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C Sanchez-Rivas

Publications and source records attributed to C Sanchez-Rivas.

At least 19 recordsLinked to original sources
PubMed2005

Cell wall modifications during osmotic stress in Lactobacillus casei.

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.

Bacteriological Techniques↗
PubMed2003

Adaptation to high salt in Lactobacillus: role of peptides and proteolytic enzymes.

AIMS: To study the influence of peptides and proteolytic enzymes in the osmotic adaptation of Lactobacillus casei. METHODS AND RESULTS: Di- and tri-peptides added individually increased the osmotolerance of Lact. casei when grown in a chemically defined medium (CDM) containing NaCl. Growth stimulation and the re-establishment in their presence of plasmid DNA supercoiling (recovery of the linking number) in hyperosmotic medium indicated that they are used as osmocompatible solutes as carnithine a known osmoprotector does. The investigation of the proteolytic system showed that in high osmolarity medium, the cell envelope-associated proteinase (PrtP), and PepX (X-prolyl-dipeptidyl aminopeptidase) increased activity and lost repression by peptides. PepI, an iminopeptidase was also derepressed. PepQ, a prolidase that specifically liberated proline from dipeptides, was almost unaffected. Derepression in the presence of peptides took place at the transcriptional level. However, the twofold activation of PrtP in CDM hyperosmotic medium was essentially through an increase of the apparent Vmax of the enzyme. CONCLUSIONS: These results strongly suggest a contribution of the proteolytic system peptide supply in the osmotic adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: Advances in understanding the role of peptides in the adaptation to high osmolarity particularly involved in dairy processes.

Culture Media↗
PubMed1998

Normal induction of the SOS response in Bacillus subtilis is prevented by the mutant repressor from phage phi 105cts23.

The presence of the phi 105cts23 mutant prophage in Bacillus subtilis induces a series of pleiotropic effects that could be ascribed to an anti-SOS activity. In order to circumvent the phage function responsible for this phenomenon, the cts23 mutant repressor was cloned and sequenced. The isolated repressor reduced the survival capacity of the host cells after mitomycin C or nalidixic acid treatments and lowered the spontaneous reversion frequency. When SOS induction kinetics were studied, low or null induction of the damage-inducible din22::LacZ fusion was observed. In contrast, the presence of the wild-type prophage amplified the SOS response. Sequencing of the mutant repressor revealed that the cts23 mutation is a T-->C transition affecting the 5' closest codon to one of the two reported DNA binding domains.

Artificial Gene Fusion↗
PubMed1998

In Bacillus subtilis DegU-P is a positive regulator of the osmotic response.

The osmosensitivity presented by spo0A and degU null mutant strains of Bacillus subtilis pointed to their protein products as essential regulators for the osmotic response. This was further investigated by analyzing their transcription activity. The results showed that both spo0A-lacZ and degSU-lacZ were induced by the hypertonic medium. The actual phosphorylation state of these proteins was also analyzed by the bias of two reporter gene promoters activity (abrB-lacZ and degQ-lacZ). The absence of repression of abrB in hypertonic conditions suggested that Spo0A was not phosphorylated while the derepression of degQ promoter suggested that DegU-P was formed. These results were in accordance with the observed absence of sporulation in hyperosmotic media and semi-constitutive osmotolerance of degUh mutant strains known to retain phosphorylated DegU. The failure to secrete proteases and to sporulate in hypertonic media suggested that Spo0A acts through abrB regulation in the prevention of alternate responses. The role of DegU-P as positive regulator of the osmotic response seems to be settled and is discussed.

Bacillus subtilis↗
PubMed1998

A novel antimicrobial activity of a Paenibacillus polymyxa strain isolated from regional fermented sausages.

