RESUMO
A genetic system was devised that positively selects for bacterial genes that are specifically induced when bacteria infect their host. With the pathogen Salmonella typhimurium, the genes identified by this selection show a marked induction in bacteria recovered from mouse spleen. Mutations in all ivi (in vivo-induced) genes that were tested conferred a defect in virulence. This genetic system was designed to be of general use in a wide variety of bacterial-host systems and has several applications in both vaccine and antimicrobial drug development.
Assuntos
Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência/genética , Animais , Cromossomos Bacterianos , Clonagem Molecular , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Proteínas Recombinantes de Fusão/metabolismo , Salmonelose Animal/microbiologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The two-component regulatory system, OmpR and EnvZ, in Escherichia coli controls the differential expression of ompF and ompC in response to medium osmolarity. Previous studies suggest that EnvZ functions as a membrane sensor relaying information to the DNA-binding protein, OmpR, which in turn activates expression of the appropriate promoter. A strategy has been devised to isolate and characterize a collection of missense mutations in ompR that alter, but do not abolish protein function. Mutants were isolated using strains that contain the ompR and envZ genes in separate chromosomal locations yet maintain the production of both regulatory proteins at physiological levels. Such an arrangement facilitates ompR diploid analysis and tests of epistasis with known envZ mutations. The data obtained indicate that OmpR works in both a positive and negative fashion to control the transcription of ompF and this result forms the basis of a model for porin regulation that explains the switch from OmpF to OmpC production in response to increasing medium osmolarity.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Genes de Troca , Fatores de Transcrição/genética , Análise Mutacional de DNA , Genes Dominantes , Óperon , Porinas , Conformação Proteica , Transdução de Sinais , Equilíbrio HidroeletrolíticoRESUMO
OmpR is a member of a family of bacterial transcriptional regulators whose activity is controlled by phosphorylation. It regulates the transcription of two genes, serving as an activator of ompC, and as both an activator and a repressor of ompF. A previously isolated collection of ompR mutations was analyzed for the effect of each on the expression of both genes simultaneously. The results of this analysis indicate that the activation, repression, and DNA binding functions of OmpR can be disrupted independently, and that mutations interfering with each of these functions cluster within the sequence of the OmpR protein. The nature of these mutations is discussed in terms of the mechanisms by which OmpR regulates transcription, and potentially similar mechanisms operating within closely related response regulators.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diploide , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Concentração Osmolar , Fosforilação , Porinas , Regiões Promotoras Genéticas/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives. We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.
Assuntos
Bacteriófago P22/genética , Proteínas de Escherichia coli , Dosagem de Genes , Plasmídeos/genética , Salmonella typhimurium/genética , Transdução Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonuclease V , Exodesoxirribonucleases/metabolismo , Modelos Genéticos , Mutação , Recombinases Rec A/metabolismo , Recombinação GenéticaRESUMO
IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect. The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. The IVET system has several applications in the area of vaccine and antimicrobial drug development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.
Assuntos
Fusão Gênica Artificial/métodos , Bactérias/genética , Bactérias/patogenicidade , Animais , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas de Ligação a DNA , Genes Bacterianos , Camundongos , Proteínas do Tecido Nervoso , Óperon , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Fatores de Transcrição , Transcrição Gênica , Virulência/genética , beta-Galactosidase/genéticaRESUMO
We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The system is based on complementation of an attenuating auxotrophic mutation by gene fusion, and it is designed to be of use in a wide variety of pathogenic organisms. In Salmonella typhimurium, we have successfully used the system to identify a number of genes that are induced in BALB/c mice, and that, when mutated, confer a virulence defect. The IVET system has several applications in the area of vaccine and antimicrobial drug development. The technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live attenuated vaccines. In addition, the identification of promoters that are optimally expressed in animal tissues provides a means of establishing in vivo regulated expression of heterologous antigens in live vaccines, an area that has been previously problematic. Finally, we expect that our methodology will be used to uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.
