GENETIC SCREENING FOR IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS WITH POTENTIAL HYPERSENSITIVITY TO SHORT-CHAIN FATTY ACIDS

This study was conducted to test if transposon footprinting could be used to identify transposon mutants of Salmonella typhimurium with growth defects in a media containing short-chain fatty acids (SCFA) as the test selective condition. High concentrations of SCFA are one of the characteristic conditions in the animal intestine that has been suggested to play a role in inhibiting colonization by nonindigenous bacterial pathogens. When the mutant pools containing 25 Tn5 mutants/pool were analyzed for transposon footprints before and after selection, a polymerase chain reaction (PCR) product could be identified that was present in an input pool, but not in a corresponding output pool. The results indicate that transposon footprinting can be used for negative screening of genes sensitive to SCFA in the S. typhimurium bacterial genome.     

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains

The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP. This has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutant Salmonella hosts [Nakayama et al., Biotechnology 6 (1988) 693-697]. We have cloned the asd gene from S. typhimurium, characterized the gene product and used this gene to construct Asd+ expression cloning vectors. In addition we have constructed an asd cassette and a transposon derived from Tn5 that allow the rapid modification of other vectors for use with delta asd vaccine strains of S. typhimurium adding versatility to the Asd+ vector/delta asd host system of plasmid maintenance.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

The traT protein is able to normalize the phenotype of a plasmid-carried permeability mutation of Salmonella typhimurium

The isolation of different classes of antibiotic-supersensitive outer membrane permeability mutants of Salmonella typhimurium has been described previously (Sukupolvi et al., 1984, Journal of Bacteriology 159, 704-712). One of these, the SS-A mutation, sensitizes the bacteria to gentian violet and to hydrophobic antibiotics. The phenotype of the SS-A mutant was restored to normal when a cloned fragment of the F plasmid, or the R plasmid R6-5, carrying the genes traS, T and D was introduced on a multicopy plasmid. The introduction of a plasmid carrying only the traT gene showed that this gene was sufficient to restore the phenotype. Only clones with functioning traT (irrespective of copy number) restored the normal antibiotic-resistant phenotype in the SS-A mutant. An incompatibility test using a donor strain which carried transposon Tn10 in the 60 MDa plasmid of S. typhimurium and a recipient in which Tn5 was placed close to the SS-A mutation indicated that the SS-A mutation was located in the 60 MDa virulence plasmid (previously called the cryptic plasmid) of S. typhimurium. The introduction of the large virulence plasmid carrying the SS-A mutant allele into wild-type S. typhimurium or Escherichia coli resulted in strains with a phenotype identical to that of the original SS-A mutant.     

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.     

A common virulence region on plasmids from eleven serotypes of Salmonella

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.     

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

Fine-structure genetic map of the maltose transport operon of Salmonella typhimurium.

We have constructed a fine-structure genetic map of the maltose transport operon in Salmonella typhimurium. We have isolated mal mutants by using indicator plates, penicillin selection, or a proton suicide technique. Mutants were obtained as spontaneous events or were induced by chemical mutagenesis and transposon insertion. Tn10 and Mu d(lac Ap)1 insertion mutations were used to create deletions. Mutations were also obtained in a gene that is equivalent to lamB in Escherichia coli, which codes for the lambda bacteriophage receptor. The gene products in the mutants were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and immunoblotting. Our data indicate that the location of this operon on the Salmonella chromosome as well as the gene order and its orientation are the same as those in E. coli. This map will be useful in studying the mechanism of periplasmic transport in S. typhimurium.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

Construction of delta aroA his delta pur strains of Salmonella typhi.

