot detection of SprB in whole-cell ex-tracts of wild-type and mutant strains of F. johnsoniae. Cells weredisrupted using a French pressure cell, and samples were boiled inSDS-PAGE loading buffer. Proteins (40 �g per lane) were separatedby electrophoresis, and SprB was detected using anti-SprB antibody.Lane 1, molecular mass markers; lane 2, streptomycin-resistant “wild-type” F. johnsoniae CJ1827; lane 3, sprB deletion mutant CJ1922; lane4, CJ1922 complemented with pSN60; lane 5, polar remG insertionmutant CJ1708; lane 6, CJ1883 [�(remF remG fjoh_0982)]; lane 7,CJ1939 [�(remF remG fjoh_0982) �remH �remI]. Strains in lanes 2, 3,5, 6, and 7 carried empty control vector pCP29, which had no effect onexpression of SprB.



.org/D

ownloaded from

http://jb.asm.org/

pRR70 restored formation of spreading colonies, whereas in-troduction of remG on pRR69 did not (Fig. 6N and O).

Effect of mutations in remF, remG, remH, and remI on glid-ing of cells on glass. Wild-type and mutant cells were examinedfor movement on glass as previously described (25). Wild-typecells attached to glass and glided at speeds of approximately 2�m/s. Cells of CJ1883 [�(remF remG fjoh_0982)], CJ1899(�remH), CJ1898 (�remI), and CJ1988 (�remH �remI) at-tached to and moved on glass as well as the wild-type cells did(Fig. 9; see also Movie S1 in the supplemental material). In

contrast, cells of CJ1922 (�sprB), CJ1913 [�(remF remGfjoh_0982) �remH], CJ1912 [�(remF remG fjoh_0982) �remI],and CJ1939 [�(remF remG fjoh_0982) �remH �remI] attachedto glass as well as wild-type cells did but were partially defec-tive in movement (Fig. 9 and 10; see also Movies S1, S2, and S3in the supplemental material). Cells of these mutants changeddirection of movement more frequently than did wild-type cellsand thus made less net progress. Changes of direction occurredeither when the leading pole became the lagging pole or whena cell pivoted or flipped, thus changing direction while main-taining the same leading pole. This behavior is similar to thatpreviously reported for cells with mutations in sprC, sprD, sprB,and sprF (28). Complementation with plasmids that restoredcolony spreading (Fig. 6 and 8) also restored normal gliding onglass (Fig. 9 and 10; see also Movies S1, S2, and S3 in thesupplemental material).

RemF, RemG, RemH, and RemI are not required for surfacelocalization or movement of SprB. A mutation in sprF, anothergene of the sprB operon, results in lack of secretion of SprBacross the outer membrane (28). We examined wild-type andmutant cells to determine whether RemF, RemG, RemH, andRemI have roles in secretion of SprB. The presence of SprB onthe cell surface was monitored by adding protein G-coatedpolystyrene spheres and anti-SprB to cells. As previously re-ported (25), antibody-coated spheres attached specifically towild-type cells expressing SprB and were rapidly propelledalong the cell surface (Table 2; see also Movie S4 in thesupplemental material). Such spheres failed to attach to cellsof the sprB deletion mutant CJ1922, and protein G-coatedspheres without antibodies failed to bind to wild-type cells.Cells of CJ1883 [�(remF remG fjoh_0982)] and CJ1939[�(remF remG fjoh_0982) �remH �remI] produced SprB (Fig.5). These cells bound to anti-SprB-coated spheres and pro-pelled them similar to wild-type cells (Table 2; see also Movie

