Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.     

Colicin E1 export inSalmonella typhimurium wild-type and lipopolysaccharide mutants

Colicin export was studied in differentSalmonella typhimurium strains lacking the O-antigen repeating units (O⁻) and different strains with different chemotypes for the lipopolysaccharide core, as well as the wild-type strain (O⁺) to determine the role of lipopolysaccharide length on colicin E1 export. While the lipopolysaccharide length influences the levels of external hemolytic activity inS. typhimurium, no effect was detected on colicin E1 export.     

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides

Summary Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effects were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.     

Interaction of 125I-Labeled Colicin E1 with Escherichia coli

Colicin E1 protein was labeled with ¹²⁵I to specific activities of up to 2 × 10⁸ cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

Summary Plasmid DNA has been isolated from five fi ⁺ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 m. A variant line of S. typhimurium type 36 which was fi ^- yeilded no plasmid DNA. These results support the hypothesis that the fi ⁺ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP10₃₆, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi ⁺ property, is correlated with loss of MP10₃₆. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP10₃₆ and K. MP10₃₆ has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP10₃₆ is autotransferable at low frequency. The significance of these findings is discussed.     

Salmonella typhimurium poultry isolate growth response to propionic acid and sodium propionate under aerobic and anaerobic conditions

Propionic acid and its sodium salt have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acids in poultry feed may have a potential role in inhibiting growth of Salmonella in the chicken intestine. In this study, we determined growth response of a Salmonella typhimurium poultry isolate to propionic acid and sodium propionate under aerobic and anaerobic conditions. Growth rate consistently decreased with the addition of greater concentrations of either propionic acid or sodium propionate. The extent of growth inhibition was much greater with propionic acid than the sodium form. Media pH decreased only with addition of propionic acid. Growth inhibition was more effective under anaerobic growth conditions with either propionic acid or sodium propionate. When determined at the same pH level, growth rate was significantly lowered by addition of 25 mM of either propionate or sodium propionate alone, and also by the decrease in pH levels (P<0.05). These results showed that growth inhibition of S. typhimurium by propionic acid or sodium propionate is greatly enhanced by pH decrease, and to lesser extent by anaerobiosis. We also found that sodium propionate was more inhibitory for growth of S. typhimurium than propionic acid when compared at the same pH levels.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition caused by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzymes synthesis as well as inhibition of activity.     

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2

We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I-CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BinI sites in glt-tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E. coli K-12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

Influence of lipopolysaccharide on external hemolytic activity ofSalmonella typhimurium andKlebsiella pneumoniae

Excretion ofEscherichia coli -hemolysin was tested in rough and smooth strains ofKlebsiella pneumoniae andSalmonella typhimurium. Smooth strains harboring the hemolytic recombinant plasmid pANN202-312 showed a five- to tenfold increase in the hemolytic activity evaluated in the external medium compared with isogenic rough strains harboring the same plasmid.     

The immunity genes of colicins E2 and E8 are closely related

We have determined the nucleotide sequence of the newly characterized colicin E8imm gene which exists in tandem with the colicin E3imm gene in the: ColE3-CA38 plasmid. Comparison of these immunity structures reveals considerable sequence divergence) but the ColE8imm gene is markedly homologous to the colicin E2imm gene from the ColE2-P9 plasmid.Issued as NRCC no. 23586 and as CBRI no. 1480.     

Virulence Increasing of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> in Balb/c Mice After Heat-Stress Induction of Phage Shock Protein A

Salmonella typhimurium is a potentially intracellular pathogen and is responsible for thousands of reported cases of acute gastroenteritis and diarrhea each year. Although many successful physiological and genetic approaches have been taken to conclude the key virulence determinants encoded by this organism, the total number of uncharacterized reading frames observed within the S. typhimurium genome suggests that many virulence factors remain to be discovered. This study was conducted to evaluate the role of heat induced phage shock protein A (PspA), in the pathogenicity of S. typhimurium. The stress proteins detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identified specifically by immunoblotting with polyclonal antibody against PspA. PspA was produced in response to heat stress at 45°C and it was over-expressed at 65°C. At this temperature, the stressed bacterial cells producing PspA were more virulent (16 folds greater) to female 6–8 week-old Balb/c mice. Correspondency between decrease in LD₅₀ and increase in PspA production during heat stress and lower pathogenicity in non-producing cells that emerged during stress at 55°C represents PspA as an important virulence factor in heat stressed S. typhimurium.     

On the mode of action of 5-diazouracil on bacterial cell division

Cell division by strains ofEscherichia coli andSalmonella typhimurium is inhibited by 5-diazouracil (5-DU). Division recovers in the presence of the inhibitor after a period which is temperature-dependent. Recovery is probably due to breakdown of 5-DU and the rate of this breakdown is apparently increased at alkaline pH. Growth with 5-DU caused only a slight reduction in the rate of murein synthesis and no alteration in the properties or composition of membranes ofS. typhimurium. The agent caused chaining inStreptococcus fecalis and inhibition of the penicillin-induced lysis ofS. typhimurium. These effects may have been due to direct inhibition of lysin activity but an indirect effect seems more likely. The most marked effect of 5-DU onS. typhimurium was to cause a transient inhibition of DNA synthesis. Since 5-DU did not stop uncoupled cell division (i.e. division occurring independently of DNA replication) and sincelon^? strains were more sensitive to 5-DU thanlon⁺ strains, it was concluded that 5-DU acts on cell division via an inhibitory effect on DNA replication.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

Molecular Cloning and Nucleotide Sequencing of a Novel Aminoglycoside 6′-N-Acetyltransferase Gene from an R-Plasmid of Salmonella typhimurium S24 Isolated in Taiwan

A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K-12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6′-N-acetyltransferase[AAC(6′)] was cloned from plasmid pST2 on a 1,393 kilobase (kb) of Sphl-SalI fragment into vector pACYC184 and pUC18. This novel A AC (6′) gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6′) gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.     

Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data

Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1.