Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.     

A motility mutant ofSpiroplasma melliferum induced with nitrous acid

The honeybee pathogen,Spiroplasma melliferum, growing on 1.2% agar medium, forms fried egg (umbonate) colonies surrounded by large numbers of satellite colonies. In liquid media, these helical, cell wall-free prokaryotes flex, twist, and rotate rapidly. A motility mutant was isolated as an umbonate colony without satellites after nitrous acid mutagenesis and an enrichment procedure with chemotaxis. The development of an efficient motility mutant enrichment procedure, easily applied to spiroplasmas, which is described here, will not only greatly expedite the accumulation of motility mutants, but will also be extremely useful for isolating chemotactic mutants.     

Disruption of a gene predicted to encode a solute binding protein of an ABC transporter reduces transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.     

The aster yellows controversy: current status

Evidence for and against the spiroplasmal etiology of aster yellows (AY) disease is examined. A spiroplasma, serologically identical to Spiroplasma citri, was cultivated by some workers from lettuce (Lactuca sativa L.) plants claimed to be naturally infected with AY. The isolated spiroplasma was shown to be infectious by injecting Macrosteles fascifrons with the cultured organisms and then confining the injected leafhoppers on healthy plants. The reports claiming that a spiroplasma is the etiological agent of AY, however, exist only in astract form, and several essential questions still need to be answered to substantiate the claim. Evidence against the claim is based on significant differences that have been observed between the behavior of S. citri and the AY agent in the leafhoppers as well as in the plant. Also, helical organisms could not be found in AY-infected plants by either scanning or immunosorbent electron microscopy, and S. citri is serologically unrelated to the mycoplasma-like organisms found in AY-infected plants. These results strongly support the conclusion that the classical AY disease is not caused by a variant of S. citri.     

Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli.

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Spiralin is not essential for helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 x 10(6) to 2.8 x 10(6) CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 x 10(6) to 1.4 x 10(7) CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.     

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations

Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site. Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA.     

Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis

Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN? Tnp transposome? system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 10(9) spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon's presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN? Tnp transposome? system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.     

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.     

Role of the dpr product in oxygen tolerance in Streptococcus mutans

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H(2)O(2), implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc(r) mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc(r) mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc(r) ahpC::Em(r)::nox-1 triple mutant nor the dpr::Spc(r) sod::Em(r) double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.     

The cytoskeleton of spiroplasma: a complex linear motor

Spiroplasma are wall-less, helical bacteria from the class Mollicutes. The Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) evolved by regressive evolution to generate one of the simplest and minimal free-living and self-replicating forms of life. The spiroplasmas are the more advanced members in the class and are the closest to their clostridial ancestors. Spiroplasmas were discovered and identified as such only in 1972 and the finding of a unique and well-defined internal cytoskeleton, believed to be uncommon in bacteria, followed in 1973. Structural analysis suggests that the core of the spiroplasmal cytoskeleton is a flat, monolayered ribbon comprised of the 59-kDa fib gene product. The ribbon follows the shortest helical line of the polar cell from end to end. The structural building blocks of the cytoskeletal ribbon are fibrils assembling into a structure with approximately 10-nm axial and lateral repeats. Differential length changes of the fibrils may generate a wide dynamic spectrum of helical and non-helical geometries allowing for directional motility in low Reynolds number environments. The presence of other cytoskeletal elements (FtsZ, FtsA, EF-TU, MreB) has been demonstrated only recently in Spiroplasma cells. The cellular and molecular structure and dynamics of spiroplasmas and their cytoskeletal elements are reviewed.     

An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts.

The pathogenicity gene, pthA, of Xanthomonas citri is required to elicit symptoms of Asiatic citrus canker disease; introduction of pthA into Xanthomonas strains that are mildly pathogenic or opportunistic on citrus confers the ability to induce cankers on citrus (S. Swarup, R. De Feyter, R. H. Brlansky, and D. W. Gabriel, Phytopathology 81:802-809, 1991). The structure and the function of pthA in other xanthomonads and in X. citri were further investigated. When pthA was introduced into strains of X. phaseoli and X. campestris pv. malvacearum (neither pathogenic to citrus), the transconjugants remained nonpathogenic to citrus and elicited a hypersensitive response (HR) on their respective hosts, bean and cotton. In X. c. pv. malvacearum, pthA conferred cultivar-specific avirulence. Structurally, pthA is highly similar to avrBs3 and avrBsP from X. c. pv. vesicatoria and to avrB4, avrb6, avrb7, avrBIn, avrB101, and avrB102 from X. c. pv. malvacearum. Surprisingly, marker-exchanged pthA::Tn5-gusA mutant B21.2 of X. citri specifically lost the ability to induce the nonhost HR on bean, but retained the ability to induce the nonhost HR on cotton. The loss of the ability of B21.2 to elicit an HR on bean was restored by introduction of cloned pthA, indicating that the genetics of the nonhost HR may be the same as that found in homologous interactions involving specific avr genes. In contrast with expectations of homologous HR reactions, however, elimination of pthA function (resulting in loss of HR) did not result in water-soaking or even moderate levels of growth in planta of X. citri on bean; the nonhost HR, therefore, may not be responsible for the "resistance" of bean to X. citri and may not limit the host range of X. citri on bean. The pleiotropic avirulence function of pthA and the heterologous HR of bean to X. citri are both evidently gratuitous.     

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.