FIG. 6. Deletion of remH and remI causes motility defects in CJ1883 [�(remF remG fjoh_0982)]. Colonies were grown for 48 h at 25°C on PY2agar medium. (A) Streptomycin-resistant “wild-type” (WT) strain CJ1827. All other strains shown were derived from CJ1827. (B) CJ1883 [�(remFremG fjoh_0982)]; (C) CJ1899 (�remH); (D) CJ1898 (�remI); (E) CJ1988 (�remH �remI); (F) CJ1913 [�(remF remG fjoh_0982) �remH];(G) CJ1913 plus pRR68 which carries remF, remG, and fjoh_0982; (H) CJ1913 plus pRR72, which carries remF and remG; (I) CJ1913 plus pRR75,which carries remH; (J) CJ1913 plus pRR75, which carries remH, and pRR70, which carries remI; (K) CJ1912 [�(remF remG fjoh_0982) �remI];(L) CJ1912 plus pRR68, which carries remF, remG, and fjoh_0982; (M) CJ1912 plus pRR72, which carries remF and remG; (N) CJ1912 pluspRR70, which carries remI; (O) CJ1912 plus pRR69, which carries remG. Bar, 1 mm (applies to all panels).

FIG. 7. Presence of remH on a multicopy plasmid in wild-type cellsinhibits colony spreading. Colonies of CJ1827 with or without plasmidswere grown for 48 h at 25°C on PY2 agar medium. Bar, 1 mm (appliesto all panels). pCP23 and pCP29 are shuttle vectors without inserts.pRR73 (remF), pRR69 (remG), and pRR70 (remI) are derived frompCP23, and pRR75 (remH) is derived from pCP29.



.org/D

ownloaded from

http://jb.asm.org/

S4 in the supplemental material), indicating that SprB waspresent on the cell surface. RemF, RemG, RemH, and RemIare clearly involved in motility, but they are not required forexpression, secretion, or movement of the cell surface motilityprotein SprB.

RemF, RemG, RemH, and RemI are not required for chitinutilization or for bacteriophage sensitivity. Many motility mu-tants of F. johnsoniae display defects in chitin utilization andresistance to bacteriophages that infect wild-type cells (5, 12,13, 22). The defects in chitin utilization are thought to arisebecause disruption of the motility genes results in defects inthe protein secretion system (PorSS), which is involved in se-cretion of surface components of the motility machinery and ofextracellular chitinase (29, 32). Strains with mutations in remF,remG, remH, and remI, including CJ1939, which lacks each ofthese genes, retained the ability to digest chitin (see Fig. S2 inthe supplemental material). This is similar to the phenotype ofcells with mutations in sprB, sprC, sprD, and sprF (28) andsuggests that RemF, RemG, RemH, and RemI are not com-ponents of the PorSS required for secretion of extracellularchitinase.

It has been suggested that F. johnsoniae bacteriophages mayuse SprB and other cell surface proteins secreted by the PorSSas receptors to infect cells (25, 28, 29). This may explain whysprB mutants are resistant to some bacteriophages and whycells with mutations in genes that are thought to encode corecomponents of the PorSS (gldK, gldL, gldM, gldN, and sprA)are resistant to most or all bacteriophages that infect F. john-soniae (4, 26, 29). Strains with mutations in remF, remG, remH,and remI, including CJ1939, which lacks each of these genes,were sensitive to infection by each of the bacteriophages tested(see Fig. S1 in the supplemental material). This suggests thatRemF, RemG, RemH, and RemI are not receptors for thesebacteriophages and are not required for secretion of the re-ceptors.

DISCUSSION

Development of techniques for the genetic manipulationof F. johnsoniae (1, 4, 16, 23, 29) has made this organism a

