Studies on ribosomal mutants of Salmonella typhimurium LT-2

Summary Mutations conferring resistance to spectinomycin, resistance to neomycinkanamycin and cold-sensitivity of ribosome biosynthesis were located on the Salmonella typhimurium chromosome in relation to the genes cysG, strA, and aroC. The effect of temperature and growth medium on the expression of these mutations, and the interactions between the mutations was determined as a prerequisite to the mapping. On the basis of chromosomal location and dominance, it is concluded that cold-sensitivity of ribosome synthesis can result from mutations in several distinct genes.This work was supported by Public Health Service research grant AI-05526.     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

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.     

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.     

Conditional mutations in the translational apparatus of Bacillus subtilis

Summary Four temperature sensitive mutants of B. subtilis were isolated by localized mutagenesis in the major ribosomal gene cluster, and characterized genetically and biochemically. Three are mutations which cause temperature sensitivity in the elongation factor Ef-G, and one which has a similar effect on the elongation factor Ef-Tu. They map in a cluster near strA, with the temperature-sensitive mutations in Ef-G mapping between the strA gene and the temperature sensitive mutation in Ef-Tu.     

Coevolution of antibiotic production and counter‐resistance in soil bacteria

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance‐only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.     

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.     

Incidence of resistance to ampicillin,chloramphenicol, kanamycin,tetracycline and trimethoprim of Salmonella strains isolated in The Netherlands during 1975–1980

From 1975–1980, about 130 000 Salmonella strains isolated from various sources were tested for resistance to ampicillin, chloramphenicol, kanamycin, tetracycline and trimethoprim. Following the ban on incorporation of tetracycline in animal feeds for nutritive purposes, tetracycline resistance in S. typhimurium and S. panama strains of porcine origin dropped from about 90% in 1974 for both species, to about 34% and 1%, respectively, in 1980. The incidence of resistance in human strains concurrently decreased from about 80% in 1974 to 25% and 1%, respectively, in 1980.The build-up of multiple resistance in bovine S. dublin and S. typhimurium strains, already started in 1973–1974, has continued. Recently, phage type 193 S. typhimurium strains have become predominant and they are invariably resistant to ampicillin, chloramphenicol, tetracycline, kanamycin, neomycin, streptomycin, sulphonamide and trimethoprim. Up to now, type 193 strains were hardly encountered in human patients, but the number of human isolates is slowly increasing.A fairly large number of multiply resistant strains belonging to S. oranienburg, S. schwarzengrund, S. typhimurium and, recently, S. krefeld have been isolated from adoptive children from the Far East.     

A genetical and biochemical study of chlorate-resistant mutants ofSalmonella typhimurium

S.typhimurium can form nitrate reductase A, chlorate reductase C, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. None of these enzymes are formed in chlorate-resistant mutants. Conjugation experiments showed the presence of a strong linkage between thechl andgal markers of the bacterial chromosome. By deletion mapping the gene ordernic A aro G gal bio chl D uvr B chl A was found. Strains with deletions terminating betweenbio anduvr B or betweenuvr B andchl A have a number of aberrant properties. Though resistant against chlorate they reduce nitrate and form gas. After growth with nitrate they form less nitrate reductase than the wild type which may explain the resistance against chlorate. After growth with thiosulfate they form small amounts of thiosulfate reductase and chlorate reductase C. In crosses between anE.coli Hfrchl ⁺ strain and aS.typhimurium chl A strain recombinants were obtained, forming nitrate reductase A and chlorate reductase C. These recombinants do not form gas, which indicates that thechl ⁺ gene fromE.coli does not function normally inS.typhimurium.The author is very gratefull to Miss C. W. Bettenhaussen, Miss W. M. C. Kapteijn and Mr. K. Pietersma for technical assistance. Helpfull suggestions of Dr. P. van de Putte (Medical Biological Laboratory of the National Defence Organization TNO, Rijswijk) are gratefully acknowledged.     

Ribosomal proteins. 33. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin

Ribosomal protein S12, the protein coded by the strA ciatron, was isolated from nine streptomycin-resistant mutants originating from various Escherichia coli strains. Analysis of the tryptic peptides revealed that each mutant had a single amino-acid replacement in one of two peptides: in mutants belonging to the allele types strA1, strA2 and strA60 the lysine residue in position 42 (peptide T6) of protein S12 is replaced by one of three ammo acids (asparagine, threonine or arginine) whereas the mutants belonging to allele type strA40 have a replacement of lysine by arginine in peptide T15. There is a good agreement between our protein-chemical data and earlier genetic data on streptomycin-resistant mutants.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

DNA restriction and modification systems in Salmonella. II. Genetic complementation between the K and B systems of Escherichia coli and the Salmonella typhimurium system SB, with the same chromosomal location

Summary E. coli x S.typhimurium partially diploid hybrids were constructed to investigate the possibility of genetic complementation between the SA and the SB restriction and modification systems of S. typhimurium and the K and B systems of E. coli.An hsdR _K ^- mutation was complemented in a stable hybrid in which the hsd _SA ⁺ -hsd _SB ⁺ -serB ⁺ portion of the S. typhimurium chromosome was integrated at a non-homologous locus. By isolating hsd ^- mutants in that hybrid, it was shown that complementation occured between K and SB, but not between K and SA.Similarly, in a set of F-prime merodiploids bearing the SA, SB and B systems, complementation was observed between B and SB, but not between B and SA.     

