Transposon Tn5 mutagenesis of Brucella abortus.

We demonstrate that transposon-mediated mutagenesis can be used to construct mutations in the pathogen Brucella abortus. We have used both a plasmid and a bacteriophage to introduce either Tn5 or Tn5 lac into the Brucella chromosome. B. abortus is naturally sensitive to kanamycin. We have selected 22 independent kanamycin-resistant colonies in strain US-19 and 19 colonies in strain 2308. We have demonstrated by Southern hybridization that Tn5 was inserted into the chromosome.     

Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori.

Helicobacter pylori now is recognized as an etiological agent in chronic superficial gastritis and peptic ulcer disease. Although only about 60% of H. pylori isolates produce an immunodominant 128-kDa antigen (CagA; cytotoxin-associated gene product), virtually all H. pylori-infected patients with duodenal ulceration develop a serologic response to the 128-kDa protein, which suggests an association of this gene with ulceration. The cloned cagA gene from H. pylori 84-183 was disrupted by insertion of a kanamycin resistance gene, and this inactivated cagA construct was introduced into H. pylori 84-183 by electrotransformation. Southern hybridization of kanamycin-resistant H. pylori transformants demonstrated that the wild-type cagA gene had been disrupted by insertion of the kanamycin cassette, and immunoblot analysis showed that the mutant strains no longer produced the 128-kDa CagA protein. Similar results were obtained when the cagA mutation was introduced by natural transformation into H. pylori 60190, a high-level toxin-producing strain. The cagA-negative H. pylori strains showed cytotoxin, urease, and phospholipase C activities, C3 binding and adherence similar to those of the isogenic wild-type strains. These findings demonstrate that the cagA gene product does not affect the vacuolating cytotoxin activity of H. pylori.     

Characterization of a sialyltransferase-deficient mutant ofNeisseria gonorrhoeaestrain F62: instability of transposon Tn1545 Δ3 in gonococci and evidence that multiple genetic loci are essential for lipooligosaccharide sialylation

Neisseria gonorrhoeaestrain JB1 was previously shown to be defective in the sialylation of lipoologosaccharide (LOS) by exogenous CMP-NANA. The LOS components synthesized by the mutant now have been shown by mass spectrometry to be similar to those in the parental strain, F62, and to include the 4.5 kDa widely conserved lacto-N-neotetraose component that can be sialylated. The same two LOS components could be sialylated on the surface of the mutant and parental strains. One major component was sialylatable after chemical extraction of the LOS from either strain. These data confirm that the mutant, JB1, retains the ability to synthesize the LOS target required for the conversion by sialylation of serum-sensitive gonococci to serum resistance. A single base frame-shift mutation was found in thelstgene from the mutant, resulting in the replacement of the final 61 amino acids at the C-terminus of the sialyltransferase by four residues. Seventeen independent clones of thelstgene were isolated from the parental strain, but none of them complemented the sialyltransferase defect of the mutant and no sialyltransferase activity expressed from the clones could be detected inEscherichia coli.Although the data suggest that the mutant might be defective in genes at more than one chromosomal locus and that multiple loci are essential for sialyltransferase synthesis and activity, the alternative possibility, that DNA adjacent to thelstgene encodes a product which is toxic toE. coli, cannot be excluded. The site of insertion of the transposon Tn1545-Δ3 in strain JB1 was cloned and sequenced. The transposon is located in an intergenic region adjacent to genes for a putative ATP-dependent transport protein, but encoding no recognizable function relevant to LOS sialylation. Evidence that transposon Tn1545-Δ3 is unstable in gonococci is presented.     

A method for allelic replacement that uses the conjugative transposon Tn916: deletion of the emm6.1 allele in Streptococcus pyogenes JRS4.

The emm6.1 allele of Streptococcus pyogenes JRS4 was deleted by using the conjugative transposon Tn916. The aphA-3 gene, conferring resistance to kanamycin, was cloned between the sequences flanking the structural gene for the type 6 M protein (emm6.1) and inserted into the BstXI site of Tn916 to generate the chimeric transposon Tn916-5K3. Because the BstXI site lies in a nonessential region of Tn916, the chimeric transposon could transfer by conjugation from Bacillus subtilis into JRS4. In some of the transconjugants, Tn916-5K3 replaced the emm6.1 locus of JRS4 by homologous recombination between the cloned emm6.1-flanking regions and the resident chromosome. One recombinant studied in detail, JRS75, was kanamycin resistant and tetracycline sensitive and lacked immunologically detectable M6 protein. Furthermore, by Southern blot analysis, the DNA region encompassing the emm6.1 structural gene was found to have been replaced by aphA-3.     