model system for studies of bacteroidete gliding motility andphysiology. However, some studies have been hampered bythe lack of a general technique for constructing unmarkedmutations, such as nonpolar gene deletions. The rpsL coun-terselection strategy described here allows construction ofsuch mutations. This approach will facilitate future studiesof gliding motility and of other aspects of F. johnsoniaebiology, and it may also be adapted for use with othermembers of the large and diverse phylum Bacteroidetes. TherpsL counterselection strategy can be used to construct in-frame deletions, allowing precise determination of functionof individual genes within operons. The large fragmentscloned (typically 2 kbp of DNA upstream and 2 kbp of DNAdownstream of the region to be deleted) result in efficientrecombination both during plasmid integration and duringresolution and loss of plasmid DNA. In most cases tens tohundreds of erythromycin-resistant colonies are obtainedfor the first step (plasmid integration) and thousands ofstreptomycin-resistant colonies are obtained for the secondstep (plasmid loss), and approximately 50% of streptomycin-resistant colonies carry the deletion. It is important to streakcolonies for isolation on selective media after both the plas-mid integration and plasmid loss steps to eliminate nonse-lected cells. Since pRR51 carries rpsL, integration of theplasmid could occur at this site during the first stage of theprocess. In this case, selection for streptomycin resistancewould not result in the desired deletion. In practice we havenot observed this event, presumably because recombinationoccurs more frequently in the large segment spanning theregion to be deleted than in the small rpsL fragment. Thepotential problem caused by plasmid integration at chromo-somal rpsL2 is easily dealt with either by screening coloniesafter the initial erythromycin selection by PCR or by select-ing several erythromycin-resistant colonies before proceed-ing to the second stage of the process (selection for strep-tomycin resistance). Another problem that could arise isspontaneous mutation of the plasmid-borne rpsL, resultingin background streptomycin-resistant colonies that have re-tained the integrated plasmid. In practice, we have not ob-served this event, presumably because recombination (re-

FIG. 8. Pairwise combinations of genes that restore colony spreading to CJ1939 [�(remF remG fjoh_0982) �remH �remI]. Colonies of CJ1939with or without plasmids were grown for 48 h at 25°C on PY2 agar medium. The plasmids contained in each strain, and the genes carried on theplasmids, are indicated in the panels. Bar, 1 mm (applies to all panels).



.org/D

ownloaded from

http://jb.asm.org/

sulting in loss of the integrated plasmid) occurs much morefrequently than the rare spontaneous mutations in rpsL.

In addition to allowing the facile construction of in-framedeletion mutants and strains with multiple deletions, theapproach described here can also be used to introduce site-directed point mutations in genes of interest or to insert anyDNA fragment of interest into a desired location on thechromosome. The ability to perform these sophisticated ge-netic manipulations will allow many questions regarding the

FIG. 10. Effects of mutations on gliding of cells on glass. Cells at-tached to a glass coverslip on a Palmer cell were observed by phase-contrast microscopy, and digital images (recorded at time zero) of cellsare shown for the wild-type CJ1827 (A), CJ1913 [�(remF remGfjoh_0982) �remH] (B), CJ1913 complemented with pRR75 and pRR70(C), CJ1912 [�(remF remG fjoh_0982) �remI] (D), CJ1912 comple-mented with pRR70 (E), CJ1939 [�(remF remG fjoh_0982) �remH�remI] (F), CJ1939 complemented with pRR72 (G), and CJ1939 com-plemented with pRR75 and pRR70 (H). Tracks illustrating the move-ments of the cells shown in panels A to H over a 60-s period wereobtained by superimposing individual digital video frames of the wild-typestrain (I), CJ1913 [�(remF remG fjoh_0982) �remH] (J), CJ1913 comple-mented with pRR75 and pRR70 (K), CJ1912 [�(remF remG fjoh_0982)�remI] (L), CJ1912 complemented with pRR70 (M), CJ1939 [�(remFremG fjoh_0982) �remH �remI] (N), CJ1939 complemented with pRR72(O), and CJ1939 complemented with pRR75 and pRR70 (P). Imageswere recorded using a Photometrics CoolSNAPcf

2 camera mounted on anOlympus BH-2 phase-contrast microscope. Bar, 40 �m.

FIG. 9. Cells with mutation(s) in remF, remG, remH, or remI glideon glass. Cells attached to a glass coverslip on a Palmer cell wereobserved by phase-contrast microscopy, and digital images of cells ofthe wild-type CJ1827 (A), sprB deletion mutant CJ1922 (B), CJ1922complemented with pSN60 (C), CJ1883 [�(remF remG fjoh_0982)](D), CJ1899 (�remH) (E), CJ1898 (�remI) (F), and CJ1988 (�remH�remI) (G) were recorded at time zero. Tracks illustrating the move-ments of the cells shown in panels A to G over a 60-s period wereobtained by superimposing individual digital video frames of the wild-type strain (H), CJ1922 (�sprB) (I), CJ1922 complemented withpSN60 (J), CJ1883 [�(remF remG fjoh_0982)] (K), CJ1899 (�remH)(L), CJ1898 (�remI) (M), or CJ1988 (�remH �remI) (N). Images wererecorded using a Photometrics CoolSNAPcf

2 camera mounted on anOlympus BH-2 phase-contrast microscope. Bar, 40 �m.



