Natural Populations of Escherichia Coli and Salmonella Typhimurium Harbor the Same Classes of Insertion Sequences

Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange. In this study, genetically diverse collections of E. coli and S. typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction. A majority of salmonellae contained IS1 or IS3, elements originally recovered from E. coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E. coli. Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E. coli and S. typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa. This suggests that copies of IS200 have not been recently transferred between E. coli and S. typhimurium and that the element was present in the common ancestor to both species. IS200 is polymorphic within E. coli but homogeneous among isolates of S. typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.     

Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate.

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.     

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.     

Stability of plasmids R1-19 and R100 in hyper-recombinant Escherichia coli strains and in Salmonella typhimurium strains.

Plasmids R1-19 and R100 dissociate in hyper-recominant Escherichia coli strains in a way that is similar to but slower than dissociation in Salmonella typhimurium. The results presented suggest that the molecular mechanism for plasmid dissociation in hyper-recombinant E. coli strains is different than that in S. typhimurium strains.     

Thermosensitive Replication of a Kanamycin Resistance Factor

A strain of Proteus vulgaris isolated from the urinary tract of a patient with postoperative pyelonephritis and resistant to sulfonamide, streptomycin, tetracycline, and kanamycin (KM) was found to transfer only KM resistance by cell-to-cell conjugation. The genetic determinant controlling the transferable KM resistance was considered to be an R factor and was designated R (KM). Successive transfer of KM resistance was demonstrated also from Escherichia coli 20S0, which received the R (KM) factor, to other substrains of E. coli K-12 or Salmonella typhimurium LT-2. The transfer of the R (KM) factor was strongly affected by the temperature at which the mating culture was kept. The transfer frequency of R (KM) at 25 C was about 10(5) times higher than at 37 C. The R (KM) factor was spontaneously eliminated from the host bacterial cells when P. vulgaris was cultured at 42 C, but no elimination occurred at 25 C. This elimination of the R (KM) factor at elevated temperature was also observed when the R (KM) factor infected E. coli and S. typhimurium. On the other hand, a normal R factor could not be eliminated from the same E. coli host strain by cultivation at the higher temperature. We consider the thermosensitive transfer and the spontaneous elimination of the R (KM) factor at higher temperature to depend upon thermosensitive replication of the R (KM) factor.     

R-Factor Transfer in Selenite and Tetrathionate Broths

After overnight incubation of R(+)Escherichia coli with R(-)Salmonella typhimurium in selenite and tetrathionate with Brilliant Green (TBG) broths, R-factor transfer was demonstrated in 10 of 12 experiments. R-factor transfer in these enrichment broths occurred at a markedly reduced frequency in comparison to that in Trypticase soy broth, apparently due to an adverse effect either on the viability of the donor E. coli or the conjugation process itself. Transfer of R factors in commonly used enrichment broths may give rise to falsely resistant antibiotic patterns in Salmonella. However, the frequency of R-factor transfer is so low that it is unlikely to affect significantly the interpretation of R-factor studies.     

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.     

Comparison of the tryptophan synthetase alpha-subunits of several species of Enterobacteriaceae

Creighton, T. E. (Stanford University, Stanford), D. R. Helinski, R. L. Somerville, and C. Yanofsky. Comparison of the tryptophan synthetase alpha subunits of several species of Enterobacteriaceae. J. Bacteriol. 91:1819-1826. 1966.-The tryptophan synthetase alpha subunits of Escherichia coli K-12, E. coli B, Shigella dysenteriae, Salmonella typhimurium, and Aerobacter aerogenes have been purified and their structures compared. Each of these alpha subunits exhibits a sedimentation coefficient of about 2.7S. Peptide patterns of trypsin plus chymotrypsin digests of the alpha subunits have indicated that all of the alpha subunits have peptide regions in common. The patterns of E. coli K-12, E. coli B, and S. dysenteriae alpha subunits appear to be nearly identical, whereas the alpha subunits from S. typhimurium and A. aerogenes differ from those of E. coli and from each other. It has also been shown that the E. coli structural gene for the alpha subunit is translated identically in E. coli and S. typhimurium.     

