Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus.

The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics on strains either containing or lacking ermB. The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS(B) antibiotics.

The role of genomics in antibacterial target discovery.

Complete DNA sequence information has now been obtained for several prokaryotic genomes, defining the entire genetic complement of these organisms. The collection of genomic data has provided new insights into the molecular architecture of bacterial cells, revealing the basic genetic and metabolic structures that support viability of the organisms. Genomic information has also revealed new avenues for inhibition of bacterial growth and viability, expanding the number of possible drug targets for antibiotic discovery. This review examines how genomic sciences and experimental tools are applied to antibacterial target discovery, the necessary first step in the development of new antibiotic classes. Significant advances have been realized in the development of functional genomic, comparative genomic, and proteomic methods for the analysis of completed genomes. The combination of these methods can be used to systematically parse the genome and identify targets worthy of inhibitor screens. Two basic categories of targets emerge from this exercise, comprising in vitro essential targets required for bacterial viability on synthetic media and in vivo essential targets required to establish and maintain infection within a host organism. Current use of genomic information is focused primarily on a definition of all in vitro essential targets that satisfy criteria of selectivity, spectrum, and novelty. As the genomes of additional bacterial pathogens are solved, it will be possible to select in vivo essential targets common to groups of select pathogens (e.g., bacterial agents of community acquired pneumonia) or even pathogen-specific targets. Consideration of host-pathogen interactions, defined at the level of gene expression for each organism, might provide novel therapeutic options in the future.

Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ.

Shigella species and enteroinvasive Escherichia coli contain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains of Shigellae. Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen from Shigella flexneri, designated IpaJ. The ipaJ gene encodes a 780 bp open reading frame (ORF), separated from the ipaR (virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoA mutagenesis of the ipaJ ORF confirmed the presence of export signal sequences in IpaJ. Unlike ipaBCDA genes, transcription analysis of ipaJ indicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as a Shigella immunogen. Construction and phenotypic characterization of ipaJ mutants in two serotypes of S. flexneri showed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, the ipaJ mutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.

A targeted mutagenesis system for Actinobacillus pleuropneumoniae.

We describe methods for the mutagenesis of cloned Actinobacillus pleuropneumoniae (Ap) genes and for the construction of Ap mutants by allelic exchange. We used these methods to construct isogenic mutants of Ap which no longer synthesized a 48-kDa outer membrane protein (AopA). The native aopA locus was replaced with a mutated locus that had been inactivated by insertion of a gene (KmR) encoding kanamycin resistance from Tn903. The inactivated aopA locus was cloned into a conjugative, R6K-derived, lambda pir-dependent suicide vector and introduced into Ap using a filtermating technique. Southern and Western blot analyses indicated that the wild-type locus was replaced by the mutated locus through either single- or double-crossover events, and that AopA was no longer produced by either type of mutant. These methods were used successfully to construct AopA- mutants in Ap serotypes 1 and 5. These methods should be generally useful in constructing mutant loci which can be used to analyze the roles of various Ap genes in the pathogenesis of contagious pleuropneumonia in swine.

Genetic polymorphism of the ipaH multicopy antigen gene in Shigella spps. and enteroinvasive Escherichia coli.

The ipaH loci comprise a multicopy antigen gene family unique to Shigella species and enteroinvasive Escherichia coli (EIEC). DNA probes derived from the Shigella flexneri serotype 5 ipaH7.8 gene were used to compare the molecular arrangement of ipaH alleles in a variety of Shigella and EIEC strains. Multiple copies of ipaH-homologous sequences were detected in all invasion plasmids examined. Oligonucleotide probes covering discrete 24 bp segments of the ipaH7.8 gene and sequences flanking the ipaH4.5 (probe H25) and ipaH2.5 (probe H24) loci were used to define the extent of homology among invasion plasmid copies of ipaH in S. flexneri serotypes 1, 2 and 5 and in S. sonnei. IpaH alleles carried by these invasion plasmids were not structurally equivalent and showed sequence divergence at their amino- and carboxy-terminal ends. The H25 probe was shown to correspond to an IS629 sequence genetically linked to the ipaH alleles, while the H24 probe defined a DNA sequence found only in Shigella invasion plasmids. Chromosomal DNA from invasion plasmid-cured S. flexneri and S. sonnei strains hybridized a core ipaH7.8 gene segment, indicating that portions of the ipaH7.8 structural gene were reiterated and contained within the shigellae chromosomes. Based on the specificity of the ipaH7.8 core probe and the detection of ipaH sequences on the invasion plasmids and chromosomes of Shigella strains, three polymorphic groups within a collection of forty S. dysenteriae 1 isolates received by the United States Centers for Disease Control in 1988 were identified using this probe. These results suggest that ipaH restriction fragment length polymorphisms may be useful in genetic lineage and epidemiologic studies of virulent shigellae.