.org/D

ownloaded from

http://jb.asm.org/

functions of the individual F. johnsoniae motility proteins tobe addressed.

The rpsL counterselection strategy was used to identifyredundant gliding motility genes. SprB is a large protein thatis involved in gliding motility and appears to move rapidlyalong the cell surface (25). sprB is transcribed as part of aseven-gene operon that also includes remF, remG,fjoh_0982, sprC, sprD, and sprF. Previous genetic analysesdemonstrated that sprC, sprD, sprB, and sprF are requiredfor normal motility, but the involvement of the three up-stream genes was not clear (28). The results presented heredemonstrate that remF, remG, and fjoh_0982 are not re-quired for normal motility or for the formation of spreadingcolonies. They also led to the identification of the remF andremG paralogs remH and remI, respectively. Analysis ofstrains with multiple mutations suggested that the paralo-gous pairs RemF/RemG and RemH/RemI perform redun-dant or semiredundant functions in gliding. The exact func-tions of RemF, RemG, RemH, and RemI in motility are notimmediately obvious. Cotranscription of remF and remGwith sprC, sprD, sprB, and sprF suggests that their functionsmay be related to those of the cell surface motility proteinSprB. SprF is thought to be required for secretion of SprBacross the outer membrane, but neither RemF nor RemGappears to have roles in this secretion, since cells lackingRemF, RemG, RemH, and RemI exhibited SprB protein onthe cell surface.

The redundant paralogous proteins identified here arenot completely interchangeable. Introduction of pairwisecombinations of the genes into CJ1939 [�(remF remGfjoh_0982) �remH �remI] revealed that the pairs RemF/RemG, RemH/RemI, and RemF/RemI allowed normal mo-tility and formation of spreading colonies, but the RemG/RemH pair did not. Although we do not know the exactfunctions of these proteins, RemF and RemG may functiontogether in support of SprB function, and RemH and RemImay perform a similar role.

Analysis of the F. johnsoniae genome suggests that redun-dancy of motility genes may not be limited to remF, remG,remH, and remI. Multiple paralogs of sprB are present, andthese may encode semiredundant cell surface adhesins in-volved in movement over different types of surfaces and mayexplain the limited residual motility that sprB mutants exhibit.Multiple paralogs of sprF are also present and are usually

located just downstream of sprB paralogs. The encoded SprF-like proteins may facilitate secretion of their cognate SprB-likeadhesins. Redundancy between gldN and gldO has also beendemonstrated (29). Further studies may identify additionalexamples of redundancy, and the genetic tools described aboveshould facilitate analysis of the functions of the individualgenes and proteins in gliding motility.

ACKNOWLEDGMENTS

This research was supported by MCB-0641366 and MCB-1021721from the National Science Foundation.

REFERENCES

1. Agarwal, S., D. W. Hunnicutt, and M. J. McBride. 1997. Cloning and char-acterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) glidingmotility gene, gldA. Proc. Natl. Acad. Sci. U. S. A. 94:12139–12144.

2. Alvarez, B., P. Secades, M. J. McBride, and J. A. Guijarro. 2004. Develop-ment of genetic techniques for the psychrotrophic fish pathogen Flavobac-terium psychrophilum. Appl. Environ. Microbiol. 70:581–587.

3. Bolivar, F., and K. Backman. 1979. Plasmids of Escherichia coli as cloningvectors. Methods Enzymol. 68:245–267.

4. Braun, T. F., M. K. Khubbar, D. A. Saffarini, and M. J. McBride. 2005.Flavobacterium johnsoniae gliding motility genes identified by mariner mu-tagenesis. J. Bacteriol. 187:6943–6952.