Different Sensitivity of Complement to Salmonella typhimurium Accounts for the Difference in Natural Resistance to Murine Typhoid between A/J and C57BL/6 Mice

The difference in natural resistance to Salmonella typhimurium between S. typhimurium-resistant A/J mice and S. typhimurium-susceptible C57BL/6 mice was analyzed. In both strains, the growth of S. typhimurium was controlled in the spleen until 48 hr of infection, while serum C3b levels were increased in A/J mice immediately after infection but not in C57BL/6 mice. Incubation of A/J mouse serum with S. typhimurium or its lipopolysaccharide (LPS) generated sufficient amounts of C3b, but that of C57BL/6 mouse serum with them did not. A/J macrophages had higher intracellular killing activity in vitro than did C57BL/6 cells against S. typhimurium pre-opsonized with each corresponding fresh serum. However, the cells from both mice exhibited a similar level of killing activity against S. typhimurium pre-opsonized with fresh A/J serum or rabbit complement. The resistance of C57BL/6 mice was significantly increased by opsonizing S. typhimurium with fresh A/J serum or rabbit complement before inoculation. The serum level of interferon-γ (IFN-γ) in A/J mice was 2.7 times as high as in C57BL/6 mice at 48 hr post-infection. Recombinant murine IFN-γ enhanced the intracellular killing activity of macrophages from both mice when S. typhimurium was pre-opsonized with fresh A/J serum but not with fresh C57BL/6 serum. These findings suggest that A/J macrophages exhibit maximal killing activity against A/J serum-opsonized S. typhimurium in vivo when the cells are activated with IFN-γ. Therefore, the rapid and sufficient activation of complement by Salmonella LPS may render A/J mice more resistant against murine typhoid.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

Summary The effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA ⁺ parent bacteria or phenotypically POL⁺ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and c1, indicating that the polA ⁺ and recA ⁺ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.     

Carbendazim resistance and dimethachlone sensitivity of field isolates of Sclerotinia sclerotiorum from oilseed rape in Henan Province,China

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases in oilseed rape‐growing areas of China. To determine the frequency of resistance of field isolates of S. sclerotiorum to carbendazim and dimethachlone, a total of 556 isolates from 10 different regions of Henan Province were obtained between 2015 and 2016. The frequency of isolates with a high‐resistance phenotype and a moderate‐resistance phenotype to carbendazim was 69.2% and 10.8%, respectively. However, S. sclerotiorum isolates resistant to dimethachlone were not detected. The baseline sensitivity of S. sclerotiorum to dimethachlone was distributed as a unimodal curve with a mean EC₅₀ value of 0.39 ± 0.09 μg ml^?1 for the inhibition of mycelial growth. Four dimethachlone‐resistant mutants were obtained from 20 wild‐type isolates induced by exposure to increasing concentrations of the fungicide in vitro. The mutants showed high levels of resistance to dimethachlone, with resistance factors that ranged from 179 to 323. Positive cross‐resistance occurred between dimethachlone and procymidone, iprodione, and fludioxonil; however, no cross‐resistance was observed for carbendazim and boscalid. The fitness of the dimethachlone‐resistant mutants was significantly lower than that of the wild‐type isolates, as measured by mycelial growth, hyphal dry weight, sclerotium number and dry weight, and pathogenicity. Additionally, based on osmotic tests, the inhibition of mycelial growth caused by NaCl applied at different concentrations was significantly higher for the dimethachlone‐resistant mutants than for their wild‐type parents.     

Nonspecific Stimulation of Antibacterial Resistance by a Synthetic Thymic Factor (FTS) in Mice

Treatment of mice with 0.01–1 μg of a synthetic serum thymic factor (FTS) significantly increased their resistance to lethal doses (1–5 × 10⁶ organisms) of Salmonella typhimurium LT2 (LT2). At least seven injections of FTS were necessary for induction of resistance, since the effects of one to three injections were variable. FTS additively potentiated the protective immunity to LT2 induced by immunization with an avirulent mutant of LT2, irrespective of the structural components of the cell wall lipopolysaccharides of the mutants, which are basically correlated with their immunogenicity and virulence. Activated macrophages may have a direct role in the induction of host resistance, since peritoneal macrophages from FTS-treated DBA/2 mice which are resistant to the doses of LT2 used could by themselves retard bacterial growth in vitro, whereas those from CBA/N mice which are relatively susceptible to LT2 did not inhibit bacterial growth without intervention of FTS-treated syngeneic T cells.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.