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.

Spontaneous insertion of an IS1-like element into the virF gene is responsible for avirulence in opaque colonial variants of Shigella flexneri 2a.

Colonial variation of Shigella flexneri serotype 2a from the translucent (2457T) to the opaque form (2457O) occurs spontaneously once in 10(4) cell divisions, with concomitant loss of ipa gene expression and virulence. The appearance of 2457O was associated with the insertional inactivation of virF, an invasion plasmid-encoded positive regulator of ipa gene expression. Plasmid pWR110, a Tn5-tagged invasion plasmid that restores the invasive phenotype to plasmid-cured Shigella derivatives, was conjugally transferred into 2457O. Synthesis of the invasion-associated IpaB and IpaC polypeptides, normally present on the surface of virulent shigellae, and the invasive phenotype were restored in 2457O(pWR110) transconjugants. Plasmid DNA restriction endonuclease patterns of 2457T and 2457O, along with hybridization analysis, showed that a SalI fragment carrying the virF gene in 2457O had increased in size relative to its counterpart in 2457T. Analysis of virF DNA sequences amplified by the polymerase chain reaction revealed that the virF sequence from 2457O was 780 bp larger than that amplified from 2457T. Moreover, the virF sequence amplified from 2457O hybridized to an IS1 DNA probe whereas the amplified 2457T virF sequence did not. DNA sequence analysis mapped the insertion element, designated IS1SFO, within an A.T-rich region of the virF open reading frame and identified a 9-bp virF target sequence that was duplicated at the insertion site of IS1SFO. The DNA sequence of IS1SFO was greater than 99% homologous to IS1F. Plasmid pWR600, carrying a 1,260-bp HpaII fragment encoding a wild-type virF gene, was able to restore the virulent phenotype and translucent colonial morphology to nine independently isolated 2457O hosts.

Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins.

Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five ipaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT-W. The primary structure of IpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in IpaH7.8 were replaced in IpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino-terminal region of IpaH4.5 displayed an internal repeat structure, also seen in IpaH7.8, characteristic of members of the leucine-rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of ipaH7.8. Western blot analysis of a lambda gt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of IpaH7.8 resides outside the leucine-rich repetitive region.

Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides.

Shigellosis is a major cause of infant morbidity and mortality in developing countries. To find immunological correlates of specific protection against shigellosis, we examined chronological samples of sera, stool extracts, duodenal aspirates, and saliva samples from 39 adults and 22 children with shigellosis from Peru for the presence of specific antibody to invasion plasmid antigens (Ipa) common to all virulent Shigella strains, by using both a whole-organism enzyme-linked immunosorbent assay (ELISA) and a Western blot (immunoblot) assay. Antibody responses to lipopolysaccharide (LPS) from Shigella serotypes both homologous and heterologous to the infecting strain were also determined by ELISA. ELISAs showed that the highest serum immunoglobulin G (IgG) antibody titers to Shigella whole organisms both with and without surface Ipa were found in adults and malnourished children, the two groups with the shortest and longest durations of disease, respectively. Mucosal IgA antibody titers to Shigella strains decreased over time to a much greater extent than serum IgG titers, and IgA to Ipa in mucosal secretions was found in adults and well-nourished children but not in malnourished children. The presence of mucosal antibody to Ipa may limit the spread and severity of the infection, as indicated by the prolonged illness observed in malnourished children who have no significant mucosal antibody to Shigella Ipa. Serum antibody titers to the Ipa antigens were high relative to anti-Shigella LPS antibody titers, especially in pediatric patients. In contrast to the anti-Ipa responses observed, no differences in antibody responses to LPS in children compared by nutritional status were found. High levels of serum and mucosal cross-reacting antibody to heterologous serotype LPS were found between Shigella flexneri serotypes 1a and 2a. Different patterns of immune response to Ipa proteins and LPS that may aid in the definition of Shigella antigens important in host protection were observed in adults, well-nourished children, and malnourished children.