A strain isolated from Argentinean regional fermented sausages was found to produce and secrete a compound that inhibited growth of Lactobacillus strains used as indicators. It was characterized as Paenibacillus polymyxa (P13). The antimicrobial activity, named polyxin, was obtained from culture supernatant fluid of late stationary phase and was inhibitory to actively growing cells. It was effective against a wide range of Gram-positive and Gram-negative bacterial species tested including food-borne pathogens. Bacteriocin-like properties such as proteinaceous nature (sensitive to proteases), insensitivity to organic solvents and chelators, stability to heat (up to 10 min at 90 degrees C), and acidic pH but instability in alkaline conditions, were determined. A molecular mass of 10 kDa was estimated by molecular gel filtration.

Animals↗
PubMed1997

DNA supercoiling and osmoresistance in Bacillus subtilis 168.

The importance of the DNA structure for the expression of the osmotic response (osmotolerance) was investigated in Bacillus subtilis 168. Plasmid pUB110 DNA was used as a reporter of the chromosomal DNA topology, and analyses were performed in chloroquine agarose gels. Plasmidic DNA obtained from cultures in Schaeffer medium (D) taken in those periods in which B. subtilis is able to express osmotolerance (early stationary phase or from germinating spores) or from adapted cultures to hyperosmotic medium (DN) presented a higher level of negative supercoiling than DNA samples from vegetative cultures, normally refractory to induction of osmotolerance. The involvement of the DNA gyrase was investigated through the sensitivity to novobiocin, an antibiotic inhibitor of its activity and the behavior of a gyrB1 mutant strain (RG1). In the wild-type strain, the addition of a sublethal concentration of novobiocin (0.5 microg/ml) to the hyperosmotic medium relaxed DNA and inhibited growth. Moreover, already growing cultures in DN medium and later submitted to the same antibiotic presented a relaxed DNA and stopped growing. The RG1 mutant strain submitted to similar novobiocin treatments displayed normal growth in DN novobiocin medium. These results pointed to the requirement of a highly negative supercoiled DNA structure involving the gyrase activity in osmotic response.

Anti-Bacterial Agents↗
PubMed1994

Osmoresistance of spores from Bacillus subtilis and the effect of ssp mutations.

Spores of Bacillus subtilis show similar plating efficiency on media with or without 1.5 M NaCl. In contrast, vegetative cells are osmosensitive unless the stationary phase has been reached. In the present work, loss of heat and osmotic resistance during germination was studied. Their kinetics and sensitivity to protein synthesis inhibition were different: heat resistance was lost first and even in the presence of chloramphenicol, whereas loss of osmotolerance occurred later and was inhibited in the presence of this antibiotic. The influence of spore-associated small acid-soluble proteins (SASPs) on spore osmotolerance was investigated using ssp mutants: all produced spores which germinated poorly and were sensitive to osmotic strength. SASP-E deficiency was particularly significant. Spore osmotolerance was largely restored in complementation assays performed with cloned ssp genes. It is possible that germination-associated degradation of SASP proteins provides osmotically significant levels of amino acids (especially glutamate).

Bacillus subtilis↗
PubMed1994

Physiological and genetic characterization of the osmotic stress response in Bacillus subtilis.

Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.

Adaptation, Physiological↗
PubMed1993

The spoOA and degU genes of Bacillus subtilis show genetic homology.

The transformation efficiency of competent Bacillus subtilis degU32(Hy) strains was found to depend on the marker that was selected. Prototrophic transformants were obtained at frequencies similar to those in the wild type control, but Spo- transformants were rare also when a spoOA::erm insertion that produces a selectable marker (ErmR) was used. The ErmR transformants obtained within the degU32(Hy) background were Spo+ and had lost the characteristics of the DegU(Hy) parental recipient strain i.e., secretion of exo-enzymes and sporulation resistance to catabolites. The spoOA::erm insertion was mapped to a location near degU. The similarities between the spoOA and degU sequences and the metabolic interferences between the mutated products which result in this unexpected recombination, are discussed.

Bacillus subtilis↗
PubMed1993

Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage.

The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.

Bacillus Phages↗
PubMed1988

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion.

Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chromosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp- recombinants (the most frequent type of a Leu- Met- Thr- x Ade- Ura- Trp- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura+ Met+ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.

Alleles↗
PubMed1984

Complementation and genetic inactivation: two alternative mechanisms leading to prototrophy in diploid bacterial clones.

