Interaction of the Gifsy-1 Xis protein with the Gifsy-1 attP sequence

The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.     

Identification of GtgE,a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.     

Distribution of Gifsy-3 and of Variants of ST64B and Gifsy-1 Prophages amongst Salmonella enterica Serovar Typhimurium Isolates: Evidence that Combinations of Prophages Promote Clonality

Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.     

Characterization of grvA, an antivirulence gene on the gifsy-2 phage in Salmonella enterica serovar typhimurium

The lambdoid phage Gifsy-2 contributes significantly to Salmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene, sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes, grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA(+) on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor the grvA(+) plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and the grvA(+) plasmid required wild-type sodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such as sodCI and antivirulence factors such as grvA.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Unsuspected prophage-like elements in Salmonella typhimurium

We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).     

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.     

Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents

The introduction of the F-like resistance plasmid R124 into an ompC mutant of Escherichia coli K12 conferred altered sensitivity to a wide range of inhibitory agents. Sensitivity to ampicillin, chloramphenicol, ethionine, copper ions, deoxycholate, two fatty acids and colicins L and M was decreased by the plasmid. In contrast the plasmid-bearing ompC derivatives were more sensitive than the plasmid-free ompC mutant to erythromycin, cetyltrimethylammonium bromide and phenol. Introduction of R124 into the ompC strain also decreased the level of the OmpF protein and some (but not all) of the changed sensitivities listed above clearly resulted from this outer membrane protein deficiency. The presence in the ompC mutant of R124 (rather than the more efficient introduction of the plasmid into variants of the ompC strain) led to at least most of the changes described above because those tested were accentuated by the presence of a copy mutant of R124 and reversed by plasmid curing.     

Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents

R ossouw F.T. & R owbury R.J.1984. Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents. Journal of Applied Bacteriology , 56 , 63–79.
The introduction of the F-like resistance plasmid R124 into an ompC mutant of Escherichia coli K12 conferred altered sensitivity to a wide range of inhibitory agents. Sensitivity to ampicillin, chloramphenicol, ethionine, copper ions, deoxycholate, two fatty acids and colicins L and M was decreased by the plasmid. In contrast the plasmid-bearing ompC derivatives were more sensitive than the plasmid-free ompC mutant to erythromycin, cetyltrimethylammonium bromide and phenol. Introduction of R124 into the ompC strain also decreased the level of the OmpF protein and some (but not all) of the changed sensitivities listed above clearly resulted from this outer membrane protein deficiency. The presence in the ompC mutant of R124 (rather than the more efficient introduction of the plasmid into variants of the ompC strain) led to at least most of the changes described above because those tested were accentuated by the presence of a copy mutant of R124 and reversed by plasmid curing.     

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.     

Nucleotide sequence of the Serratia marcescens SR50 chromosomal ampC β-lactamase gene

A novel type of osmoregulatory mutant of Escherichia coli K-12 exhibiting constitutive expression of the ompC gene was isolated and characterized at the molecular level In this particular mutant ( cec ; c onstitutive e xpression of Omp C ). an insertion sequence (IS-1) was found to be located at right upstream of the regulatory sequence for the ompC promoter. We demonstrate that the IS1 insertion observed in the cec mutant does not provide the ompC gene with an artificial promoter, but rather perturbs normal regulation of the ompC promoter, which is mediated by the regulatory gene, ompR .     

Lignin biodegradation by cultures of Rigidoporus lignosus in solid state conditions

A novel type of osmoregulatory mutant of Escherichia coli K-12 exhibiting constitutive expression of the ompC gene was isolated and characterized at the molecular level. In this particular mutant (cec; constitutive expression of OmpC), an insertion sequence (IS-1) was found to be located at right upstream of the regulatory sequence for the ompC promoter. We demonstrate that the IS1 insertion observed in the cec mutant does not provide the ompC gene with an artificial promoter, but rather perturbs normal regulation of the ompC promoter, which is mediated by the regulatory gene, ompR.     

Isolation and characterization of a substitution mutation in the ompR gene of Salmonella typhimurium LT2.

The expression of the genes ompC and ompF encoding major outer membrane proteins is dependent on the ompR-envZ operon. Here we describe the isolation and characterization of an ompR mutation, a single-base-pair change, that results in an Arg-to-Cys substitution. When present in multiple copies, the mutant allele conferred a dominant OmpC- OmpF+ phenotype. Furthermore, the mutant allele exhibited allele-specific negative complementation with other ompR mutations. This ability, together with its dominant character, suggested that the OmpR protein is capable of multimerization.     

Interaction of OmpR, a positive regulator, with the osmoregulated ompC and ompF genes of Escherichia coli. Studies with wild-type and mutant OmpR proteins

The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompC and ompF genes that specify the major outer membrane proteins OmpC and OmpF, respectively. We purified the OmpR protein not only from wild-type cells but also from two ompR mutants (ompR2 and ompR3) exhibiting quite different phenotypes as to osmoregulation of the ompC and ompF genes. The OmpR2 protein has an amino acid conversion in the C-terminal portion of the OmpR polypeptide, whereas the OmpR3 protein has one in the N-terminal portion. Comparative studies on these purified OmpR proteins were carried out in terms of their interaction with the ompC and ompF promoters. The nucleotide sequences involved in OmpR-binding were determined in individual promoter regions by deoxyribonuclease I footprinting. The OmpR3 protein as well as the wild-type OmpR protein appeared to bind, to similar extents, to both the ompC and ompF promoters. In contrast, the OmpR2 protein bound preferentially to the ompF promoter and failed to protect the ompC promoter against DNAse I digestion. These results support the view that the C-terminal portion of the OmpR protein is responsible for the binding of the OmpR protein to the ompC and ompF promoter DNAs. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.