DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic') plasmid.     

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.     

In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of na?ve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.     

Genomic structure of the Salmonella enterica serovar Typhimurium DT 64 bacteriophage ST64T: evidence for modular genetic architecture

The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined. The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22. Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins. The putative immunity genes were highly related to those of Salmonella enterica serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of S. enterica serovar Typhimurium PS3.     

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.     

Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions

A Caenorhabditis elegans-Salmonella enterica host-pathogen model was used to identify both novel and previously known S. enterica virulence factors (HilA, HilD, InvH, SptP, RhuM, Spi4-F, PipA, VsdA, RepC, Sb25, RfaL, GmhA, LeuO, CstA, and RecC), including several related to the type III secretion system (TTSS) encoded in Salmonella pathogenicity island 1 (SPI-1). Mutants corresponding to presumptive novel virulence-related genes exhibited diminished ability to invade epithelial cells and/or to induce polymorphonuclear leukocyte migration in a tissue culture model of mammalian enteropathogenesis. When expressed in C. elegans intestinal cells, the S. enterica TTSS-exported effector protein SptP inhibited a conserved p38 MAPK signaling pathway and suppressed the diminished pathogenicity phenotype of an S. enterica sptP mutant. These results show that C. elegans is an attractive model to study the interaction between Salmonella effector proteins and components of the innate immune response, in part because there is a remarkable overlap between Salmonella virulence factors required for human and nematode pathogenesis.     

Salmonella phage ST64B encodes a member of the SseK/NleB effector family

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.     

Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways.

The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.     

Salmonella enterica serovar Enteritidis dam mutant induces low NOS-2 and COX-2 expression in macrophages via attenuation of MAPK and NF-kappaB pathways

Although dam mutants of Salmonella have been proposed as live vaccines, their capacity to trigger cell inflammatory cascades has not been fully elucidated. We investigated in detail the ability of Salmonella enterica dam mutant to activate the signalling pathways of the inflammatory response in RAW 264.7 cells. Apoptosis in macrophages treated with Salmonella dam mutant was low. Similarly, the expression of both NOS-2 and COX-2 and subsequently the production of NO and PGE(2) was significantly reduced. Also, Salmonella dam mutant induced an attenuated activation of the inflammatory signalling pathway as indicated by the reduced degradation of IkappaBalpha and IkappaBbeta and the low IkappaBalpha phosphorylation found. In addition, translocation of p65 to the nucleus was notably impaired and the amount of phosphorylated p44, p42 and p38 MAPKs was clearly reduced in extracts from dam-infected macrophages. These results indicate that the lack of ERK and p38 phosphorylation at the proper time in dam-infected cells notably reduces the engagement of subsequent signalling pathways involved in the full activation of NF-kappaB in response to infection. Taken together, these results suggest that Salmonella activation of both signalling cascades in the inflammatory response is a mechanism requiring Dam protein participation.     

Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence

Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors.     

Phospholipid changes in seqA and dam mutants of Escherichia coli

SeqA and Dam proteins were known to be responsible for regulating the initiation of replication and to affect the expression of many genes and metabolisms. We have examined here the fatty acids composition and phospholipids membrane in dam and/or seqA mutants. The dam mutant showed an accumulation of the acidic phospholipids cardiolipin, whereas, the seqA mutant showed a higher proportion of phosphatidylglycerol compared with the wild-type strain. The seqA dam double mutant showed an intermediate proportion of acidic phospholipids compared with the wild-type strain. Based on these observations, we discuss the role of Dam and SeqA proteins in the regulation of phospholipids synthesis.     