Assuntos
Bactérias/patogenicidade , Técnicas Bacteriológicas , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Virulência/genética , Adenilossuccinato Sintase/biossíntese , Adenilossuccinato Sintase/genética , Animais , Bactérias/genética , Sequência de Bases , Genes Sintéticos , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Óperon , Proteínas Recombinantes de Fusão/biossíntese , Salmonella typhimurium/genética , Seleção GenéticaRESUMO
In order to understand the genetic regulation of bacterial genes whose products are important for pathogenesis, one needs to measure the expression of the genes during the infection process. We have devised a method to measure the transcriptional activity of such genes from bacteria recovered directly from infected host tissue. Starting with bacterial strains containing lacZ transcriptional fusions to the genes of interest, animals can be infected, with subsequent isolation of infected host tissue. Here we describe the separation of bacterial cells away from a particular host tissue and the subsequent measurement of the activity of beta-galactosidase, the product of the lacZ gene, in the bacterial cells. This assay is sensitive enough to compensate for the potentially low number of bacteria recovered from the infection site.
Assuntos
Bactérias/genética , Infecções Bacterianas/microbiologia , Transcrição Gênica , Animais , Bactérias/isolamento & purificação , Fluoresceínas/metabolismo , Galactosídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Espectrometria de Fluorescência , Baço/microbiologia , beta-Galactosidase/análise , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Antibodies directed against lipopolysaccharide (LPS) O-antigen are often critical in the immune response to Gram-negative pathogens. Mice were orally immunized with isogenic strains of Salmonella typhimurium that differ only in a minor modification of O-antigen, namely acetylation, mediated by the oafA locus. To specifically examine the effect of acetylation on the antibody response to O-antigen, antibody titers were determined against both acetylated and unacetylated LPS by ELISA. In mice immunized with an oafA+ strain, the median titer against acetylated LPS was 32-fold higher than the titer against unacetylated LPS. Mice immunized with the oafA- strain had an 8-fold higher titer against unacetylated LPS. Thus, acetylation of O-antigen alters recognition by the vast majority of individual antibodies. This differential antibody recognition of O-antigen had a statistically significant correlation with protection against subsequent challenge with virulent S. typhimurium.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias , Antígenos O/metabolismo , Salmonella typhimurium/imunologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Administração Oral , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Antígenos O/imunologia , Salmonelose Animal/imunologia , Salmonelose Animal/prevenção & controle , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , VacinaçãoRESUMO
OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Genes Bacterianos , Mutação , Regiões Promotoras Genéticas , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenótipo , Porinas , Recombinação Genética , Transcrição GênicaRESUMO
Mutations in the Salmonella enterica serovar Typhimurium ompC gene conferred resistance to Gifsy-1 and Gifsy-2 bacteriophages. Selection for complementing plasmids yielded clones of ompC. Introduction of an ompC clone into Escherichia coli conferred the ability to adsorb Gifsy phage. These data show that OmpC is the receptor for Gifsy-1 and Gifsy-2 phages.
Assuntos
Porinas/genética , Receptores Virais/genética , Fagos de Salmonella/patogenicidade , Salmonella typhimurium/virologia , Adsorção , Teste de Complementação Genética , Plasmídeos/genéticaRESUMO
The lambdoid phage Gifsy-2 contributes significantly to Salmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene, sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes, grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA(+) on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor the grvA(+) plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and the grvA(+) plasmid required wild-type sodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such as sodCI and antivirulence factors such as grvA.
Assuntos
Genes Virais , Fagos de Salmonella/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/virologia , Proteínas Virais/química , Animais , Dosagem de Genes , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Mutagênese Insercional , Provírus/genética , Espécies Reativas de Oxigênio/metabolismo , Fagos de Salmonella/patogenicidade , Análise de Sequência de DNA , Superóxido Dismutase/genética , Proteínas Virais/metabolismoRESUMO
Salmonella typhimurium is an invasive pathogen that causes diseases ranging from mild gastroenteritis to enteric fever. During the infection process, S. typhimurium induces a number of virulence genes required to circumvent host defences and/or acquire nutrients in the host. We have used the in vivo expression technology (IVET) system to select for S. typhimurium genes that are induced after invasion of a murine cultured cell line. We have characterized a putative iron transporter in Salmonella pathogenicity island 1, termed sitABCD. The sitABCD operon is induced under iron-deficient conditions in vitro and is repressed by Fur. This locus is induced in the animal specifically after invasion of the intestinal epithelium. We show that a sit null mutant is significantly attenuated in BALB/c mice, suggesting that SitABCD plays an important role in iron acquisition in the animal.