A Streptococcus mutans mutant that synthesizes elevated levels of intracellular polysaccharide is hypercariogenic in vivo.

We used the streptococcal transposon, Tn916 to identify and isolate mutants of Streptococcus mutans with altered intracellular polysaccharide (IPS) accumulation. We report on the isolation and characterization of S. mutans SMS202, a transposon mutant which accumulated the glycogen-like IPS in excess of wild-type levels. Southern blot analysis confirmed a single Tn916 insertion into the SMS202 chromosome. Moreover, quantitative ultrastructural analysis revealed significantly increased concentrations of IPS in SMS202 relative to those of the wild-type progenitor strain, UA130. The activities of ADPglucose pyrophosphorylase (GlgC) and glycogen synthase (GlgA), enzymes required for the biosynthesis of bacterial IPS, were also elevated in the IPS excess mutant. Furthermore, SMS202 was significantly more cariogenic on the molar surfaces of germ-free rats than the wild type (P < 0.01), thus confirming a central role for IPS in S. mutants-induced caries formation. We propose that the increased cariogenic potential of SMS202 is due to constitutive expression of genes which encode glycogen biosynthesis in this oral pathogen. The coordinate expression of GlgC and GlgA along with the results of ongoing nucleotide sequence analysis and Northern hybridization experiments support an operon-like arrangement for the glg genes of this oral pathogen.     

Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants.

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.     

Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide.

We recently found that the 16.4-kb conjugative transposon Tn916 could be introduced into Neisseria meningitidis by transformation and that it appeared to transpose to many different sites in the chromosome of recipient meningococci. In order to identify transposon-induced alterations of specific meningococcal virulence determinants, a library of meningococcal Tetr transformants containing Tn916 was made and screened for those altered in the production of group B capsular polysaccharide. A capsule-defective mutant, M7, was identified by using monoclonal and polyclonal antisera to group B polysaccharide in immunoblot and agar antiserum procedures. Growth of M7 was similar to that of the parent strain. M7 produced no group B capsular polysaccharide by rocket immunoelectrophoresis, and the mutation was stable during laboratory passage. The capsule-defective phenotype was linked to Tetr, as demonstrated by immunoblot and Southern blot analysis of progeny Tetr transformants (transformants of the parent strain obtained with DNA from M7). A capsule-deficient mutant, O8, was identified by using a similar approach. Analysis of the Tn916 insertions in M7 and O8 indicated that a significant portion of the transposon on either side of the tetM determinant had been lost. The ability of Tn916 to generate defined, stable mutations in meningococcal virulence determinants is demonstrated by our study.     

Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.     

Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.     

Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396–402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.     

Identification of a possible cytadherence regulatory locus in Mycoplasma pneumoniae.

Transposon mutagenesis was used to analyze Mycoplasma pneumoniae cytadherence. Mycoplasmas were electroporated with Tn4001, and transformants were identified by antibiotic selection using gentamicin. The resulting colonies were screened for hemadsorption (HA) as an indicator for cytadherence. Six HA- colonies from independent transformations were isolated, filter cloned, and characterized in more detail. Southern hybridization analysis revealed that all six transposon insertions mapped to the same 252-kbp ApaI fragment and 19.5-kbp XhoI fragment. More detailed analysis localized the insertion to two adjacent EcoRI fragments. This site is distinct from the locus containing the genes for the high-molecular weight cytadherence-accessory proteins HMW1 and HMW3, and yet these proteins were absent from the protein profiles of all six transformants. To determine if transposon insertion was responsible for the HA- phenotype, reversion frequencies of the transformants were assessed after passage in the presence of antibiotic selection. In contrast to a spontaneously arising HMW-deficient variant, which reverted to an HA+ phenotype readily, no HA+ revertants were identified for any of the six transformants. These observations suggest that a potential regulatory locus that may be important in the expression of the HMW cytadherence-accessory proteins has been identified.     

Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases.