A dam mutant of Yersinia pestis is attenuated and induces protection against plague

We have constructed a dam mutant of Yersinia pestis GB. In BALB/c mice inoculated subcutaneously, the median lethal dose of the mutant was at least 2000-fold higher than the wild type. Mice inoculated with sub-lethal doses of the mutant were protected against a subsequent challenge with virulent Y. pestis. The effect of dam inactivation on gene expression was examined using a DNA microarray, which revealed increased expression of a number of genes associated with the SOS response. These results confirm the key role of Dam in the regulation of virulence, and its potential role as a target for the generation of attenuated strains of pathogenic bacteria.     

The related effector proteins SopD and SopD2 from Salmonella enterica serovar Typhimurium contribute to virulence during systemic infection of mice

Salmonella resides within host cells in a vacuole that it modifies through the action of virulence proteins called effectors. Here we examined the role of two related effectors, SopD and SopD2, in Salmonella pathogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) mutants lacking either sopD or sopD2 were attenuated for replication in the spleens of infected mice when competed against wild-type bacteria in mixed infection experiments. A double mutant lacking both effector genes did not display an additive attenuation of virulence in these experiments. The double mutant also competed equally with both of the single mutants. Deletion of either effector impaired bacterial replication in mouse macrophages but not human epithelial cells. Deletion of sopD2 impaired Salmonella's ability to form tubular membrane filaments [Salmonella-induced filaments (Sifs)] in infected cells; the number of Sifs decreased, whereas the number of pseudo-Sifs (thought to be a precursor of Sifs) was increased. Transfection of HeLa cells with the effector SifA induced the formation of Sif-like tubules and these were observed in greater size and number after co-transfection of SifA with SopD2. In infected cells, SifA and SopD2 were localized both to Sifs and to pseudo-Sifs. In contrast, deletion of sopD had no effect on Sif formation. Our results indicate that both SopD and SopD2 contribute to virulence in mice and suggest a functional relationship between these two proteins during systemic infection of the host.     

Identification,cloning and characterization of rfaE of Actinobacillus pleuropneumoniae serotype 1, a gene involved in lipopolysaccharide inner-core biosynthesis

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and its lipopolysaccharides (LPS) have been identified as important adhesins involved in adherence to host cells. To better understand the role of LPS core in the virulence of this organism, the aim of the present study was to identify and clone genes involved in LPS core biosynthesis by complementation with Salmonella enterica serovar Typhimurium mutants (rfaC, rfaD, rfaE and rfaF). Complementation with an A. pleuropneumoniae 4074 genomic library was successful with Salmonella mutant SL1102. This Salmonella deep-rough LPS mutant is defective for the rfaE gene, which is an ADP-heptose synthase. Novobiocin was used to select transformants that had the smooth-LPS type, since Salmonella strains with wild-type smooth-LPS are less permeable, therefore more resistant to hydrophobic antibiotics like novobiocin. We obtained a clone that was able to restore the wild-type smooth-LPS Salmonella phenotype after complementation. The wild-type phenotype was confirmed using phage (Felix-O, P22c.2 and Ffm) susceptibility and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the open reading frames contained in the 3.3-kb insert in the plasmid encoded a 475-amino-acid protein with 71% identity and 85% similarity to the RfaE protein of S. enterica. We then attempted to generate an A. pleuropneumoniae rfaE mutant by gene replacement. The rfaE gene seems essential in A. pleuropneumoniae viability as we were unable to isolate a heptose-less knockout mutant.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL, encoded within ssrA, serves as the site of integration of a region of Salmonella-specific sequence; adjacent to the 5' end of ssrA is another region of Salmonella-specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella, serves as an attachment site for Salmonella-specific sequences, and is required for the growth of phage P22.     

Resuscitation of a defective prophage in Salmonella cocultures

Widely studied Salmonella enterica serovar Typhimurium strains ATCC 14028s and SL1344 harbor a cryptic ST64B prophage unable to produce infectious virions. We found that coculturing either strain with an isogenic sibling lacking the prophage leads to the appearance of active forms of the virus. Active phage originates from reversion of a +1 frameshift mutation at a monotonous G:C run in a presumptive tail assembly pseudogene.