Site-directed (IS30-FljA) transposon mutagenesis system to produce nonflagellated mutants of Salmonella Enteritidis

Site-directed integration/mutagenesis systems are used to carry out targeted transpositions on DNA. The well-characterized IS30-element and its transposase have numerous advantages that predestine it to be a good candidate for such applications. In order to generate nonflagellated mutants of Salmonella Enteritidis, a new site-directed mutagenesis system has been developed and applied. The system was constructed based on the assumption that the DNA-binding FljA component of the fusion transposase would bind to its target (the operator of fliC), and as a consequence, insertions could be concentrated in the flagellin operon. The system consists of two components: one expresses the fusion transposase and the other is an integration donor plasmid harbouring the (IS30)(2) reactive structure. The application of this site-directed mutagenesis system on a strain of S. Enteritidis 11 (SE11) resulted in several nonmotile mutants with fliD insertion that could serve as negatively markered vaccine candidates. Analysis of less motile mutants generated by the fusion transposase revealed further hot spot sequences preferred by the fusion construct.     

Conversion of the Salmonella phase 1 flagellin gene fliC to the phase 2 gene fljB on the Escherichia coli K-12 chromosome.

The Escherichia coli-Salmonella typhimurium-Salmonella abortus-equi hybrid strain EJ1420 has the two Salmonella flagellin genes fliC (antigenic determinant i) and fljB (determinant e,n,x) at the same loci as in the Salmonella strains and constitutively expresses the fliC gene because of mutations in the genes mediating phase variation. Selection for motility in semisolid medium containing anti-i flagellum serum yielded 11 motile mutants, which had the active fliC(e,n,x) and silent fljB(e,n,x) genes. Genetic analysis and Southern hybridization indicated that they had mutations only in the fliC gene, not in the fljB gene or the control elements for phase variation. Nucleotide sequence analysis of the fliC(e,n,x) genes from four representative mutants showed that the minimum 38% (565 bp) and maximum 68% (1,013 bp) sequences of the fliC(i) gene are replaced with the corresponding sequences of the fljB(e,n,x) gene. One of the conversion endpoints between the two genes lies somewhere in the 204-bp homologous sequence in the 5' constant region, and the other lies in the short homologous sequence of 6, 8, or 38 bp in the 3' constant region. The conversions include the whole central variable region of the fljB gene, resulting in fliC(e,n,x) genes with the same number of nucleotides (1,503 bp) as the fljB gene. We discuss the mechanisms for gene conversion between the two genes and also some intriguing aspects of flagellar antigenic specificities in various Salmonella serovars from the viewpoint of gene conversion.     

Association of attenuated mutants of Salmonella enterica serovar Enteritidis with porcine peripheral blood leukocytes

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a lambda-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells

A nonflagellated mutant of Salmonella enterica serotype Enteritidis was constructed by disrupting the flagellin gene (fliC). Northern blot analysis indicated that the mutation did not affect expression of the downstream fliU gene. Infection experiments with differentiated Caco-2 cells revealed that the mutant was about 50-fold less invasive than the wild-type strain, while bacterial adherence was unaffected. Complementation of the mutant with an intact fliC copy restored flagella formation and efficient bacterial invasion. Our data demonstrate that the fliC gene of S. enterica serotype Enteritidis is essential for the invasion of Caco-2 cells.     

Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coli.

In a search for genes that produce hypersensitivity to cadmium salts in Escherichia coli, random transposon mutagenesis with TnphoA was used. One of the mutant strains obtained was sensitive to Cd2+ and Zn2+. Sequence analysis showed that the TnphoA insertion was located in the dsbA gene coding for a periplasmic protein required for disulfide bond formation.     

TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins.

A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.     

Analysis of protein localization by use of gene fusions with complementary properties.

This report describes a new transposon designed to facilitate the combined use of beta-galactosidase and alkaline phosphatase gene fusions in the analysis of protein localization. The transposon, called TnlacZ, is a Tn5 derivative that permits the generation of gene fusions encoding hybrid proteins carrying beta-galactosidase at their C termini. In tests with plasmids, TnlacZ insertions that led to high cellular beta-galactosidase activity were restricted to sequences encoding either cytoplasmic proteins or cytoplasmic segments of a membrane protein. The fusion characteristics of TnlacZ are thus complementary to those of TnphoA, a transposon able to generate alkaline phosphatase fusions whose high-activity insertion sites generally correspond to periplasmic sequences. The structure of TnlacZ allows the conversion of a TnlacZ fusion into the corresponding TnphoA fusion (and vice versa) through recombination or in vitro manipulation in a process called fusion switching. Fusion switching was used to generate the following two types of fusions with unusual properties: a low-specific-activity beta-galactosidase-alkaline phosphatase gene fusion and two toxic periplasmic-domain serine chemoreceptor-beta-galactosidase gene fusions. The generation of both beta-galactosidase and alkaline phosphatase fusions at exactly the same site in a protein permits a comparison of the two enzyme activities in evaluating the subcellular location of the site, such as in studies of membrane protein topology. In addition, fusion switching makes it possible to generate gene fusions whose properties should facilitate the isolation of mutants defective in the export or membrane anchoring of different cell envelope proteins.     

Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165.

Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.     

Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis.

We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. USA 82:8129-8133, 1985), in Rhizobium meliloti. Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins. By confining our screening to 1,250 TnphoA-generated mutants of R. meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa. Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion. Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci. One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein. Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport. The remaining six loci were located on the R. meliloti chromosome. One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure. Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis. The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.     

Isolation, characterization, and complementation of a paralyzed flagellar mutant of Rhodobacter sphaeroides WS8.

A paralyzed Rhodobacter sphaeroides mutant strain (PARA1) was isolated by a motility screening procedure following mutagenesis of wild-type R. sphaeroides WS8-N with the transposable element TnphoA (Tn5 IS50L::phoA). PARA1 synthesized a wild-type level of flagellin, as detected by Western immunoblotting with antiflagellar antiserum. Flagellar staining showed that flagellin was assembled into apparently normal external flagellar filaments. Electron micrographs of basal body structures from PARA1 showed that some ring structures that were present were similar to those in wild-type R. sphaeroides WS8-N. PARA1 cells were nonmotile under all growth conditions. No pseudorevertants to motility were seen when PARA1 was grown in the presence of kanamycin to select for the presence of the transposon. The presence of the single copy of TnphoA in the PARA1 chromosome was demonstrated by Southern blotting. Western blotting of cytoplasmic, periplasmic, and membrane fractions of PARA1 with anti-alkaline phosphatase antiserum showed that the transposon had been inserted in-frame into a gene encoding a membrane protein. A SalI restriction endonuclease fragment was cloned from the chromosome of PARA1; this fragment contained a portion of the transposon and R. sphaeroides DNA sequence 5' of the site of insertion. This flanking R. sphaeroides DNA sequence was used to probe an R. sphaeroides WS8 cosmid library. A cosmid designated c19 hybridized to the probe, and a SalI restriction endonuclease fragment derived from this cosmid restored wild-type motility to PARA1 when introduced into this mutant strain by conjugation. The significance of this finding in a bacterium with unidirectionally rotating flagella is discussed.     

Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli.

In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.     

Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes

Each of the two mutants isolated from a fliC (= hag, flagellin-deficient) Escherichia coli strain made motile by a plasmid carrying the fliC gene of Salmonella muenchen by selection for motility in the presence of anti-d (Salmonella flagellar antigen) serum had both lost and gained one or more subfactors of the wild-type antigen. In one mutant codon 246 was GAC (alanine) instead of GCC (asparagine); the other had a deletion of 105 base pairs, explicable by a 10bp direct repeat, starting at bases 782 and 887. The in vitro removal of a 48bp EcoRV(631)/EcoRV(679) fragment produced plasmid pLS408, which was found to lack a subfactor of wild-type antigen d but able to confer motility on flagellin-negative Salmonella sp. (and used for insertion of epitope-specifying oligonucleotides at its EcoRV site). Immunoblotting with absorbed and unabsorbed sera from rabbits immunized with E. coli with wild-type or mutated antigen d showed that the fusion proteins specified by lambda gt11 with the N-terminal part of gene lacZ joined to a restriction fragment coding for residues 145-391 of flagellin gave the same pattern of parent-specific and mutant-specific reactions as the flagellate bacteria. Four out of five similarly selected mutants had the same 105 bp deletion as the first-isolated mutant; the fifth had a 72 bp deletion made possible by a 7-base pair direct repeat, starting at positions 649 and 721. All these changes in serological character without loss of function affected segment IV, specifying residues 182 to 308 of the total of 505, where there is little homology between different flagellar-antigen alleles.     

Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.     

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.     

Identification by a multiplex PCR-based assay of Salmonella Typhimurium and Salmonella Enteritidis strains from environmental swabs of poultry houses

A multiplex-PCR-based assay (m-PCR) was developed for the detection of Salmonella and for the identification of the two serotypes Enteritidis and Typhimurium. Three sets of primers selected from different genomic sequences amplified a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, a 559 bp target specific for Salmonella Typhimurium within the fliC gene and a 312 bp fragment specific for Salmonella Enteritidis within the sefA gene. The m-PCR-based assay was used for detecting Salmonella from 1078 environmental swabs of poultry houses. Prior to PCR, these swabs were pre-enriched in phosphate-buffered peptone water for 18-20 h and then sub-cultured on a Modified Semi-solid Rappaport Vassiliadis medium (MSRV) for 18-20 h. The m-PCR combined with MSRV had a better sensitivity (95%) than the bacteriological method (92.5%). The MSRV-m-PCR assay and the bacteriological method had an agreement rate of 95.6%.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.     

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.