Salmonella typhi strains with two deletion mutations, each causing an attenuating auxotrophy, have been constructed from strains Ty2 and CDC 10-80 for possible use as oral-route live vaccines. An aroA(serC)::Tn10 transposon insertion was first transduced from a Salmonella typhimurium donor into each wild-type S. typhi strain. Transductants of the Aro- SerC- phenotype were treated with transducing phage grown on an S. typhimurium strain with an extensive deletion at aroA; selection for SerC+ yielded transductants, some of which were delta aroA. A his mutation was next inserted into a delta aroA strain in each line by two steps of transduction. Two deletions affecting de novo purine biosynthesis were used as second attenuating mutations: delta purHD343, causing a requirement for hypoxanthine (or any other purine) and thiamine, and delta purA155, causing an adenine requirement. The purHD343 deletion was introduced into the delta aroA his derivatives of each strain by cotransduction with purH::Tn10, and the purA155 deletion was introduced into the CDC 10-80 delta aroA his derivative by cotransduction with an adjacent silent Tn10 insertion by selection for tetracycline resistance. Tetracycline-sensitive mutants of each of the three delta aroA his delta pur strains were isolated by selection for resistance to fusaric acid. The tetracycline-sensitive derivative of the CDC 10-80 delta aroA his delta purA155 strain, designated 541Ty, and its Vi-negative mutant, 543Ty, constitute the candidate oral-route live-vaccine strains used in a recent volunteer trail (M. M. Levine, D. Herrington, J. R. Murphy, J. G. Morris, G. Losonsky, B. Tall, A. A. Lindberg, S. Stevenson, S. Baqar, M. F. Edwards, and B. A. D. Stocker, J. Clin. Invest. 79:885-902, 1987). Tetracycline-sensitive mutants of the delta aroA his delta purHD derivative of strains Ty2 and CDC 10-80 may also be appropriate as live vaccines but have not been tested as such.     

Genetic map of the opp (Oligopeptide permease) locus of Salmonella typhimurium.

The uptake of peptides by Salmonella typhimurium is mediated by three apparently independent transport systems. One of these systems, the oligopeptide permease, is encoded by a genetic locus (opp) which has been mapped at 34 min on the S. typhimurium chromosomal map. We accurately mapped the location of opp by cotransduction frequencies and by deletion analysis and show that the gene order for this region of the chromosome is cysB-trp-tonB-opp-galU-tdk. All opp mutants, independently isolated by a variety of means, mapped at this one locus, between tonB and galU. Spontaneous and transposon Tn10-generated deletions were used to construct a fine-structure genetic map of opp. Evidence is presented which indicates that opp covers a 5- to 6-kb segment of DNA and is therefore likely to consist of more than one gene.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells

During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection. This host-pathogen interaction requires several S. typhimurium genes. To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S. typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells. The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae. Unlike wild-type S. typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils. Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S. typhimurium phenotype. Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S. typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses. We therefore conclude that S. typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S. typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S. typhimurium flagellin-induced inflammation.     

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Integration of transposon Tn10 into phage L (Salmonella typhimurium)

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.     

Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II.

Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium. Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with Tn10. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The Tn10 insertion in strain SMS409 was mapped by conjugation and transduction to the region between aroA and pncB at 20 min on the chromosome of S. typhimurium; this location is similar to the genetic location of aspC in Escherichia coli. The specific activity of the aspC product, aspartate aminotransferase, was severely reduced in strain SMS409. This indicated that the Tn10 insertion in strain SMS409 inactivated aspC. An aspC mutant of E. coli was also inhibited by either sulfometuron methyl or tyrosine. We present a hypothesis which relates the observed alpha-ketobutyrate accumulation in sulfometuron methyl-inhibited cultures of strain SMS409 to aspartate starvation.     

以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究──Tn5的水平转移及其对R．fredii Tn5突变株运动的影响

将一株弗氏中华根瘤菌（Ｒ．ｆｒｅｄｉｉ）ＱＢ１１３０的Ｔｎ５插入突变株ＯＮ－２用于生态学研究,以评估Ｔｎ５在自然环境中的水平转移以及各种水势下Ｔｎ５对突变株ＯＮ－２在土壤中运动的影响．试验表明,在自然潮湿的土壤中,Ｔｎ５本身的水平转移频率很低,且与Ｔｎ５插入相关的突变株卡那霉素抗性表型标记在非选择性平板上连续传４０代后仍然稳定．突变株ＯＮ－２与相对应的野生型菌株ＱＢ１１３０在各种相同水势的土壤中的运动无明显差异（Ｐ＝０．０１）,表明Ｔｎ５的插入不影响突变株的运动．因此,Ｔｎ５可作为研究Ｒ．ｆｒｅｄｉｉ基因工程菌大回应用的一个稳定有效的生态学标记．     

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.