Biomedical subjects
Publications and source records attributed to P Boutibonnes.
The complete nucleotide sequence of rpoD gene from Lactococcus lactis has been determined. The nucleotide data have indicated the presence of an open reading frame of 1020 base pairs encoding a polypeptide which shares the framework structure for principal sigma factors of eubacteria strains.
We have characterized the general properties of the heat shock response of the Gram-positive hardy bacterium Enterococcus faecalis. The heat resistance (60 degrees C or 62.5 degrees C, 30 min) of log phase cells of E. faecalis grown at 37 degrees C was enhanced by exposing cells to a prior heat shock at 45 degrees C or 50 degrees C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72kDa) and GroEL (63kDa) heat shock proteins of Escherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La) E. coli proteins and to the Bacillus subtilis sigma 43 factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.
With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of an rpoD-like gene of Lactococcus lactis subsp. lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical "RpoD box" of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs in Pediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.
Exposure of Lactococcus lactis subsp. lactis cells to a heat shock at 40 degrees C for 30 min induces thermotolerance, the increased ability of bacterial cells to survive exposure to lethal temperature (52 degrees C for 25 min). This transient state of thermal resistance is accompanied, as in Escherichia coli, by the synthesis of a new set of specific proteins termed heat-shock proteins (Hsps). Pre-treatment of the bacterial cells by antibiotics (streptomycin, spiramycin, kanamycin and erythromycin) known to act on translation, induces the major Hsps synthesis but no thermal protection; conversely, puromycin and amino acid analogues treatments, known to produce abnormal and incomplete peptides, triggers the thermotolerance state without inducing significant Hsps synthesis. These results demonstrate that heat-shock response and induced thermotolerance are not tightly correlated phenomena in L. lactis subsp. lactis.
In this preliminary work, the heat shock response of lactic acid bacteria was investigated and characterized. Log-phase Lactococcus lactis cells pre-incubated at 40 degrees C before heat challenge at 52 degrees C for 30 min demonstrated increased thermotolerance as compared with cells pre-incubated at 30 degrees C. The response persisted for at least 60 min. Additionally, we demonstrated that: (i) the physiological expression of the heat shock response is temperature dependent; (ii) ethanol 4.0% (v/v) caused, to a lesser extent, a response similar to the heat shock; and (iii) hydrogen peroxide failed to induce a detectable response. Furthermore, we suggest that the induction of the heat shock response increases the resistance of a lysogenic strain of L. lactis, treated by mitomycin C (1.25 micrograms/ml), to lysis by the bacteriophage.
Pretreatment of cells of Lactococcus lactis subsp. lactis with low levels of N-methyl-N'-nitro-N-nitrosoguanidine does not reduce the cytotoxic and mutagenic effects caused by high concentration of this agent. This observation indicates that there is no efficient inducible error-free repair system for alkylation damage similar to the 'adaptive response' described in detail for Escherichia coli.
The lethal and mutagenic effects of various mutagens on three strains of Streptococcus lactis were investigated. Lethality studies demonstrated that S. lactis was relatively sensitive to UV irradiation, methyl methanesulphonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and, to a lesser extent, to ethyl methanesulphonate (EMS). A spontaneous derivative Lac-, which has lost a 37-Md plasmid, was slightly more resistant and much less mutable than the wild-type after UV irradiation. Although the three strains were strongly mutated by EMS for the genetic marker assayed (Rifr), an increase in the mutation frequency was also observed after MMS and MNNG treatments.
The genotoxic activity of 11 mycotoxins was investigated in Escherichia coli K 12. The induction of the SOS function sfi A whose level of expression is monitored by means of a sfi A::lac Z operon fusion was assayed by measuring the beta-galactosidase activity in the PQ 37 strain. Most of these fungal metabolites did not induce SOS response in this bacterial test. Only aflatoxicol, a reduced metabolite of aflatoxin B1 was well detected as an SOS inducer if metabolic activation was performed. Patulin, penicillic acid and viomellein are only weak inducing agents. The other fungal compounds tested failed to demonstrate a positive SOS inducing activity. Relationship between SOS chromotest, mutagenicity to Salmonella typhimurium and in vivo carcinogenicity was discussed.
Bacillus thuringiensis is shown to have an inducible error-free repair system for alkylation damage as found in Escherichia coli and Bacillus subtilis. Growth of cells in the presence of low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces an adaptive response which is characterized by an increase in resistance to killing and mutagenesis by challenge with higher concentrations of MNNG. In addition, we have noted with interest that adaptive low doses seem to produce lesions at a rate sufficient to induce an increase of mutation frequency, and inhibition of cell division. The possibility of an interaction between SOS and adaptive responses with these low doses of MNNG is discussed.
Weigle reactivation of ultraviolet-irradiated luminal diameter 8 bacteriophage was observed after ultraviolet treatment of Bacillus thuringiensis cells. A slight increased frequency of clear plaque mutants was detected among the survivors. The kinetics of induction of the phage reactivation and phage mutagenesis have been determined. The presence of chloramphenicol before and after irradiation abolished the induction of repair and mutagenesis. These experiments suggest that, in spite of the relatively small mutagenic response in bacteriophage progeny, B. thuringiensis has an inducible repair system responsible to the significant Weigle reactivation of irradiated phage.