Virulence phenotype and genetic characteristics of the T32-ISTRATI Shigella flexneri 2a vaccine strain.

The T32-ISTRATI strain, which has been used as an oral attenuated Shigella flexneri 2a vaccine, has lost the invasive phenotype due to a spontaneous deletion in the shigella virulence plasmid. This deletion has eliminated three plasmid loci (ipaBCDA, invA and virG) that are necessary for production of a positive Sereny test by Shigella species. Virulence in the Sereny test was reconstituted in the T32-ISTRATI strain by the conjugal transfer of an intact 140 M Da virulence plasmid from S. flexneri 5. The T32-ISTRATI vaccine is safe when given orally in multiple doses of 50-100 x 10(9) organisms, and both homologous and heterologous protection has been reported in large Romanian and Chinese field trials. Although the protective antigen(s) in this vaccine have not been identified, the potential use of non-invasive plasmid deletion mutants as living shigella vaccines is illustrated by the T32-ISTRATI vaccine.

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.

Molecular characterization of a trans-acting, positive effector (ipaR) of invasion plasmid antigen synthesis in Shigella flexneri serotype 5.

A trans-acting, positive effector of invasion plasmid antigen (Ipa) synthesis has been identified and mapped on the pWR100 invasion plasmid of Shigella flexneri serotype 5 (strain M90T-W). Recombinant plasmids carrying this regulatory gene, designated ipaR, were found to restore full virulence to a non-invasive ipaR::Tn5 insertion mutant [M90T-W(pHS1042)] that had lost the ability to synthesize four Ipa antigens (IpaA, 70 kDa; IpaB, 62 kDa; IpaC, 42 kDa; and IpaD, 37 kDa). Genetic mapping of the ipaR gene positioned the locus on a 2.6 kb PstI-AccI fragment contained within a larger 8.0 kb EcoRI molecule that also encoded IpaD, IpaA, and two small proteins (27 kDa and 28 kDa). The trans regulatory effect of the ipaR product on ipaB, ipaC, ipaD, and ipaA expression was demonstrated by transforming compatible ipaBC, ipaDA, ipaR and ipaDAR plasmid recombinants, in various combinations, into M90T-A3, an isogenic invasion plasmid mutant of M90T-W that contained a deletion of the pWR100 ipaBCDA and ipaR loci; such transformants produced wild type levels of the IpaB, IpaC, IpaD and IpaA antigens only in the presence of IpaR+ plasmids. DNA sequence analysis of the ipaR region established that the intiation codon for ipaR is 459 bp from the 3'-end of the ipaA gene and that ipaR encodes a 309 amino acid residue protein. An interesting feature of the IpaR polypeptide was its strong sequence homology with the bacteriophage P1 partition protein ParB, consisting of a 42.8% amino acid identity over a 278 residue section of the aligned proteins.

Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli.

The products of the ipaB, ipaC, and ipaD genes are involved in the expression of the invasive phenotype in all species of Shigella and enteroinvasive Escherichia coli (EIEC). DNA probes derived from these genes are accurate indicators of the invasive phenotype (M. Venkatesan, J. M. Buysse, E. V. Vandendries, and D. J. Kopecko, J. Clin. Microbiol. 26:261-266, 1988); however, spontaneous loss of the invasion plasmid or selective deletion of invasion-associated genes may restrict the usefulness of such probes as general diagnostic tools. In this study, we report that laboratory-passaged strains of Shigella spp. and EIEC that were invasion and Sereny test negative were unable to hybridize to the ipaC DNA probe. However, a second DNA probe, derived from the Shigella flexneri ipaH gene, a multiple-copy element found on the chromosome and invasion plasmid that encodes a 60-kilodalton antigen, was more sensitive in its ability to detect virulent as well as avirulent shigellae and EIEC. Analysis of colony blots and stool blots from pediatric patients with diarrhea indicated that the ipaH probe was more effective in detecting shigellae and EIEC than was either the ipaC or 17-kilobase EcoRI fragment probe.

Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri.