Assuntos
Proteínas de Transporte/genética , Genes Bacterianos , Ferro/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Transporte Biológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Mutação , Óperon , Infecções por Salmonella/genética , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , VirulênciaRESUMO
We have isolated mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that specifically affect transcriptional control by OmpR and EnvZ, the two-component regulatory system that controls porin gene expression in Escherichia coli. Characterization of these mutations and a previously isolated rpoA allele suggests that both positive and negative regulation of porin gene transcription involves a direct interaction between OmpR and RNA polymerase through the alpha subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the alpha protein, further suggesting that it is this domain of alpha that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Transcrição Gênica , Alelos , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Genótipo , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Porinas , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid. Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment. Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient. However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection. In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced. In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.
Assuntos
Bacteriófago P22/genética , Clonagem Molecular , Salmonella typhimurium/genética , Transdução Genética , Integração Viral , Escherichia coli , Genes Bacterianos , Genótipo , Mutagênese Insercional , Plasmídeos , Recombinases Rec A/metabolismo , Recombinação Genética , Transcrição GênicaRESUMO
A new paradigm, termed two-component regulatory systems, is emerging from the study of signal transduction in bacteria. A simple example of such a system is provided by the Omp regulon of Escherichia coli. This regulon, which controls the expression of the major outer membrane porin proteins OmpF and OmpC in response to changes in osmolarity, includes the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR (a transcriptional activator). Although we do not know what "ligand" is sensed in the Omp system, we can trace the signal transduction pathway from the receptor at the cell surface directly to regulatory sequences within the DNA. Perhaps signal transduction in bacteria can serve as a simple archetype for understanding certain functions performed by receptor kinases and phosphorylated DNA-binding proteins in higher organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Complexos Multienzimáticos , Proteínas Quinases/fisiologia , Transdução de Sinais , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Células Eucarióticas/fisiologia , Genes Bacterianos , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
In vivo expression technology was used to identify Salmonella enterica serovar Typhimurium genes that are transcriptionally induced when the bacteria colonize the small intestines of mice. These genes were subsequently screened for those that are transcriptionally inactive during the systemic stages of disease. This procedure identified gipA, a gene that is specifically induced in the small intestine of the animal. The gipA gene is carried on the lambdoid phage Gifsy-1. Consistent with the expression profile, the sole defect conferred by a gipA null mutation is in growth or survival in a Peyer's patch. The gipA strain is wild type in its ability to initially colonize the small intestine and invade the intestinal epithelium. The mutant also survives and propagates at wild-type levels during the systemic stages of disease. The gipA open reading frame is homologous to a family of putative insertion sequence elements, although our evidence shows that transposition is not required for gipA function in the Peyer's patch. These results suggest that the bacteria sense and respond to the particular environment of the Peyer's patch, a critical site for the replication of Salmonella serovar Typhimurium.
Assuntos
Nódulos Linfáticos Agregados/microbiologia , Fagos de Salmonella/genética , Salmonella enterica/genética , Salmonella enterica/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Feminino , Intestino Delgado/microbiologia , Lisogenia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Salmonelose Animal/microbiologia , Salmonella enterica/patogenicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , VirulênciaRESUMO
The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Genes Bacterianos , Mutação , PorinasRESUMO
The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment. The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism. Antibodies directed against O antigen are vital to the immune response to infection. In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium. Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S. typhimurium infection, both in vivo and in vitro. Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope. Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes. This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants. We have shown that a change in the acetylation state of LPS has no effect on virulence. However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Polissacarídeos Bacterianos/química , Salmonella typhimurium/imunologia , Acetilação , Mapeamento Cromossômico , Genes Bacterianos , Antígenos O , Salmonella typhimurium/genéticaRESUMO
Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.