Transforming activity of R- and Lac-plasmids of clinical strains of Gram-negative bacteria]

The isolated plasmid DNA of clinical strains of Gram-negative bacteria were shown to have transforming activity when E. coli strain 0600 and S. typhimurium strain LT-2 were used as recipients. The frequency of transformation depended on the recipient strain and the character of the plasmids. The presence of deletion mutants was revealed among the transformants. Such mutants occurred with varying frequency, most often in S. typhimurium strain LT-20; the reason for this phenomenon is at present under discussion. The transformation of plasmids controlling lactose splitting and their conjugation transfer into recipient S. typhimurium strain LT-2 is possible only under condition of using recipient (R+). The possibility of the formation of the cointegrate (R and lac plasmids) in recipient S. typhimurium strain LT-2 is discussed.     

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.     

Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.     

Effect of the R plasmids of Salmonella typhimurium strains of various origins on salmonella virulence

Antibiotic-multiresistant S. typhimurium strains, phagovars 20 and 25, of different origin (isolated in hospital infections transferred through everyday contacts and by alimentary route, as well as from samples taken from open water basins and sewage) have been found to carry both transmissible and nontransmissible R plasmids. The frequency of the transfer of R plasmids to Escherichia coli K-12 C 600 rif varies within 0.6 X 10(-5) and 0.7 X 10(-3) per donor cell. The direct transfer of plasmids to S. typhimurium has been mostly realized only in the presence of the mobilizing plasmid, the transfer frequency being 1.0 X 10(-7) to 2.2 X 10(-4) per donor cell. S. typhimurium transconjugates show decreased virulence in both enteral and intraperitoneal infection of mice.     

Transduction of Various R Factors by Phage P1 in Escherichia coli and by Phage P22 in Salmonella typhimurium

R factors fi(+) and fi(-), with various combinations of drug-resistance markers and isolated from independent sources, were transduced by phage P1kc in Escherichia coli and by phage P22 in Salmonella typhimurium. Usually the entire R factor was transduced by P1kc in E. coli, as indicated by the absence of segregation of the drug-resistance markers from their conjugal transferability. In contrast, the patterns of segregation of the drug-resistance markers and their conjugal transferability differed considerably among various R factors after transduction by P22 in S. typhimurium. Transduction frequencies varied among R factors in both transduction systems.     

Intergeneric conjugational crossing of Escherichia coli with Salmonella typhimurium. II. Transfer of a polA1 mutation from Escherichia coli to Salmonella typhimurium and its phenotypic expression in the salmonella genome]

The Escherichia coli structural gene for DNA polymerase I was inserted into Salmonella typhimurium chromosome by conjugal transfer. The genetic analysis of P1-mediated transduction of obtained hybrid showed that polA gene is located in it between metE and rha loci and is cotransduced with metE (about 50%) and rha (12%). The phenotypic properties of polA1 hybrid E. coliXS. typhimurium concerning UV-MMS-NG and gamma-ray sensitivity are similar to the polA1 mutants of E. coli.     

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.     

Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.     

Comparison of the Activities of Extracts of Escherichia coli and Salmonella typhimurium in Amino Acid Incorporation

We have compared the amino acid incorporating activities of extracts of Escherichia coli and Salmonella typhimurium in in vitro protein-synthesizing systems directed by bacterial messenger ribonucleic acid (mRNA) of both species and by the genomes of coliphages Qbeta and f2. E. coli and S. typhimurium extracts translate both homologous and heterologous bacterial mRNAs at comparable rates. S. typhimurium extracts translate phage RNAs only 10 to 15% as fast as E. coli extracts do. The presence of glucose in the growth medium increases the activity of S. typhimurium extracts three- to fourfold in the phage RNA-directed systems. Glucose has a much more limited effect on the activities of E. coli extracts. We show that similar amounts of phage RNA-ribosome complexes are formed in both the E. coli and the S. typhimurium systems, indicating that the different activities observed may be attributed to different rates of peptide elongation or to the formation of complexes at different sites on the RNA strand.