The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

Characterization of a WaaF (RfaF) Homolog Expressed by Haemophilus ducreyi

Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601–2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290–4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to the protein product of the rfaF (waaF) gene of Salmonella typhimurium. The H. ducreyi waaF gene was able to complement an S. typhimurium rfaF (waaF) mutant, a result which confirmed the identity of this gene. In contrast to the rfaF (waaF) gene of enteric bacteria, the H. ducreyi waaF gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of the H. ducreyi waaF gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type H. ducreyi LOS. Testing of the wild-type H. ducreyi strain and its isogenic waaF mutant in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi revealed that this waaF mutant was less virulent than the wild-type parent strain. Complementation of the H. ducreyi waaF mutant with the wild-type H. ducreyi waaF gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Interruption of capsule production in Streptococcus pneumonia serotype 3 by insertion of transposon Tn916.

Transposon Tn916 mutagenesis was used to produce mutant strains of Streptococcus pneumoniae serotype 3 that lacked only a polysaccharide capsule. Southern blotting, DNA-DNA hybridization, and immunochemical analyses demonstrated that the presence of a single copy of Tn916 was sufficient to produce unencapsulation. The 50% lethal dose for such mutants was greater than 5 x 10(7) CFU, as opposed to a 50% lethal dose of 1 CFU for wild-type strains. These experiments outline an effective method for targeting genes in S. pneumoniae by transposon interruption and provide molecular evidence to support the longstanding hypothesis that the capsule is the principal virulence factor in this pathogen.     

Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

Haemophilus ducreyi, a cause of genital ulcer disease in developing countries, appears to facilitate the heterosexual transmission of the human immunodeficiency virus in Africa. Despite an increase in studies of this gram-negative human pathogen, little is known about the pathogenesis of chancroid. Our studies have shown that the lipooligosaccharides (LOS) of H. ducreyi may play an important role in ulcer formation. Monoclonal antibody and mass spectrometric analyses identified a terminal trisaccharide present on H. ducreyi LOS that is immunochemically similar to human paragloboside. This epitope is present on the LOS of Neisseria gonorrhoeae, and it may be the site of attachment for pyocin lysis. We have used pyocin, produced by Pseudomonas aeruginosa, to select LOS variants with sequential saccharide deletions from N. gonorrhoeae. On the basis of the similarities between N. gonorrhoeae and H. ducreyi LOS, we employed the same technique to determine if H. ducreyi strains were susceptible to pyocin lysis. In this study, we report the generation of a pyocin N-resistant H. ducreyi strain which synthesizes a truncated version of the parental LOS. Further studies have shown that this H. ducreyi variant has lost the terminal LOS epitope defined by monoclonal antibody 3F11. This report demonstrates that H. ducreyi is sensitive to pyocins and that this technique can be used to generate H. ducreyi LOS variants. Such variants could be used in comparative studies to relate LOS structure to biologic function in the pathogenesis of chancroid.     

Transposon mutagenesis in Halomonas eurihalina

We have established a transposon mutagenesis procedure for the moderate halophile Halomonas eurihalina, a bacteria that produces an exopolysaccharide (EPS) of considerable biotechnological interest. We used suicide plasmids pUT and pSUP102 to introduce the transposons mini-Tn5 and Tn1732 into H. eurihalina via Escherichia coli mediated conjugation. Southern hybridization analysis demonstrated that insertions of the transposon mini-Tn5 into H. eurihalina occurred randomly at single sites in the chromosome, whereas Tn1732 insertion also took place at random, but simultaneously, at several sites. Phenotypic analysis revealed that different mutants were generated by using mini-Tn5. The isolation of exopolysaccharide-defective strains is the first stage towards carrying out genetic studies on EPS production by this microorganism.     

Expression of sialylated or paragloboside-like lipooligosaccharides are not required for pustule formation by Haemophilus ducreyi in human volunteers

The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.     

Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae.

A library of Tn916-generated, tetracycline-resistant (Tc) mutants of the group B Neisseri meningitidis strain NMB was screened by using monoclonal antibodies (MAbs) that recognize structural differences in neisserial lipooligosaccharide (LOS). The LOS of parental strain NMB had a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were identified by colony immunoblots, electrophoresis, and Western immunoblots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6E4 but not MAb 3E11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa and reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSs of the R6 and SS3 mutants were predicted to contain different truncations of the core oligosaccharide. The LOS phenotype of each mutant was linked to Tc(r), as determined by transformation of the parent strain with DNA from the mutant. Southern hybridizations and single-specific-primer PCR revealed in each mutant a single truncated tn916 insertion which had lost genes required for mobilization. Tn916 mutagenesis was used to identify two distinct genetic sites in the meningococcal chromosome involved in biosynthesis of the oligosaccharide chain of LOS and to create genetically defined LOS mutants of N. meningitidis and Neisseria gonorrhoeae.