Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes.

Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract. Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified. The C. trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins. Here we show that C. trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells. The protein was present transiently in infected cells during the period of cytotoxicity. Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis diseases.

Homologue scanning mutagenesis reveals CD66 receptor residues required for neisserial Opa protein binding.

The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae. N. gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66-Opa binding is mediated by the NH(2)-terminal domain of the receptor and occurs through protein-protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa-CD66 interaction.

Proteoglycan receptor binding by Neisseria gonorrhoeae MS11 is determined by the HV-1 region of OpaA.

The interaction of the OpaA protein of Neisseria gonorrhoeae MS11mk with heparan sulphate-containing proteoglycan receptors on Chang conjunctiva epithelial cells was examined using isolated receptor binding and cell adherence/internalization assays. OpaA deletion proteins, in which the four surface-exposed regions of the protein were deleted individually, and chimeric OpaA/B proteins, in which the surface-exposed regions of the OpaA and OpaB proteins were exchanged, were expressed in N. gonorrhoeae. The recombinant deletion proteins and the chimeric OpaA/B proteins were surface exposed in the outer membrane of N. gonorrhoeae. Isolated receptor-binding assays and Chang cell infection assays with OpaA deletion variants indicated that hypervariable region 1 was essential for the interaction of N. gonorrhoeae with the proteoglycan receptor. Expression of chimeric OpaA/B proteins confirmed the central role of hypervariable region 1 in receptor binding and demonstrated that this domain alone confers the invasive biological phenotype in a non-heparan sulphate proteoglycan-binding Opa protein. The other variable regions of OpaA enhanced receptor binding in the presence of region 1, but did not constitute binding domains on their own. The results indicate that proteoglycan receptor binding results from a hierarchical interaction between the variable domains of the OpaA protein of MS11mk.

CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains.

Neisseria gonorrhoeae strain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed in Escherichia coli reflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants, suggesting that the differential recognition of CD66 receptors by Opa variants is dictated by the presence of specific binding regions on the N-domain of the receptor.

The ihf mRNA levels decline as Neisseria gonorrhoeae enters the stationary growth phase.

Integration host factor (IHF) is a small heterodimeric DNA binding protein found in all Gram-negative bacteria and is implicated as a transcription cofactor of pilE in Neisseria gonorrhoeae (Hill, S.A., Samuels, D.S., Carlson, J.H., Wilson, J., Hogan, D., Lubke, L., Belland, R.J., 1997. Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae. Mol. Microbiol. 23, 649-656). The ihf genes (ihfA and ihfB) were cloned from N. gonorrhoeae through functional complementation of defined Escherichia coli ihf mutants for plating of phage lambda. The predicted aa sequences of each gonococcal IHF polypeptide showed extensive homology to other reported IHF polypeptide sequences. Northern blotting and primer extension analysis defined the tsp for each gene and indicated a disparity in ihfA and ihfB message levels over time, with ihfB mRNA being more abundant throughout the entire growth cycle. Furthermore, both the ihfA and ihfB message levels declined as cells entered the stationary growth phase. Overall, this study reveals several unique features of ihf transcription in the gonococcus which questions whether certain aspects if ihf transcriptional regulation are universally shared by all Gram-negative bacteria.

Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11, the same 4 recognized CGM1a and NCA. CGM6, however, was not recognized by any Opa variant of MS11. CEA was recognized by at least 9 of 11 Opa variants, and the BGP transfectants specifically bound and internalized 10 of 11 Opa variants and also bound Opa-negative gonococci. Immunofluorescence experiments showed that clustering of CEA-like molecules occurred upon infection of HeLa transfectants with those Opa variants that interacted specifically with the CEA family member. Together these data show that CEA family members are differentially recognized by gonococcal Opa variants, suggesting that this phenomenon may contribute to cell tropism displayed by gonococci.

Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel-retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF-binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF-binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel-shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild-type pilE locus or a deleted pilE locus where the IHF-binding domain was removed. Primer-extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10-fold. Together, these data indicate that IHF is a transcriptional co-activator of pilE.

Selection of Opa+ Neisseria gonorrhoeae by limited availability of normal human serum.

Experimental infections of human male volunteers with Neisseria gonorrhoeae have provided valuable insights into the early stages of gonorrheal disease. Bacterial variants expressing outer membrane opacity (Opa) proteins appear to be selected from the inoculum during a period in which total recoverable numbers of bacteria decrease rapidly. This apparent survival advantage occurs simultaneously with the onset of an inflammatory response, characterized by local production of interleukin 6 (IL-6) and IL-8 and the appearance of leukocytes in urine. Since the inflammatory response may also result in the presence of serum factors on the mucosal surface, we investigated the possibility that killing in normal human serum (NHS) leads to the selection of Opa+ variants. We therefore studied killing of separate populations and mixtures of Opa- and Opa+ N. gonorrhoeae MS11mk in NHS. Expression of an Opa protein conferred a survival advantage upon the organism; i.e., the Opa+ variants were more serum resistant than their isogenic Opa- counterparts, resulting in a selection for Opa+ phenotypes when a mixture of Opa+ and Opa- gonococci (GC) was exposed to submaximal doses of NHS. This selection was observed in three different lipooligosaccharide (LOS) backgrounds, indicating that it was not due to a difference in LOS expression between Opa- and Opa+ phenotypes. Incubation in NHS of sialylated GC resulted in a similar selection for Opa+ variants. The presence of normal human urine during the serum killing assay had no effect on the selection phenomenon but drastically depleted NHS of bactericidal activity, which was found to be at least partly due to complement inhibition. The results suggest that serum killing may contribute to the transition from Opa- to Opa+ phenotypes during the early stages of infection of the male urethra.

Promoter strength influences phase variation of neisserial opa genes.

The opa multigene family of Neisseria gonorrhoeae encodes 11 related outer-membrane proteins which phase vary in vitro and in vivo. Illegitimate recombination within direct pentameric DNA repeats, encoding the signal-peptide region of pre-Opas, leads to switches in expression states. Despite the conserved nature of the variation mechanism, specific genes are expressed at high frequencies in the transition from Opa- to Opa+. The genes which are expressed at elevated frequencies differ from the rest of the family with respect to promoter structure, based on sequence comparisons between the opa genes of strain MS11mk. We have analysed transcription of the opa gene family of N. gonorrhoeae MS11mk, focussing on the different promoters found among the 11 genes to determine whether increased levels of expression are associated with increased phase-variation rates. Primer extension and Northern blotting was used to assess the levels of transcription of three representative opa genes (opaA, B and C) in 'on' and 'off' states. Full-length opa mRNA was detected primarily in strains expressing the homologous gene. Truncated opa mRNA was constitutively expressed from all opa genes regardless of their expression state. Quantitative comparisons in N. gonorrhoeae were complicated by the simultaneous expression of all 11 genes and the cross-reactivity of mRNA probes. Expression levels from the individual promoters were therefore assessed by creating transcriptional and translational lacZ fusions to each of the representative opa promoters which lacked the DNA repeats responsible for variation. The expression levels were compared to the phase-variation rates of translational opa::phoA fusions containing the same promoters in addition to the corresponding coding repeat regions. A strong correlation was found between expression levels from the different promoters and the variation rates at which 'on' variants appeared from an 'off' population (i.e. opaA > opaB > opaC). These results provide an explanation for the favoured expression of specific Opa proteins and indicate that expression of opa genes may be regulated at the level of transcription.

Identification of an EF-Tu protein that is periplasm-associated and processed in Neisseria gonorrhoeae.

A 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae gamma phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful.

Adherence of pilus- Opa+ gonococci to epithelial cells in vitro involves heparan sulfate.

