James L. Doran

The location of four fimbrin-encoding genes, agfA, fimA, sefA and sefD, on the Salmonella enteritidis and/or S. typhimurium XbaI-BlnI genomic restriction maps

Four fimbrin-encoding genes, fimA (type-1 or SEF21 fimbriae), agfA (thin aggregative or SEF17 fimbriae), sefA (SEF14 fimbriae and sefD (SEF18 fimbriae) from Salmonella enteritidis (Se) 27655-3b were located onto the XbaI-BlnI genomic restriction maps of Salmonella typhimurium (St) LT2 and Se strains SSU7998 and 27655-3b. The XbaI or BlnI genomic fragments carrying these genes were identified by hybridization with labeled oligodeoxyribonucleotides or fimbrin-encoding genes. The fimbrin-encoding genes were not encoded by the virulence plasmids, but were located on chromosomal DNA fragments. The position of each gene on a given XbaI fragment was determined by hybridization of a series of XbaI-digested genomic DNA samples from previously characterized Tn10 mutants of Se and St with its respective probe. The fimA gene mapped near 13 centisomes (Cs) between purE884::Tn10 at 12.6 Cs (11.8 min) and apeE2::Tn10 at 12.8 Cs (12.3 min) beside the first XbaI site at 13.0 Cs in St or between purE884::Tn10 at 12.6 Cs and the XbaI site at 13.6 Cs in Se. The agfA gene mapped near 26 Cs between putA::Tn10 and pyrC691::Tn10 in St, but near 40 Cs between pncX::Tn10 and the XbaI site at 43.3 Cs in Se. This difference in map position was due to the location of agfA near one end of the 815-kb chromosomal fragment inverted between Se and St. The sefA and sefD genes mapped precisely at 97.6 Cs in Se, but were absent from the genome of St LT2. To verify the mapping procedures used herein, tctC was also mapped in both Salmonella serovars. As expected, tctC mapped near 60 Cs in both St and Se, thereby confirming previous studies.

Production of Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli using two-cistron expression systems

SummaryStreptomyces clavuligerus isopenicillin N synthase (IPNS) gene expression was achieved inEscherichia coli by the construction of two-cistron expression systems formed in the high copy number plasmid vector pUC119. These two-cistron constructions were composed of the IPNS gene and its flanking sequences which encoded an upstream open reading frame (ORF), the IPNS ribosome binding site and a putative transcription terminator. NoE. coli- likeStreptomyces promoter motif was present upstream of the IPNS gene therefore transcriptional regulation of the two-cistron system was provided by thelac promoter of pUC119. Enzymatically active IPNS was detected inE. coli cells harboring the recombinant plasmids thereby providing evidence for the activity of the IPNS ORF and for the feasibility of production ofS. clavuligerus IPNS inE. coli. These results indicate that simple two-cistron constructions involving foreign gene flanking sequences may be used to express foreign proteins inE. coli.

Plasmid Transformation of Azotobacter vinelandii OP

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

Role of calcium in assembly of the Azotobacter vinelandii surface array

Azotobacter vinelandii grown in a defined medium lacking calcium did not exhibit the tetragonally-arranged surface layer present on calcium-sufficient cells, although each cell type possessed equivalent amounts of surface-localized S-protein. The addition of Ca2+ or Sr2+ to calcium-limited cells suspended in buffer resulted in formation of the S-layer, whereas a similar addition of Mg2+ or Be2+ did not. Incubation of cells with 35SO2- 4 during Ca2+-mediated in vivo reassembly of the S-layer confirmed that the array was formed from previously synthesized, surface-localized S-protein. Rate-zonal sedimentation of S-protein extracted from calcium-limited cells demonstrated tetrameric S-protein subunits characteristic of the native array. S-protein on the surface of calcium-limited cells was not more susceptible to iodination or proteolytic degradation than that on calcium-sufficient cells. These data suggested minimal alteration of the surface layer beyond disorganization of the S-protein subunits. Calcium limitation caused only a minor perturbation of the outer membrane and did not prevent the outer membrane from serving as a template for the in vitro reassembly of externally supplied S-protein subunits. Notably, Mg2+ or Ca2+ mediated in vitro reassembly of the S-layer and produced a layer that was more loosely attached than the native array. These data support the hypothesis that calcium is specifically required for the in vivo assembly of S-protein subunits into a tetragonal surface array.

CHARACTERIZATION OF THE AGFBA FIMBRIAL OPERON ENCODING THIN AGGREGATIVE FIMBRIAE OF SALMONELLA ENTERITIDIS

Of the several fimbrial types produced by the human enteropathogen Salmonella enteritidis, the thin aggregative fimbriae, SEF17, are particularly interesting being highly immunogenic, aggregative, extremely stable, cell surface structures which are widely distributed among Salmonella serovars (Collinson et al., 1991; Doran et al., 1993). These fimbriae are composed of a 17,000 Da fimbrin, AgfA, and require treatment with 90% formic acid before depolymerization occurs (Collinson et al., 1991). Diarrheagenic Escherichia coli strains produce biochemically and serologically related, thin, aggregative fimbriae (Collinson et al., 1992) called curli which are composed of polymerized CsgA fimbrins (Arnqvist et al., 1994). An agfA-specific, oligonucleotide probe capable of distinguishing agfA from csgA forms the basis for a valuable, new, Salmonella-specific, diagnostic test (Doran et al., 1993). The role(s) of SEF17 in Salmonella pathogenesis remain(s) to be elucidated but purified SEF17 binds fibronectin in vitro suggesting a possible role in facilitating bacterial-tissue interactions (Collinson et al., 1993).