The lethal and mutagenic effects of u.v. light and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on Bacillus thuringiensis were investigated. Lethality studies demonstrated that B. thuringiensis was relatively sensitive to these agents. This bacterium was mutated at the rifampicin resistance marker by u.v. light and to a lesser extent by the direct acting alkylating agent MNNG. One mutant selected for its greater sensitivity to u.v. light expressed a higher frequency of mutagenesis after u.v. light treatment and appeared to be defective in an excision repair pathway. However, this mutant was only slightly mutable by MNNG in comparison with the wild-type strain. This unusual phenotype does not yet have a parallel among the radiation sensitive mutants described in other bacterial species.
Cultures of the lysogenic strain of Bacillus thuringiensis var. tolworthi were made in the presence of various drugs. The determination of bacterial size and plaque forming units (by using an indicator strain of B. thuringiensis var. galleriae) as well as colony forming units were then performed. Treatment of lysogenic cells by aflatoxin B1: provokes the formation of elongated cells (filamentation); induces a pathway that leads to the induction of prophage. Results of the present study indicated that filament formation and bacteriophage induction are two commonplace effects that occur in virtually every member of this cellular population exposed to low doses of certain drugs such as aflatoxin B1 (10 micrograms/ml); all of which have in common the ability to produce damaging changes in DNA. The following findings support the hypothesis that error-prone repair mechanisms seem to be present in B. thuringiensis as in Escherichia coli.
Comparison between about 80 strains of Aspergillus flavus, belonging to the series flavus and oryzae, obtained from international collections but also isolated from French or African substrates revealed the following observations: 1. Cultural and morphological characteristics of toxicogenic and atoxicogenic strains of A. flavus are similar. However, the former produce a diffusible yellow pigment in 83% of isolates. 2. The two groups of conidiospores have the same resistance to UV irradiation (254 nm, 5 and 10 min). All the strains are equally sensitive to 4 antifungal antibiotics: nystatine, ketoconazole, clotrimazole and amphotericine. 3. A difference was seen in the capacity to produce enzymes as alpha-galactosidase, beta-galactosidase and beta-glucosidase, implicated in the glucid metabolism. The specific hydrolytic activity has been confirmed by the characterization of a large amount of beta-galactosidase and by a diauxic growth on glucose medium supplemented by lactose. Possible relationship between these characters and aflatoxin B1 production by A. flavus strains is discussed.
Most of the 33 fungal metabolites tested provoke: Bacterial growth inhibition of Bacillus thuringiensis similar to lethal effect of antibiotics. Positive response in the 'Rec' assay using strains of Bacillus subtilis; this fact shows that these toxins are DNA modifying agents. Enlargement of cell volume in the first bacteria species; this cell-abnormality induction resembles those obtained with mitomycin C. Correlation between elongation of cells (filamentation) and in vivo carcinogenicity of mycotoxins is discussed. The filamentation should be an expression of a perturbated DNA replication (S.O.S.-error prone repair) as the consequence of DNA damages induced by genotoxic agents (i.e. carcinogens).
Zearalenone (F-2 toxin) an estrogenic mycotoxin secreted by some species of Fusarium exhibits an antibacterial activity which can be easily demonstrated in Gram-positive spore forming bacteria, especially in Bacillus sp. Morphologically abnormal cells were produced by Bacillus thuringiensis (Berliner) in response to zearalenone. The most significant ultrastructural alterations obtained with the mycotoxin at 2.5 or 5.0 micrograms/ml, are: increase of number of mesosomes which are hypertrophied, decrease of ribosomes, formation of imperfect cell septa and unknown 'crystalloïd inclusion'. These effects which provoke induction of atypical long cells, resemble those obtained with one other mycotoxin which possesses as zearalenone an unsaturated lactone structure: aflatoxin B1.
Antibacterial activity of pure preparations of mycotoxins, was studied against strains of Bacillus thuringiensis (Berliner). Most of fungal metabolites used provokes: 1) Growth bacterial inhibition similar to lethal effect of antibiotics 2) Enlargment of cell volume; this cell abnormality induction resembles those obtained with mitomycin C. The bioassay of fungal toxins with B. thuringiensis can be recommended as a sensitive technique to supplement chemical characterization. By using specific cell lines to such a mycotoxin and specific inactivators as coumarin for aflatoxin, or sulfhydryl compounds for penicillic acid and patulin.
Zearalenone, a mycotoxin secreted by Fusarium sp. and Gibberella zeae, shows a narrow range of antibacterial activity limited to some Gram-positive aerobic spore-forming bacteria. In Bacillus thuringiensis (Berliner), a highly sensitive species, this activity is characterized by a decrease of cellular division and induction of atypical cells. These effects resemble those obtained with two other mycotoxins which possess a lactone structure: aflatoxin B1 and patulin.
The antibiotic activity of these two strains of S. albus is facilitated by culture on slices of Potatoes. Streptomyces extracts show an antibiotic activity against Gram-positive and Gram-negative bacteria and an antifongic activity aginst nearly all Aspergillus. These antimicrobial effects bring hope of a utilization against some mycotoxin pollution, or mold attacks.