5. Braun, T. F., and M. J. McBride. 2005. Flavobacterium johnsoniae GldJ is alipoprotein that is required for gliding motility. J. Bacteriol. 187:2628–2637.

6. Chang, L. Y. E., J. L. Pate, and R. J. Betzig. 1984. Isolation and character-ization of nonspreading mutants of the gliding bacterium Cytophagajohnsonae. J. Bacteriol. 159:26–35.

7. Chen, S., M. Bagdasarian, M. G. Kaufman, A. K. Bates, and E. D. Walker.2007. Mutational analysis of the ompA promoter from Flavobacterium john-soniae. J. Bacteriol. 189:5108–5118.

8. Drecktrah, D., J. M. Douglas, and D. S. Samuels. 2010. Use of rpsL as acounterselectable marker in Borrelia burgdorferi. Appl. Environ. Microbiol.76:985–987.

9. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containingderivative of plasmid RK2 dependent on a plasmid function provided intrans. Proc. Natl. Acad. Sci. U. S. A. 76:1648–1652.

10. Funatsu, G., and H. G. Wittmann. 1972. Ribosomal proteins. XXXIII. Lo-cation of amino acid replacements in protein S12 isolated from Escherichiacoli mutants resistant to streptomycin. J. Mol. Biol. 68:547–550.

11. Holland, I. B. 2010. The extraordinary diversity of bacterial protein secretionmechanisms. Methods Mol. Biol. 619:1–20.

12. Hunnicutt, D. W., M. J. Kempf, and M. J. McBride. 2002. Mutations inFlavobacterium johnsoniae gldF and gldG disrupt gliding motility and inter-fere with membrane localization of GldA. J. Bacteriol. 184:2370–2378.

13. Hunnicutt, D. W., and M. J. McBride. 2001. Cloning and characterization ofthe Flavobacterium johnsoniae gliding motility genes gldD and gldE. J. Bac-teriol. 183:4167–4175.

14. Hunnicutt, D. W., and M. J. McBride. 2000. Cloning and characterization ofthe Flavobacterium johnsoniae gliding motility genes gldB and gldC. J. Bac-teriol. 182:911–918.

15. Jarrell, K. F., and M. J. McBride. 2008. The surprisingly diverse ways thatprokaryotes move. Nat. Rev. Microbiol. 6:466–476.

16. Kempf, M. J., and M. J. McBride. 2000. Transposon insertions in the Fla-

TABLE 2. Effects of mutations in remF, remG, remH, and remI on binding of protein G-coatedpolystyrene spheres carrying antibodies against SprB

Strain Description Antibodyadded

Avg % (SD) ofcells with spheres

attacheda

CJ1827 � control plasmid pCP29 Wild type No antibody 0.3 (0.6)CJ1827 � pCP29 Wild type Anti-SprB 38.3 (7.1)CJ1922 � pCP29 �sprB Anti-SprB 0 (0)CJ1922 � pSN60 �sprB complemented with pSN60 Anti-SprB 51.7 (3.8)CJ1883 � pCP29 �(remF remG fjoh_0982) Anti-SprB 53.3 (4.9)CJ1939 � pCP29 �(remF remG fjoh_0982) �remH �remI Anti-SprB 44.3 (4.0)

a Purified anti-SprB and 0.5-�m-diameter protein G-coated polystyrene spheres were added to cells as described in Materials and Methods. Samples were spottedon a glass slide, covered with a glass coverslip, incubated for 1 min at 25°C, and examined using a phase-contrast microscope. Images were recorded for 30 s, and 100randomly selected cells were examined for the presence of attached spheres during this period. Numbers in parentheses are standard deviations calculated from threemeasurements.



.org/D

ownloaded from

http://jb.asm.org/

vobacterium johnsoniae ftsX gene disrupt gliding motility and cell division. J.Bacteriol. 182:1671–1679.