Neisseria gonorrhoeae attaches to host epithelial cells via pili and opacity-associated (Opa) outer membrane proteins. Pilus- gonococci (Gc) of strain MS11 adhere to both human and nonhuman cells, but only when particular Opa proteins are expressed; OpaA+ variants adhere best, OpaC+ variants are next best, and the seven other Opa+ variants adhere poorly or not at all. The adherence of OpaA+ Gc to Chinese hamster ovary (CHO) cells is inhibited by heparin or heparan sulfate (HS), but not by chondroitin sulfate. OpaA+ Gc do not adhere to CHO cells devoid of HS proteoglycans; low concentrations of heparin restore OpaA+ Gc adherence to these HS-deficient CHO cells and high concentrations inhibit it. 3H-heparin binding to whole Gc parallels their adherence abilities (OpaA+ > OpaC+ > OpaH+ >> Opas B, D, E, F, G, I = Opa- = 0). Opa proteins separated by SDS-PAGE also bind 3H-heparin. These data suggest that adherence of pilus-, Opa+ Gc involves HS-proteoglycan of eukaryotic cells.

Heparin protects Opa+ Neisseria gonorrhoeae from the bactericidal action of normal human serum.

The pathobiological significance of lipooligosaccharide (LOS) and outer membrane opacity protein (Opa) changes in gonorrheal disease are poorly understood. We assessed variants of strain MS11mk with different LOS and Opa phenotypes for their liability to killing by normal human sera. LOS differences correlated with strikingly disparate susceptibilities to serum killing; LOSa variants were serum resistant, LOSb variants were serum sensitive, and sialylation of LOSb variants enhanced their survival (as reported previously). Opa phenotype had little influence on the killing of serum-sensitive LOSb cells that were incubated directly in normal human sera, but preincubation of Opa+ LOSb variants in heparin increased their serum resistance whereas Opa- LOSb variants showed no change. Some Opa proteins conferred slightly higher resistance than others, but heparin preincubation increased serum resistance for variants expressing each of seven Opa proteins. These in vitro phenomena may relate to conditions within the male urethra where sulfate-containing proteoglycans are abundant and where antibody and complement may transude from blood plasma. The results suggest that the selective advantage for Opa+ Neisseria gonorrhoeae bacteria observed in vivo may reflect their ability to utilize host cell components to resist killing by host defenses.

Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates.

Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the C-terminus of the predicted open reading frame. A series of ciprofloxacin-resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10,000-fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high-level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are involved in the establishment of extreme levels of ciprofloxacin resistance.

Neisserial surface variation: how and why?

Neisseria gonorrhoeae exhibits striking variability in several of its surface components (pili, Opa proteins and lipooligosaccharide) in vivo and in vitro. Such flagrant variation of this mucosal pathogen's surface components contrasts sharply with changes in single surface components of blood-borne trypanosomes and borreliae. Despite these differences, similar molecular events are sometimes involved.

Human neutrophil response to recombinant neisserial Opa proteins.

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.

H-DNA formation by the coding repeat elements of neisserial opa genes.

The coding repeat region of opa genes from Neisseria gonorrhoeae and Neisseria meningitidis determines the expression state of their respective genes through high-frequency addition of deletion of pentanucleotide coding repeat units (CRs; CTTCT). In vitro analyses of cloned opa gene CR regions using single-strand specific nucleases, oligonucleotide protection experiments, and modifications of non-B-DNA residues indicate that the regions form structures resembling H-DNA under acidic conditions in the presence of negative supercoiling. The purine/pyrimidine strand bias and H-palindromic nature of the repeat region are consistent with sequence requirements for H-DNA formation. Sequences flanking the repeat elements are required to form the H-DNA structure in vitro as judged by the pattern of exposed non-B-DNA residues in CR sequences synthesized as oligonucleotides to form beta-galactosidase::CR translational fusions. The fusions phase vary by addition and deletion of CR elements and the rate of phase variation increases upon induction of the fusion genes. The opa gene CR region is the first reported bacterial H-DNA structure and is unique in that it lies within the coding sequence for the gene.

Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein.

Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis. The pathogenic process(es) that cause these sequelae are thought to be immunological. A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection. This protein is thought to mediate the immunopathology that follows chlamydial infection. To more thoroughly characterize this chlamydial component, we cloned its gene from a C. psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide. The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae. Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed. These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins. Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins. Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms.

Expression and phase variation of gonococcal P.II genes in Escherichia coli involves ribosomal frameshifting and slipped-strand mispairing.

Expression and phase variation of Neisseria gonorrhoeae P.II genes in Escherichia coli were studied using TnphoA fusions. Fusions were created in the P.IIc gene of N. gonorrhoeae JS3 using lambda TnphoA-1 and were characterized by restriction digestion and dideoxy sequencing. Three fusions were chosen for further study; Tnp7 (fusion junction at mature amino acid 7), Tnp57 (amino acid 57), Tnp66 (amino acid 66). All fusions were in frame with the P.IIc coding sequence but were out of frame with the purported initiation codon. All fusion constructions were shown to phase vary in E. coli in an analogous fashion to that seen in N. gonorrhoeae, i.e. phase changes (in a recA background) at a frequency of c. 10(-3) accompanied by an alteration at the DNA level of the number of coding repeats (CRs). In vitro mutagenesis of the fusion constructions indicated that expression of out of frame P.II genes in E. coli was probably the result of ribosomal frameshifting within the run of 'A' residues immediately preceding the CR region and not due to low-level false initiation at codons other than the ATG initiation codon (as had previously been suggested). The mechanism for P.IIc::phoA phase variation appears to be related to the 'slipped-strand mispairing' mechanism responsible for frameshift mutations in a number of other bacterial genes containing short, direct, tandem repeats.

DNA:DNA reassociation analysis of Aeromonas salmonicida.

DNA from 26 Aeromonas salmonicida strains, namely 11 'typical' and 15 so-called 'atypical' strains, was used to assess the taxonomic relatedness within the species. The genomes were characterized by determination of DNA base composition, DNA:DNA reassociation, calculation of sequence divergence following reassociation, and by genome size estimations. By comparison with DNA obtained from controls and the Aeromonas hydrophila group, A. salmonicida strains were determined to be correctly placed with respect to genus and species. A. salmonicida subspecies salmonicida (the 'typical' group) was an extremely homogeneous taxon. The 'atypical' strains were more diverse, but distinct biotypes were recognizable. The first biotype included several geographically diverse isolates from goldfish. The second recognizable biotype included strains isolated from European carp. Other 'atypical' isolates could not be grouped but showed enough internal homology to be retained within the species. The A. salmonicida subspecies achromogenes and masoucida were found to be closely related to the motile aeromonads. It is considered that the present classification of A. salmonicida is unsuitable and should be restructured to include A. salmonicida subspecies salmonicida, subspecies achromogenes (to include the present subspecies masoucida), and the reintroduced subspecies nova.

Cloning of the gene for the surface array protein of Aeromonas salmonicida and evidence linking loss of expression with genetic deletion.

A gene bank of DNA from the fish pathogenic bacterium Aeromonas salmonicida was constructed in the bacteriophage lambda gt11. Phage lambda gt11/10G, a recombinant carrying a 4.0-kilobase fragment of A. salmonicida DNA, was found to express the surface array protein (A protein) in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein expressed from the cloned gene had a subunit molecular weight of 49,000, which was identical to that of subunits in the native assembled A layer. Genomic Southern analysis showed that the gene coding for this predominant cellular protein was in a single copy on the chromosome and was conserved among a wide range of A. salmonicida strains with different phenotypic characteristics and isolated from diverse geographic locations, fish species, and means of pathogenesis. Results of genomic blotting experiments also showed that loss of expression of the A layer resulting from growth at 30 degrees C was accompanied by genetic rearrangement in which N-terminal sequences of the gene for A protein were lost by deletion.