Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Effect of Exposure to Air on 84 strains of Bifidobacteria

Among 84 strains of bifidobacteria tested, 62%, belonging to animal species, decreased in count by less than one log 10 unit after 4 days of contact with air at a temperature of 20°C. When 10 strains each of B. pseudolongum subsp. pseudolongum and B. thermophilum were tested at 4°C under the same conditions, eight of the former and five of the latter survived without any decline during 4 days.     

Sensitivity to oxidative damage of two Deinococcus radiodurans strains

The action of reactive oxygen species, i.e. hydrogen peroxide, cupric sulphate and ascorbic acid, added at different concentrations to culture media, has been studied in two strains of Deinococcus radiodurans (red-pigmented parental and colourless mutant strains) in relation to their defense antioxidant systems. While the pigmented bacteria were more resistant to elevated concentrations of the different oxidants, the colourless bacteria were more sensitive and their sensitivity was dose-dependent. Reactive oxygen species induced oxidative damage, particularly to the polyethylenic fatty acids, which were more abundant in the mutant strain. Similarly, a significant increase in lipid peroxide levels was observed, whatever the chemical added during the growth of the mutant bacteria. The parental strain required high concentrations of oxidants to shorten its survival. Vitamins A and E, carotenoids and enzymes, largely present in the parental strain, could be responsible for its higher resistance to the lethal effects of radicals generated within the cells.     

Sensitivity of various escherichia coli strains to 2,4,6-trinitrotoluene

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100-200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.     

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. Methods and Results: Phage–host interactions between eight EPS‐producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (φYsca, φ3, φ5, φ6, φ8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular‐producing strains were more sensitive to phage attacks than the noncapsular‐producing strains. Streptococcus thermophilus CRL1190 (capsular‐producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular‐negative, EPS low‐producing mutant of strain CRL1190 was reduced, especially for φYcsa and φ8. Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. Significance and Impact of the Study: Capsular‐producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.     

Sensitivity to antibiotics of Clostridium difficile toxigenic nosocomial strains

Clostridium difficile is the etiological agent of diarrhoea and colitis, especially in elderly patients. The incidence of these diseases has increased during the last 10 years. Emergence of so-called hypervirulent strains is considered as one of the main factors responsible for the more severe disease and changed profile of sensitivity to antimicrobial agents. The aim of this work was to determine the sensitivity profile of toxigenic strains of C. difficile in the Czech Republic in 2011–2012 to selected antibiotics. The antibiotics clindamycin, metronidazole, vancomycin and amoxicillin with clavulanic acid were used for this purpose. Isolates cultured on Brazier's C. difficile selective agar were analysed for the presence of toxin genes using Xpert detection system. Xpert analysis revealed that 33 strains carried the genes for toxins tcdB, cdt and tcdCΔ117, thus showing characteristics typical for the hypervirulent ribotype 027/PFGE type NAP1/REA type B1. The remaining 29 strains carried only the gene for toxin B (tcdB) and not cdt and tcdCΔ117. Our results indicate the higher susceptibility of C. difficile hypertoxigenic strains to three out of four tested antibiotics (except vancomycin) than it is for the other toxigenic strains. We found that only 10.34 % of other toxigenic strains were resistant to clindamycin, and no resistance was found in all other cases. All the isolates were sensitive to amoxicillin/clavulanic acid in vitro. However, its use is not recommended for therapy of infections caused by C. difficile.     

Sensitivity of DNA-repair-deficient strains of Escherichia coli to rifampicin killing

We have analyzed the role of RNA polymerase in DNA repair using the antibiotic rifampicin which binds specifically to the beta subunit of the enzyme. Several DNA-repair-deficient strains such as recA, uvr, and polA, and their isogenic parents were used for this study. All repair-deficient strains were found to be hypersensitive to rifampicin killing. Compared to the isogenic parent strains, recA strains are about 50 times more sensitive and the polA strain is about 100 times more sensitive to rifampicin killing. UvrA and uvrB strains are slightly more sensitive to rifampicin than the wild-type strains. The hypersensitivity of repair-deficient strains to rifampicin killing is totally abolished by the introduction of rifampicin-resistant mutations into these strains. We have examined the effect of rifampicin on RNA and protein synthesis in repair-deficient and -proficient strains. RNA and protein synthesis were found to be inhibited by rifampicin to the same extent among all the strains tested. The results also show that the resumption of DNA synthesis was significantly disrupted in DNA-repair-deficient strains following drug removal. Taken together these results suggest that RNA polymerase plays an essential role in DNA metabolism and such function may be replaced by polA and recA gene products and to a lesser extend by uvrA and uvrB gene products.