17. Li, L.-Y., N. B. Shoemaker, and A. A. Salyers. 1995. Location and charac-terization of the transfer region of a Bacteroides conjugative transposon andregulation of the transfer genes. J. Bacteriol. 177:4992–4999.

18. Liu, J., M. J. McBride, and S. Subramaniam. 2007. Cell surface filaments ofthe gliding bacterium Flavobacterium johnsoniae revealed by cryo-electrontomography. J. Bacteriol. 189:7503–7506.

19. Matsuoka, M., K. Takahama, and T. Ogawa. 2001. Gene replacement incyanobacteria mediated by a dominant streptomycin-sensitive rps12 genethat allows selection of mutants free from drug resistance markers. Micro-biology 147:2077–2087.

20. McBride, M. J. 2001. Bacterial gliding motility: multiple mechanisms for cellmovement over surfaces. Annu. Rev. Microbiol. 55:49–75.

21. McBride, M. J., and T. F. Braun. 2004. GldI is a lipoprotein that is requiredfor Flavobacterium johnsoniae gliding motility and chitin utilization. J. Bac-teriol. 186:2295–2302.

22. McBride, M. J., T. F. Braun, and J. L. Brust. 2003. Flavobacterium john-soniae GldH is a lipoprotein that is required for gliding motility and chitinutilization. J. Bacteriol. 185:6648–6657.

23. McBride, M. J., and M. J. Kempf. 1996. Development of techniques for thegenetic manipulation of the gliding bacterium Cytophaga johnsonae. J. Bac-teriol. 178:583–590.

24. McBride, M. J., et al. 2009. Novel features of the polysaccharide-digestinggliding bacterium Flavobacterium johnsoniae as revealed by genome se-quence analysis. Appl. Environ. Microbiol. 75:6864–6875.

25. Nelson, S. S., S. Bollampalli, and M. J. McBride. 2008. SprB is a cell surface

component of the Flavobacterium johnsoniae gliding motility machinery. J.Bacteriol. 190:2851–2857.

26. Nelson, S. S., P. P. Glocka, S. Agarwal, D. P. Grimm, and M. J. McBride.2007. Flavobacterium johnsoniae SprA is a cell-surface protein involved ingliding motility. J. Bacteriol. 189:7145–7150.

27. Pate, J. L., S. J. Petzold, and L.-Y. E. Chang. 1979. Phages for the glidingbacterium Cytophaga johnsonae that infect only motile cells. Curr. Microbiol.2:257–262.

28. Rhodes, R. G., S. S. Nelson, S. Pochiraju, and M. J. McBride. 2011. Flavo-bacterium johnsoniae sprB is part of an operon spanning the additionalgliding motility genes sprC, sprD, and sprF. J. Bacteriol. 193:599–610.

29. Rhodes, R. G., et al. 2010. Flavobacterium johnsoniae gldN and gldO arepartially redundant genes required for gliding motility and surface localiza-tion of SprB. J. Bacteriol. 192:1201–1211.

30. Russell, C. B., and F. W. Dahlquist. 1989. Exchange of chromosomal andplasmid alleles in Escherichia coli by selection for loss of a dominant anti-biotic sensitivity marker. J. Bacteriol. 171:2614–2618.

31. Sander, P., A. Meier, and E. C. Bottger. 1995. rpsL�: a dominant selectablemarker for gene replacement in mycobacteria. Mol. Microbiol. 16:991–1000.

32. Sato, K., et al. 2010. A protein secretion system linked to bacteroidetegliding motility and pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 107:276–281.

33. Stibitz, S., W. Black, and S. Falkow. 1986. The construction of a cloningvector designed for gene replacement in Bordetella pertussis. Gene 50:133–140.

34. Wolkin, R. H., and J. L. Pate. 1985. Selection for nonadherent or nonhy-drophobic mutants co-selects for nonspreading mutants of Cytophagajohnsonae and other gliding bacteria. J. Gen. Microbiol. 131:737–750.



.org/D

ownloaded from

http://jb.asm.org/

Documents

Development and Use of a Gene Deletion Strategy for … › content › jb › 193 › 10 › 2418.full.pdf · genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for