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The facultative intracellular pathogen Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. Inside epithelial cells, Salmonella replicates in vacuoles, which localize in the perinuclear area in close proximity to the Golgi apparatus. Among the effector proteins translocated by the Salmonella pathogenicity island 2-encoded type III secretion system, SifA and SseG have been shown necessary but not sufficient to ensure the intracellular positioning of Salmonella vacuoles. Hence, we have investigated the involvement of other secreted effector proteins in this process. Here we show that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles. The observations show that the intracellular positioning of Salmonella vacuoles is a complex phenomenon resulting from the combined action of several effector proteins.

Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus pneumoniae operon, named pit , encoding proteins with similarity to components of a putative Brachyspira hyodysenteriae iron uptake ABC transporter, Bit. An S. pneumoniae strain containing a defined mutation in pit has impaired growth in medium containing the iron chelator ethylenediamine di- o -hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of S. pneumoniae systemic infection. Furthermore, addition of a mutation in pit to a strain containing mutations in the two previously described S. pneumoniae iron uptake ABC transporters, piu and pia , resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual pit , piu , and pia mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that pia is the dominant iron transporter during in vitro and in vivo growth.

To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg −1 . While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC . PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox −/− mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.

We have previously described a 27-kb pathogenicity island of Streptococcus pneumoniae , termed pneumococcal pathogenicity island 1 (PPI1), which contains iron uptake locus piaABCD , required for full virulence in mice, and a further 28 previously uncharacterized genes. We have investigated one of these, Sp1051, which encodes a protein of unknown function. Disruption of Sp1051 does not affect growth in laboratory broth, serum, or blood but impairs virulence in mouse models of infection. When S. pneumoniae capsular serotypes were analyzed by PCR and Southern hybridization, it was found that 33% did not contain Sp1051. Analysis of other genes within PPI1 demonstrated that, compared to the serotype 4 genome published by The Institute for Genome Research (TIGR), the genomes of many strains contain deletions of a variable number of genes between Sp1046 and Sp1064, conforming to one of six different patterns. Amplification by PCR of this PPI1 variable region from a capsular serotype 17 strain and comparison of the sequence to TIGR serotype 4 strain sequence showed that Sp1051 is contained within an 11.3-kb segment of DNA flanked by 7-bp direct repeats within the serotype 4 strain which is not present in the serotype 17 strain. Further comparison of the sequences of this region between the three published S. pneumoniae genomes demonstrated that serotype 19F and strain R6 contain novel complements of genes not present in the serotype 4 strain. These data indicate that there is striking variation in gene content and structure of the 3′ region of PPI1 among strains and that this region includes at least one virulence determinant. Gene variation within horizontally acquired DNA such as that of PPI1 may be one factor modulating differences in virulence among strains.