Steffen Porwollik

Complete genome sequence of Salmonella enterica serovar Typhimurium LT2

Salmonella enterica subspecies I, serovar Typhimurium (S. typhimurium), is a leading cause of human gastroenteritis, and is used as a mouse model of human typhoid fever. The incidence of non-typhoid salmonellosis is increasing worldwide, causing millions of infections and many deaths in the human population each year. Here we sequenced the 4,857-kilobase (kb) chromosome and 94-kb virulence plasmid of S. typhimurium strain LT2. The distribution of close homologues of S. typhimurium LT2 genes in eight related enterobacteria was determined using previously completed genomes of three related bacteria, sample sequencing of both S. enterica serovar Paratyphi A (S. paratyphi A) and Klebsiella pneumoniae, and hybridization of three unsequenced genomes to a microarray of S. typhimurium LT2 genes. Lateral transfer of genes is frequent, with 11% of the S. typhimurium LT2 genes missing from S. enterica serovar Typhi (S. typhi), and 29% missing from Escherichia coli K12. The 352 gene homologues of S. typhimurium LT2 confined to subspecies I of S. enterica—containing most mammalian and bird pathogens—are useful for studies of epidemiology, host specificity and pathogenesis. Most of these homologues were previously unknown, and 50 may be exported to the periplasm or outer membrane, rendering them accessible as therapeutic or vaccine targets.

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their ∼4,400 protein coding sequences: 173 in Paratyphi A and ∼210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Evolutionary genomics of Salmonella: Gene acquisitions revealed by microarray analysis

The presence of homologues of Salmonella enterica sv. Typhimurium LT2 genes was assessed in 22 other Salmonella including members of all seven subspecies and Salmonella bongori. Genomes were hybridized to a microarray of over 97% of the 4,596 annotated ORFs in the LT2 genome. A phylogenetic tree based on homologue content, relative to LT2, was largely concordant with previous studies using sequence information from several loci. Based on the topology of this tree, homologues of genes in LT2 acquired by various clades were predicted including 513 homologues acquired by the ancestor of all Salmonella, 111 acquired by S. enterica, 105 by diphasic Salmonella, and 216 by subspecies 1, most of which are of unknown function. Because this subspecies is responsible for almost all Salmonella infections of mammals and birds, these genes will be of particular interest for further mechanistic studies. Overall, a high level of gene gain, loss, or rapid divergence was predicted along all lineages. For example, at least 425 close homologues of LT2 genes may have been laterally transferred into Salmonella and then between Salmonella lineages.

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI‐1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI‐1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non‐motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2‐propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co‐ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2‐propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co‐ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species.

Background The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. Methodology/Principal Findings We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. Conclusions/Significance CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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).

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 × g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies.

Lateral gene transfer in Salmonella

Comparative genomics and microarrays reveal that the genomes of different Salmonella enterica serovars are distinguished from each other by the presence or absence of hundreds of genes. The distribution of these variable genome regions is often not clonal. Therefore, lateral gene transfer (LGT) plays an important role in diversity among Salmonella. Overall, almost one quarter of the entire S. enterica sv Typhimurium genome may have been introduced by LGT.

Low-shear modeled microgravity alters the Salmonella enterica serovar Typhimurium stress response in an RpoS-independent manner

ABSTRACT We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.