We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (Kan^R) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (Cam^R) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a Kan^R cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a Cam^R cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC. The bacterial genus Salmonella consists of two species: Salmonella enterica and Salmonella bongori. Salmonella are common causes of food poisoning in humans and can also cause more severe disease such as typhoid fever. Most of the Salmonella that cause disease in humans and animals are members of S. enterica. On the other hand S. bongori, is largely associated with reptiles but can cause disease in humans, albeit rarely. We have determined genomes for S. bongori isolates representing its known diversity. Using this, and existing genome information for a large number of different members of S. enterica, we were able to identify functions found in both species, and therefore likely to be ancestral, and differentiate them from those that have been more recently acquired. This information gives us more perspective on how pathogens evolve over the longer-term and allows us to identify functions that are associated exclusively with isolates that commonly cause disease in humans. Our analysis suggests that when S. bongori and S. enterica diverged they evolved to occupy very different niches.

Background: The recently described Type VI Secretion System (T6SS) represents a new paradigm of protein secretion in bacteria. A number of bioinformatic studies have been conducted to identify T6SS gene clusters in the available bacterial genome sequences. According to these studies, Salmonella harbors a unique T6SS encoded in the Salmonella Pathogenicity Island 6 (SPI-6). Since these studies only considered few Salmonella genomes, the present work aimed to identify novel T6SS loci by in silico analysis of every genome sequence of Salmonella available. Results: The analysis of sequencing data from 44 completed or in progress Salmonella genome projects allowed the identification of 3 novel T6SS loci. These clusters are located in differentially-distributed genomic islands we designated SPI-19, SPI-20 and SPI-21, respectively. SPI-19 was identified in a subset of S. enterica serotypes including Dublin, Weltevreden, Agona, Gallinarum and Enteritidis. In the later, an internal deletion eliminated most of the island. On the other hand, SPI-20 and SPI-21 were restricted to S. enterica subspecies arizonae (IIIa) serotype 62:z4,z23:-. Remarkably, SPI-21 encodes a VgrG protein containing a C-terminal extension similar to S-type pyocins of Pseudomonas aeruginosa. This is not only the first evolved VgrG described in Salmonella, but also the first evolved VgrG including a pyocin domain described so far in the literature. In addition, the data indicate that SPI-6 T6SS is widely distributed in S. enterica and absent in serotypes Enteritidis, Gallinarum, Agona, Javiana, Paratyphi B, Virchow, IIIa 62:z4,z23:- and IIIb 61:1,v:1,5,(7). Interestingly, while some serotypes harbor multiple T6SS (Dublin, Weltvreden and IIIa 62:z4,z23:-) others do not encode for any (Enteritidis, Paratyphi B, Javiana, Virchow and IIIb 61:1,v:1,5,(7)). Comparative and phylogenetic analyses indicate that the 4 T6SS loci in Salmonella have a distinct evolutionary history. Finally, we identified an orphan Hcp-like protein containing the Hcp/COG3157 domain linked to a C-terminal extension. We propose to designate this and related proteins as "evolved Hcps". Conclusion: Altogether, our data suggest that (i) the Salmonella T6SS loci were acquired by independent lateral transfer events and (ii) evolved to contribute in the adaptation of the serotypes to different lifestyles and environments, including animal hosts. Notably, the presence of an evolved VgrG protein related to pyocins suggests a novel role for T6SS in bacterial killing. Future studies on the roles of the identified T6SS loci will expand our knowledge on Salmonella pathogenesis and host specificity.

Significance: We conducted a genome-wide screen to identify bacterial factors required for Vibrio parahaemolyticus , an important cause of seafood-borne gastroenteritis, to survive in vitro and colonize the mammalian intestine. Our analysis revealed uncharacterized components of a horizontally acquired type III secretion system linked to virulence (T3SS2) and hundreds of genes that likely contribute to colonization independent of T3SS2. Our work revealed that toxR , a conserved gene in vibrios that governs expression of horizontally acquired virulence factors in Vibrio cholerae , was critical for expression of T3SS2. Thus, expression of disparate virulence-linked elements, acquired via lateral gene transfer in independently evolved pathogenic vibrios, is controlled by a common ancestral transcription factor.

A recent outbreak of lymphogranuloma venereum (LGV) within the men who have sex with men (MSM) community and their requirement for extended therapy has highlighted the need for laboratory tests that differentiate LGV- from non-LGV-associated serovars of Chlamydia trachomatis. Two previously described methods were evaluated against 495 clinical specimens referred to the Sexually Transmitted Bacteria Reference Laboratory (London, UK): (i) PCR amplification of a 1.1 kb region of the ompI gene followed by restriction enzyme digestion (ompI RFLP-PCR); and (ii) real-time PCR targeting a 36 bp deletion present within the polymorphic membrane protein H gene of LGV-associated serovars (pmpH real-time PCR). For specimens that could be categorized using both methods, a 94.7 % (390/412) concordance was achieved. Eighty-three specimens were found to be untypeable by ompI RFLP-PCR due to a failure to amplify the 1.1 kb fragment. Of these 83 untypeable specimens, 19 were determined to be an LGV-associated serovar by pmpH real-time PCR. Despite the high level of concordance, there were differences found in the technical complexity of the two methods. The pmpH real-time PCR exhibited greater sensitivity, a more rapid turnaround time and a lower technical requirement. Whilst the ompI RFLP-PCR was not as robust as a laboratory diagnostic method, it did enable serovar-level identification. LGV infection remains an important threat to the health of high-risk MSM in Europe. In conclusion, the two methods for the detection of LGV from clinical samples were found not only to have a high concordance (94.7 %) but also to be complementary, and could be used in an integrated way to aid LGV detection.

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro , rfa , rfb , phoP , and phoQ ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system ( SEN4290 to SEN4292 ), the peg fimbrial operon ( SEN2144A to SEN2145B ), a putative pathogenicity island ( SEN1970 to SEN1999 ), and a type VI secretion system remnant SEN1001 , encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A Δ SEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro . Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro . The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.