Flavobacterium johnsoniae sprB is part of an operon spanning the additional gliding motility genes sprC, sprD, and sprF

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces.     

SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces by an unknown mechanism. Transposon insertions in sprB resulted in cells that were defective in gliding. SprB is a highly repetitive 669-kDa cell surface protein, and antibodies against SprB inhibited the motility of wild-type cells. Polystyrene microspheres coated with antibodies against SprB attached to and were rapidly propelled along the cell surface, suggesting that SprB is one of the outermost components of the motility machinery. The movement of SprB along the cell surface supports a model of gliding motility in which motors anchored to the cell wall rapidly propel cell surface adhesins.     

Development and use of a gene deletion strategy for Flavobacterium johnsoniae to identify the redundant gliding motility genes remF, remG, remH, and remI

Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmid restored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The gene deletion 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 formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had no effect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The rpsL counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of F. johnsoniae physiology.     

Cytophaga-flavobacterium gliding motility

Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility. Cells of F. johnsoniae glide over surfaces at rates of up to 10 microm/s. Latex spheres added to F. johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding. Genetic analyses have identified a number of gld genes that are required for gliding. Three Gld proteins are thought to be components of an ATP-binding-cassette transporter. Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane. Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin. Two models that attempt to incorporate the available data to explain the mechanism of F. johnsoniae gliding are presented.     

Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.     

Cloning and Characterization of the Flavobacterium johnsoniae Gliding Motility Genes gldD and gldE

Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of F. johnsoniae propel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for F. johnsoniae gliding motility, gldA, gldB, and ftsX, have recently been described. Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding. Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection. gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function. gldE, which lies immediately upstream of gldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression of gldE partially suppressed the motility defects of a gldB point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.     

Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis

Cells of Flavobacterium johnsoniae glide rapidly over surfaces. The mechanism of F. johnsoniae gliding motility is not known. Eight gld genes required for gliding motility have been described. Disruption of any of these genes results in complete loss of gliding motility, deficiency in chitin utilization, and resistance to bacteriophages that infect wild-type cells. Two modified mariner transposons, HimarEm1 and HimarEm2, were constructed to allow the identification of additional motility genes. HimarEm1 and HimarEm2 each transposed in F. johnsoniae, and nonmotile mutants were identified and analyzed. Four novel motility genes, gldK, gldL, gldM, and gldN, were identified. GldK is similar in sequence to the lipoprotein GldJ, which is required for gliding. GldL, GldM, and GldN are not similar in sequence to proteins of known function. Cells with mutations in gldK, gldL, gldM, and gldN were defective in motility and chitin utilization and were resistant to bacteriophages that infect wild-type cells. Introduction of gldA, gldB, gldD, gldFG, gldH, gldI, and gldJ and the region spanning gldK, gldL, gldM, and gldN individually into 50 spontaneous and chemically induced nonmotile mutants restored motility to each of them, suggesting that few additional F. johnsoniae gld genes remain to be identified.     

Flavobacterium johnsoniae GldK,GldL, GldM,and SprA Are Required for Secretion of the Cell Surface Gliding Motility Adhesins SprB and RemA

Flavobacterium johnsoniae cells move rapidly over surfaces by gliding motility. Gliding results from the movement of adhesins such as SprB and RemA along the cell surface. These adhesins are delivered to the cell surface by a Bacteroidetes-specific secretion system referred to as the type IX secretion system (T9SS). GldN, SprE, SprF, and SprT are involved in secretion by this system. Here we demonstrate that GldK, GldL, GldM, and SprA are each also involved in secretion. Nonpolar deletions of gldK, gldL, or gldM resulted in the absence of gliding motility and in T9SS defects. The mutant cells produced SprB and RemA proteins but failed to secrete them to the cell surface. The mutants were resistant to phages that use SprB or RemA as a receptor, and they failed to attach to glass, presumably because of the absence of cell surface adhesins. Deletion of sprA resulted in similar but slightly less dramatic phenotypes. sprA mutant cells failed to secrete SprB and RemA, but cells remained susceptible to some phages and retained some limited ability to glide. The phenotype of the sprA mutant was similar to those previously described for sprE and sprT mutants. SprA, SprE, and SprT are needed for secretion of SprB and RemA but may not be needed for secretion of other proteins targeted to the T9SS. Genetic and molecular experiments demonstrate that gldK, gldL, gldM, and gldN form an operon and suggest that the proteins encoded by these genes may interact to form part of the F. johnsoniae T9SS.     