The large invasion plasmid of Shigella flexneri M9OT-W was used to generate recombinant plasmids carrying the ipaA, -B, -C, and -D genes, whose products are associated with the entry of the bacteria into colonic epithelial cells. Complete DNA sequences of ipaB, -C, and -D were determined. The proteins predicted (62, 42, and 37 kDa, respectively) from the nucleotide sequences lack a signal-peptide sequence. Hydrophilic segments of the IpaB and IpaC proteins were found to overlap known epitopic domains of these membrane antigens. Analysis of total RNA demonstrated that temperature control of ipa gene expression occurs at the level of transcription. Multiple mRNA bands were detected by using ipa gene fragments as hybridization probes, and a putative transcript map for the ipa genes was constructed. Comparison of this map with the DNA sequence reveals a complex system of ipa gene regulation.

Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies.

Invasion plasmid antigens B (IpaB) and C (IpaC) are associated with the ability of shigellae to invade cultured mammalian cells. Monoclonal antibodies against IpaB and IpaC polypeptides were produced and used in a whole-cell enzyme-linked immunosorbent assay to show that both IpaB and IpaC polypeptides were exposed on the surface of virulent shigellae. Moreover, these surface epitopes were shown to be highly conserved among different serotypes of Shigella spp. and enteroinvasive Escherichia coli. Cross-reactive epitopes were not found on noninvasive Shigella strains or on other enteric bacteria including Salmonella, Yersinia, Campylobacter, Vibrio, and Aeromonas spp. and various pathogenic strains of E. coli. The monoclonal antibodies were used in competitive binding assays to define three unique epitopes of the IpaB polypeptide and four unique epitopes of the IpaC polypeptide. Epitope locations and their corresponding DNA-encoding regions were defined by examining the IpaB and IpaC products expressed by lambda gt11 recombinants and by constructing a genetic map of the insert DNAs of these recombinants. Three IpaB epitopes (2F1, 1H4, 4C8) were found to be encoded on three contiguous DNA regions comprising a 700-base-pair (bp) segment that corresponded to the amino-terminal end of the IpaB polypeptide. Similarly, a 640-bp DNA segment that corresponded to the amino-terminal end of the IpaC polypeptide was found to encode three clustered IpaC epitopes (5H1, 9B6, 5B1). Approximately 50 bp downstream from this region a fourth IpaC epitope-encoding region (2G2) was found. The effect of the monoclonal antibodies on plaque formation by virulent Shigella flexneri on a monolayer of cultured mammalian cells (a sensitive measure of invasiveness) was determined. Only the IpaB-specific monoclonal antibody 2F1 was able to reduce the plaque-forming capacity by greater than 50%, suggesting that this epitope of the IpaB polypeptide is involved in the invasion process.

Development and testing of invasion-associated DNA probes for detection of Shigella spp. and enteroinvasive Escherichia coli.

Genetic determinants of the invasive phenotype of Shigella spp. and enteroinvasive Escherichia coli (EIEC), two common agents of bacillary dysentery, are encoded on large (180- to 210 kilobase), nonconjugative plasmids. Several plasmid-encoded antigens have been implicated as important bacterial ligands that mediate the attachment and invasion of colonic epithelial cells by the bacteria. Selected invasion plasmid antigen (ipa) genes have recently been cloned from Shigella flexneri serotype 5 into the lambda gt11 expression vector. Portions of three ipa genes (ipaB, ipaC, and ipaD) were tested as DNA probes for diagnostic detection of bacillary dysentery. Under stringent DNA hybridization conditions, all three DNA sequences hybridized to a single 4.6-kilobase HindIII fragment of the invasion plasmids of representative virulent Shigella spp. and EIEC strains. No hybridization was detected in isogenic, noninvasive Shigella mutants which had lost the invasion plasmid or had deleted the ipa gene region. Furthermore, these probes did not react with over 300 other enteric and nonenteric gram-negative bacteria tested, including Salmonella, Yersinia, Edwardsiella, Campylobacter, Vibrio, Klebsiella, Aeromonas, Enterobacter, Rickettsia, and Citrobacter spp. and various pathogenic E. coli strains. The use of unique invasion-essential gene segments as probes for the specific detection of invasive dysentery organisms should benefit both epidemiologic and diagnostic analyses of Shigella spp. and EIEC.

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.