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.

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS). One strategy to define bacterial genes with a role during infection involves the screening of a pool of random transposon insertion mutants, where each mutant is identifiable by a specific motif or unique transcript. Changes in the survival of each mutant indicate a role of the mutated region during infection. To ensure coverage of most genes of interest, a large number of random transposon mutants would be needed. However, when a pool of bacterial mutants spreads into different sites in an animal host, some mutants are lost at random if the founder population (i.e. the bacteria initially reaching this site, before expansion) is smaller than the number of mutants introduced into the animal. This random loss severely obscures mutants that are truly at a disadvantage. In order to minimize the number of mutants to be screened and thus minimize random loss of mutants from an infecting pool, we generated targeted specific deletions in each gene of interest. Furthermore, we inserted a promoter in each mutant that allows simultaneous monitoring of a pool of these mutants on a novel inexpensive microarray. To demonstrate the utility of our technique, a pool of over 1,000 gene-targeted Salmonella mutants was injected into mice and recovered from spleens. We identified mutants that were less fit than wild-type Salmonella in this model. Eleven mutants in genes that were not previously known to affect Salmonella fitness in intraperitoneal infection were confirmed using the individual mutants in competition with wild-type bacteria. These new phenotypes include the first two examples of attenuation and one of hypervirulence in Salmonella due to mutations in small stable RNAs, a class of regulators that bind to other RNAs and proteins.

Salmonella entericaserovar Enteritidis is often transmitted into the human food supply through eggs of hens that appear healthy. This pathogen became far more prevalent in poultry following eradication of the fowl pathogenS. entericaserovar Gallinarum in the mid-20th century. To investigate whether changes in serovar Enteritidis gene content contributed to this increased prevalence, and to evaluate genetic heterogeneity within the serovar, comparative genomic hybridization was performed on eight 60-year-old and nineteen 10- to 20-year-old serovar Enteritidis strains from various hosts, using aSalmonella-specific microarray. Overall, almost all the serovar Enteritidis genomes were very similar to each other. Excluding two rare strains classified as serovar Enteritidis in theSalmonellareference collection B, only eleven regions of the serovar Enteritidis phage type 4 (PT4) chromosome (sequenced at the Sanger Center) were absent or divergent in any of the other serovar Enteritidis strains tested. The more recent isolates did not have consistent differences from 60-year-old field isolates, suggesting that no large genomic additions on a whole-gene scale were needed for serovar Enteritidis to become more prevalent in domestic fowl. Cross-hybridization of phage genes on the array with related genes in the examined genomes grouped the serovar Enteritidis isolates into two major lineages. Microarray comparisons of the sequenced serovar Enteritidis PT4 to isolates of the closely related serovars Dublin and Gallinarum (biovars Gallinarum and Pullorum) revealed several genomic areas that distinguished them from serovar Enteritidis and from each other. These differences in gene content could be useful in DNA-based typing and in understanding the different phenotypes of these related serovars.