Cloning and characterization of the Flavobacterium johnsoniae gliding-motility genes gldB and gldC

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) with defects in gliding motility have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nongliding mutant of F. johnsoniae (UW102-99) with a library of wild-type DNA by using the shuttle cosmid pCP26. The complementing plasmid (pCP200) contained an insert of 26 kb and restored gliding motility to 4 of 50 independently isolated nongliding mutants. A 1.9-kb fragment which encompassed two genes, gldB and gldC, complemented all four mutants. An insertion mutation in gldB was polar on gldC, suggesting that the two genes form an operon. Disruption of the chromosomal copy of gldB in wild-type F. johnsoniae UW101 eliminated gliding motility. Introduction of the gldBC operon, or gldB alone, restored motility. gldB appears to be essential for F. johnsoniae gliding motility. It codes for a membrane protein that does not exhibit strong sequence similarity to other proteins in the databases. gldC is not absolutely required for gliding motility, but cells that do not produce GldC form colonies that spread less well than those of the wild type. GldC is a soluble protein and has weak sequence similarity to the fungal lectin AOL.     

GldI is a lipoprotein that is required for Flavobacterium johnsoniae gliding motility and chitin utilization

Cells of Flavobacterium johnsoniae glide rapidly over surfaces by an unknown mechanism. Seven genes (gldA, gldB, gldD, gldF, gldG, gldH, and ftsX) that are required for gliding motility have been described. Complementation of the nonmotile mutants UW102-41, UW102-85, and UW102-92 identified another gene, gldI, that is required for gliding motility. gldI mutants formed nonspreading colonies, and individual cells were completely nonmotile. They were also resistant to bacteriophages that infect wild-type cells, and they failed to digest chitin. Introduction of wild-type gldI on a plasmid restored colony spreading, cell motility, phage sensitivity, and the ability to digest chitin to the gldI mutants. gldI encodes a predicted 199-amino-acid protein that localized to the membrane fraction. Labeling studies with [(3)H]palmitate indicated that GldI is a lipoprotein. GldI is similar to peptidyl-prolyl cis/trans-isomerases of the FK506-binding protein family and may be involved in folding cell envelope protein components of the motility machinery.     

Mutations in Flavobacterium johnsoniae gldF and gldG disrupt gliding motility and interfere with membrane localization of GldA

Flavobacterium johnsoniae moves rapidly over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Four genes that are required for F. johnsoniae gliding motility, gldA, gldB, gldD, and ftsX, have recently been described. GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters. Tn4351 mutagenesis was used to identify two additional genes, gldF and gldG, that are required for cell movement. gldF and gldG appear to constitute an operon, and a Tn4351 insertion in gldF was polar on gldG. pMK314, which carries the entire gldFG region, restored motility to each of the gldF and gldG mutants. pMK321, which expresses GldG but not GldF, restored motility to each of the gldG mutants but did not complement the gldF mutant. GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters. GldG is similar to putative accessory proteins of ABC transporters. It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm. GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter. F. johnsoniae gldA is not closely linked to gldFG, but the gldA, gldF, and gldG homologs of the distantly related gliding bacterium Cytophaga hutchinsonii are arranged in what appears to be an operon. The exact roles of F. johnsoniae GldA, GldF, and GldG in gliding are not known. Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.     

Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Transposon mutagenesis was used to identify sprE, which is involved in gliding. Mutations in sprE resulted in the formation of nonspreading colonies on agar. sprE mutant cells in wet mounts were almost completely deficient in attachment to and movement on glass, but a small percentage of cells exhibited slight movements, indicating that the motility machinery was not completely disrupted. SprE is a predicted lipoprotein with a tetratricopeptide repeat domain. SprE is similar in sequence to Porphyromonas gingivalis PorW, which is required for secretion of gingipain protease virulence factors. Disruption of F. johnsoniae sprE resulted in decreased extracellular chitinase activity and decreased secretion of the cell surface motility protein SprB. Reduced secretion of cell surface components of the gliding machinery, such as SprB, may account for the defects in gliding. Orthologs of sprE are found in many gliding and nongliding members of the phylum Bacteroidetes, suggesting that similar protein secretion systems are common among members of this large and diverse group of bacteria.     

Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization

Cells of Flavobacterium johnsoniae move rapidly over surfaces by gliding motility. The mechanism of this form of motility is not known. Six genes (gldA, gldB, gldD, gldF, gldG, and ftsX) that are required for gliding have been described. Tn4351 mutagenesis was used to identify another gene, gldH, which is required for cell movement. GldH mutants formed nonspreading colonies, and individual cells lacked the cell movements and ability to propel latex spheres along their surfaces that are characteristic of wild-type cells. gldH mutants also failed to digest chitin and were resistant to bacteriophages that infect wild-type cells. Introduction of pMM293, which carries wild-type gldH, restored to the gldH mutants colony spreading, cell motility, the ability to move latex spheres, phage sensitivity, and the ability to digest chitin. gldH encodes a predicted 141-amino-acid protein that localized to the membrane fraction. Labeling studies with [3H]palmitate demonstrated that GldH is a lipoprotein. GldB and GldD, which were previously described, also appear to be lipoproteins. GldH does not exhibit significant amino acid similarity to proteins of known function in the databases. Putative homologs of gldH of unknown function are found in motile (Cytophaga hutchinsonii) and apparently nonmotile (Bacteroides thetaiotaomicron, Bacteroides fragilis, Tannerella forsythensis, Porphyromonas gingivalis, and Prevotella intermedia) members of the Cytophaga-Flavobacterium-Bacteroides group.     

Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division

Flavobacterium johnsoniae is a gram-negative bacterium that exhibits gliding motility. To determine the mechanism of flavobacterial gliding motility, we isolated 33 nongliding mutants by Tn4351 mutagenesis. Seventeen of these mutants exhibited filamentous cell morphology. The region of DNA surrounding the transposon insertion in the filamentous mutant CJ101-207 was cloned and sequenced. The transposon was inserted in a gene that was similar to Escherichia coli ftsX. Two of the remaining 16 filamentous mutants also carried insertions in ftsX. Introduction of the wild-type F. johnsoniae ftsX gene restored motility and normal cell morphology to each of the three ftsX mutants. CJ101-207 appears to be blocked at a late stage of cell division, since the filaments produced cross walls but cells failed to separate. In E. coli, FtsX is thought to function with FtsE in translocating proteins involved in potassium transport, and perhaps proteins involved in cell division, into the cytoplasmic membrane. Mutations in F. johnsoniae ftsX may prevent translocation of proteins involved in cell division and proteins involved in gliding motility into the cytoplasmic membrane, thus resulting in defects in both processes. Alternatively, the loss of gliding motility may be an indirect result of the defect in cell division. The inability to complete cell division may alter the cell architecture and disrupt gliding motility by preventing the synthesis, assembly, or functioning of the motility apparatus.     

Flavobacterium johnsoniae SprA is a cell surface protein involved in gliding motility

Flavobacterium johnsoniae cells glide rapidly over surfaces by an unknown mechanism. Transposon-induced sprA mutants formed nonspreading colonies on agar, and the cells examined in wet mounts were deficient in attachment to surfaces and were almost completely nonmotile. Exposure of intact cells to proteinase K cleaved the 270-kDa SprA into several large peptides, suggesting that it is partially exposed on the cell surface.     

Characterization and structure of genes for proteases A and B from Streptomyces griseus.

Protease A and protease B are extracellular proteins which are secreted by Streptomyces griseus. The genes encoding protease A (sprA) and protease B (sprB) were isolated from an S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing sprA and sprB were characterized by hybridization and demonstration of proteolytic activity in Streptomyces lividans. Each DNA sequence contains a large open reading frame with the coding region of the mature protease situated at its carboxy terminus. The amino terminus of each reading frame appears to encode a 38-amino-acid signal peptide followed by a 76- or 78-amino-acid polypeptide, a propeptide, which is joined to the mature protease. Strong homology between the coding regions of the protease genes suggests that sprA and sprB originated by gene duplication.     

Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Mutations in gldN cause a partial defect in gliding. A novel bacteriophage selection strategy was used to aid construction of a strain with a deletion spanning gldN and the closely related gene gldO in an otherwise wild-type F. johnsoniae UW101 background. Bacteriophage transduction was used to move a gldN mutation into F. johnsoniae UW101 to allow phenotypic comparison with the gldNO deletion mutant. Cells of the gldN mutant formed nonspreading colonies on agar but retained some ability to glide in wet mounts. In contrast, cells of the gldNO deletion mutant were completely nonmotile, indicating that cells require GldN, or the GldN-like protein GldO, to glide. Recent results suggest that Porphyromonas gingivalis PorN, which is similar in sequence to GldN, has a role in protein secretion across the outer membrane. Cells of the F. johnsoniae gldNO deletion mutant were defective in localization of the motility protein SprB to the cell surface, suggesting that GldN may be involved in secretion of components of the motility machinery. Cells of the gldNO deletion mutant were also deficient in chitin utilization and were resistant to infection by bacteriophages, phenotypes that may also be related to defects in protein secretion.Cells of Flavobacterium johnsoniae, and of many other members of the phylum Bacteroidetes, crawl over surfaces at approximately 2 μm/s in a process called gliding motility. F. johnsoniae cells glide on agar, glass, polystyrene, Teflon, and many other surfaces (16, 22). Cells suspended in liquid also bind and propel added particles such as polystyrene latex spheres (23). The mechanism of this form of cell movement is not well understood despite decades of research (15). Genome analyses suggest that F. johnsoniae gliding is genetically unrelated to other well-studied forms of bacterial movement such as bacterial flagellar motility, type IV pilus-mediated twitching motility, myxobacterial gliding motility, and mycoplasma gliding motility (10, 20, 21). Genes and proteins required for F. johnsoniae motility have been identified (1-3, 7-9, 17, 18). GldA, GldF, and GldG appear to form an ATP-binding cassette transporter that is required for gliding (1, 7). Eight other Gld proteins (GldB, GldD, GldH, GldI, GldJ, GldK, GldL, and GldM) are also required for movement (2, 3, 8, 9, 17, 18). Many of these are unique to members of the phylum Bacteroidetes. Disruption of the genes encoding any of these 11 proteins results in complete loss of motility. The mutants form nonspreading colonies, and individual cells exhibit no movement on agar, glass, Teflon, and other surfaces tested. The Gld proteins are associated with the cell envelope and presumably constitute the gliding motor, but none of them appear to be exposed on the cell surface. Mutations in sprA and sprB, which encode cell surface proteins, result in partial motility defects. Cells form nonspreading colonies, but some of the cells exhibit limited movement in wet mounts. SprA is required for efficient attachment to glass (22), and SprB appears to be a mobile adhesin that is propelled along the cell surface by the gliding motor and thus transmits the force generated by the motor to the surface over which cells crawl (10, 21). The surface localization of SprA and SprB and the phenotypes of sprA and sprB mutants suggest that the gliding motor is at least partially functional in these mutants but that force is inefficiently transmitted to the substratum. Analysis of the F. johnsoniae genome revealed the presence of multiple paralogs of sprB, which may explain the residual motility of sprB mutants (20).gldN lies downstream of gldL and gldM, and the three genes constitute an operon (2). Cells with transposon insertions in gldN form nonspreading colonies that are indistinguishable from those of other gld mutants. However, unlike other gld mutants, gldN mutants exhibit some residual ability to glide in wet mounts (2). One possible explanation for this phenotype is that GldN may have a peripheral and nonessential role in gliding. Alternatively, GldN may perform a critical function in gliding, but in its absence another cellular protein may compensate for the missing GldN function. F. johnsoniae has a gldN paralog, gldO, that is located downstream of gldN but is transcribed independently (2). The GldN and GldO proteins are 85% identical over their entire lengths, making GldO a prime candidate for a protein that might compensate for lack of GldN.Recent results suggest that some of the F. johnsoniae Gld and Spr proteins, including GldN, may be components of a novel bacteroidete protein translocation apparatus referred to as the Por secretion system (PorSS) (28). This conclusion emerged from studies of gingipain protease secretion by the distantly related nonmotile bacteroidete Porphyromonas gingivalis. P. gingivalis is a human periodontal pathogen, and gingipain proteases are important virulence factors. Gingipains have signal peptides that allow export across the cytoplasmic membrane via the Sec machinery, but they rely on components of the PorSS for secretion across the outer membrane (27-29). P. gingivalis cells with mutations in genes homologous to F. johnsoniae gldK, gldL, gldM, gldN, and sprA are defective in gingipain secretion across the outer membrane (28). F. johnsoniae has a homologue to another P. gingivalis gene required for gingipain secretion, porT. Disruption of the F. johnsoniae porT homologue (referred to as sprT) results in motility defects and defects in surface localization of SprB (28).This study was designed to identify possible roles for GldN in motility and to determine whether GldN and GldO are partially redundant components of the motility apparatus. The results demonstrate that F. johnsoniae GldN has an important function in motility and that GldO can replace GldN in this role. They suggest that GldN is needed for efficient secretion of the cell surface motility protein SprB, which may explain some of the motility defects of the gldN mutants.     

Mutations in Flavobacterium johnsoniae secDF result in defects in gliding motility and chitin utilization

secDF mutants of Flavobacterium johnsoniae were deficient in gliding motility and chitin utilization. Cells of the mutants had reduced levels of GldJ protein, which is required for both processes. SecDF is similar to Escherichia coli SecD and SecF, which are involved in protein secretion.     

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.