Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae

Abstract

Salmonella enteritidis produces thin, aggregative fimbriae, named SEF17, which are composed of polymerized AgfA fimbrin proteins. DNA sequence analysis of a 2-kb region of S. enteritidis DNA revealed three contiguous genes, agfBAC. The 453-bp agfA gene encodes the AgfA fimbrin, which was predicted to be 74% identical and 86% similar in primary sequence to the Escherichia coli curli structural protein, CsgA. pHAG, a pUC18 derivative containing a 3.0-kb HindIII fragment encoding agfBAC, directed the in vitro expression of the major AgfA fimbrin, with an M r of 17,000, and a minor AgfB protein, with an M r of 16,000, encoded by the 453-bp agfB gene. AgfA was not expressed from pDAG, a pUC18 derivative containing a 3.1-kb DraI DNA fragment encoding agfA but not agfB. Primer extension analysis identified two adjacent transcription start sites located immediately upstream of agfB in positions analogous to those of the E. coli curlin csgBA operon. No transcription start sites were located immediately upstream of agfA or agfC. Northern (RNA) blot analysis confirmed that transcription of agfA was initiated from the agfB promoter region. Secondary-structure analysis of the putative mRNA transcript for agfBAC predicted the formation of a stem-loop structure (⌬G؇, ؊22 kcal/mol [؊91 kJ/mol]) in the intercistronic region between agfA and agfC, which may be involved in stabilization of the agfBA portion of the agfBAC transcript. agfBAC and flanking regions had a high degree of sequence similarity with those counterparts of the E. coli curlin csgBA region for which sequence data are available. These data are demonstrative of the high degree of similarity between S. enteritidis SEF17 fimbriae and E. coli curli with respect to fimbrin amino acid sequence and genetic organization and, therefore, are indicative of a common and relatively recent ancestry.

AgfC and AgfE facilitate extracellular thin aggregative fimbriae synthesis in Salmonella Enteritidis

“…Media were supplemented with 100 mg ampicillin ml 21 and 0.5 % (w/v) L-arabinose. E. coli XL-1 Blue (Stratagene) harbouring pBCKS (Stratagene), pHAG (Collinson et al, 1996), pGEM-T Easy (Promega) or pHSG415 (Hashimoto-Gotoh et al, 1981) was grown at 28 or 37 uC for 24 or 48 h with agitation in Luria-Bertani (LB) broth supplemented with 50 mg chloramphenicol ml 21 or 100 mg ampicillin ml 21 and 40 mg X-Gal ml 21 , in addition to 1 mM IPTG, when required.…”

“…The genes involved in Tafi production are organized into two adjacent divergently transcribed operons, agfBAC and agfDEFG (Collinson et al, 1996). Both operons are required for biosynthesis and assembly (Collinson et al, 1993).…”

“…CsgE and -F have uncharacterized roles in assembly, but CsgE at least has been called a chaperone (Chapman et al, 2002). The agfC (csgC) gene has not been previously characterized, and has been referred to as orfC due to the lack of evidence for transcription or a distinct phenotype accompanying inactivation (Collinson et al, 1996;Hammar et al, 1995).…”

Evolutionary loss of the rdar morphotype in Salmonella as a result of high mutation rates during laboratory passage

“…Rdar formation is the result of coordinated expression of genes required for biosynthesis of fimbriae (Collinson et al, 1993(Collinson et al, , 1996Rö mling andRohde, 1999, 2000), cellulose (Rö mling et al, 2000;Zogaj et al, 2001) and additional polysaccharides . s 38 is involved in the gene regulatory network governing the rdar phenotype; in early stationary phase, it activates the transcription of a key regulator in rdar formation, agfD.…”

“…s 38 is involved in the gene regulatory network governing the rdar phenotype; in early stationary phase, it activates the transcription of a key regulator in rdar formation, agfD. AgfD (CsgD) is a transcriptional regulator that initiates transcription in the agfBAC operon, which encodes the main structural proteins for fimbrial biosynthesis (Collinson et al, 1993(Collinson et al, , 1996Rö mling et al, 1998Rö mling et al, , 2000. agfD is also regulated by the transcriptional regulators OmpR, MlrA and CpxR-P (Gerstel and Rö mling, 2003;.…”