||The group has earlier published on the possible importance of type IVB pili in the pathogenesis of typhoid fever in humans. Salmonella enterica serovar Typhi (S. typhi) uses type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. Due to the exceptional host restriction of S. typhi, there is no animal model for serovar Typhi systemic infection of humans. It was decided, therefore, to seek other evidence that might point to an important role for the type IVB pili in the pathogenesis of enteric fever. There are two other Salmonella serovars, Dublin and Paratyphi, which sometimes express the Vi antigen, and which may therefore also be pil+. However, neither serovar Dublin nor serovar Paratyphi are as pathogenic for humans as is serovar Typhi. If either or both of serovars Dublin or Paratyphi could be shown to differ from serovar Typhi in the structure or functions of pil operon genes, this would constitute some evidence that pil might be of importance in pathogenesis. First, serovar Dublin was investigated. Some strains of S. enterica serovar Dublin are Vi antigen-positive. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi+ serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggested that the serovar Dublin Type IVB pil operon might increase the human-invasiveness of serovar Dublin, compared to pil-free strains. It appeared, however, that the differences in the degree of human pathogenicity between the pil+ serovars Dublin and Typhi might not lie in the pil operon. Next, serovar Paratyphi was investigated. It was initially shown that S. enterica serovar Paratyphi C strains harboured a pil operon very similar to that of serovar Typhi. An important difference, however, was located in the shufflon which concludes the pil operon. In serovar Typhi, the Rci recombinase acts upon two 19 bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is essentially inactive because each of the Rci 19 bp substrates has acquired a single basepair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. The data thus suggested that serovar Typhi bacterial self-association using Type IVB pili and regulated by PilV protein might, in fact, be important in the pathogenesis of epidemic enteric fever. Taken together, this work suggested that a novel means of control of protein expression was operative in the shufflons of serovars Typhi and Dublin, and efforts then turned to elucidation of the molecular details of this mechanism. The data suggested that the rate of Rci-catalyzed inversion of DNA encoding the C-terminal portions of the PilV proteins controls PilV protein synthesis. This potentially represents a novel means of transcriptional control. Here, it was initially shown that DNA inversion per se is required for inhibition of gene expression from invertible DNA. Binding, without DNA scission, of Rci to its substrate sequences on DNA could not explain the data obtained. Next, it was shown that inversion frequencies of xylE-encoding DNA, bracketed by Rci substrate sequences, are affected by changes in the 19 bp consensus sequences which are essential components of Rci substrate DNA. The affinity of Rci for these sequences affects inversion frequencies, so that a greater affinity is predictive of faster inversion and therefore less synthesis of product encoded by invertible DNA. Inversion events may inhibit transcription of DNA from external promoters. In vivo, the frequency of Rci-mediated inversion is influenced by the extent of DNA supercoiling, with increasing levels of expression of invertible genes as novobiocin inhibits DNA supercoiling and thus Rci action. This inhibition of DNA supercoiling results in increased synthesis of PilV proteins as Rci activity decreases, and, in turn, bacterial self-association (particularly in serovar Dublin) decreases. In summary, the work to date has examined shufflons in two Salmonella serovars other than serovar Typhi, and, as shufflon inversion appeared to be of possible pathogenic importance, the mechanism of this control on protein expression was investigated in detail. Most importantly, shufflon inversion activity and, in turn, PilV expression are regulated by the extent of DNA supercoiling, which responds to environmental cues. This potentially represents a novel means of control of bacteria aggregation and dispersal, which play an important role in development of bacterial infections.