Evidence for diploidy at loci located all around the Bacillus subtilis chromosome previously led us to refer to the prototrophic bacterial clones produced by fusion of polyauxotrophic protoplasts as complementing diploid clones (Lévi-Meyrueis et al. 1980; Sanchez-Rivas 1982). In this paper, evidence is presented that gene inactivation may occur in such clones, as judged from the unequal expression of three unselected markers and their low transforming activity in cell lysates, an established property of inactivated genes (Bohin et al. 1982). The insensitivity to protease treatment of the lysates and also the low transforming activity observed with purified DNA may indicate that chromosome inactivation does not necessarily result from the mere attachment of proteins to DNA. Cotransfer by transformation of similarly expressed genes, initially located on separate chromosomes, suggests that genetic recombination has taken place, resulting in the reassortment of active and inactive genes on separate chromosomes. Several genetic structures compatible with the observations are presented which illustrate that prototrophy may result from such reassortment as well as from functional complementation.

Bacillus subtilis↗
PubMed1983

Absence of functional RNA encoded by a silent chromosome in non-complementing diploids obtained from protoplast fusion in Bacillus subtilis.

The molecular basis for lack of phenotypic expression of one chromosome in Bacillus subtilis non-complementing diploid clones (Ncd cells) was investigated. Correlations between chromosomal inactivation and absence of functional transcripts were determined with wild-type prophage phi 105 or a thermoinducible mutant phi 105 cts23, on either the expressed or the silent chromosome. It appears that no significant amount of phage mRNA is detectable in Ncd cells carrying the prophage in the inactive chromosome. However, phi 105 mRNA represents 0.23% of total cellular mRNA in an Ncd strain with the prophage in the expressed chromosome and 0.28% in the parental lysogenic strain. The lack of an mRNA repressor of phi 105 prophage from the silent chromosome was confirmed by the absence of repressor activity in Ncd clones with a temperature sensitive mutant phi 105 located in the silent chromosome. After heat induction, no phage production was observed. As expected these clones do not exhibit phi 105 immunity when superinfected with the same phage. The combined data of the present and previous work suggest that control of phenotypic suppression of Ncd strains should, at the transcription level, involve a different DNA tertiary organisation in one of the two chromosomes.

Bacillus subtilis↗
PubMed1982

Direct selection of complementing diploids from PEG-induced fusion of Bacillus subtilis protoplasts.

A minimal medium containing horse serum is described on which Bacillus subtilis protoplasts revert to bacillary forms at high frequency (ca. 30%). Used as a plating medium for a mixture of polyethyleneglycol-treated protoplasts from two complementary polyauxotrophic parental strains, it selects the prototrophic fusion products efficiently, and also allows isolation of various auxotrophic recombinants. These prototrophs and recombinants amount respectively to 1% and 10% of the regenerated bacteria. We confirm that two types of prototrophs can be isolated after fusion: stable recombinants and complementing diploids, the latter segregating into various types of recombinants. Based on easily recognized colonial aspects, an approximate estimation of the proportion of the two types becomes possible when a spoOA mutation has been introduced in one of the parents. At least 50% of the prototrophic fusion products are complementing diploids. Incidently, the data also settle a controversy by showing the dominance of spoOA mutations in heterozygotic bacteria.

Bacillus subtilis↗
PubMed1982

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis.

After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN+ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between ø105-lysogenic and ø105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the trpF+ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.

Bacillus subtilis↗
PubMed1980

[Formation of stable diploid bacteria by fusion of protoplasts of Bacillus subtilis and the effect of rec- mutations on the fusion products formed].

A minority among the prototrophic clones formed when protoplasts from two polyauxotrophic strains of B. subtilis are fused has been shown to be diploid, carrying the parental deficient alleles in their DNA, and generally segregating auxotrophs when grown in nutrient medium. This is true whether the strains being fused are Rec+ or Rec-, since the rec- mutations used hardly affect recombinations occurring between chromosomes in diploid Bacteria.

Alleles↗