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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.

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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.

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The behaviour and state of the envelopes from B. subtilis cultures grown in Luria Bertani (LB) medium with and without 1.5 M NaCl are compared. Under hypertonic conditions, the hydrophobicity of the cultures increases. The phospholipid and fatty acid (FA) compositions show important differences: a higher cardiolipin (CL) content [at the expense of phosphatidylglycerol (PG)], and a higher unsaturated and straight chain FA content. The fluidity of the membranes, determined with fluorescent probes, indicates an increase in viscosity of the cytoplasmic membrane. The consequences of these variations in membrane permeability and osmotolerance are discussed.

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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.

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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.

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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.

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In Bacillus subtilis, osmotolerance is a stationary phase-dependent, adaptive response inhibiting sporulation and sharing common regulators with this process. The extent of this inhibition was determined by measuring transcription activity of promoter lacZ fusions to early sigma genes (spoIIG and spoIIA coding for precursors of sigmaE and sigmaF) and to reporters of them (spoIID, spoIIQ and spoIIIG), in the absence and presence of 0.6 M or 1 M NaCl. The transcription activity of these sigma precursors, normally occurring at the onset of the stationary phase, was reduced to 30-50% of their maximal expression in hyperosmotic conditions; expression of genes under their control was, however, more inhibited (<10%). Therefore, sporulation was blocked at the sigma sigmaE and sigmaF activation steps. This assumption was confirmed by electron microscopic examinations of hyperosmotic cultures, which presented asymmetric septa characteristic of stage II mutants. Discussion was focused on the particular composition and/or structure of membranes during hyperosmotic growth and their involvement in the arrest of sporulation.

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The envelope properties of B. subtilis cultures grown in LB and LBN hyperosmotic media (LB + 1.5 M NaCl) were compared. Since hypertonic cultures showed a Spo-phenotype, a Spo-mutant grown in LB was also analyzed. LBN cultures showed extensive filamentation and presented different sensitivities toward phage infection (phi29 and phi105), or antibiotics whose targets are at wall (lysozyme, penicillin G) or membrane level (polymyxin B, phosphonomycin). Results of the biochemical composition revealed that during hyperosmotic growth, the cell wall increased in thickness, and among the membrane lipids, glycolipid and cardiolipin increased in parallel with a decrease in phosphatidylglycerol. The fatty acid composition was also modified, and an increase in saturated straight chain with a decrease of saturated iso-branched fatty acids was observed. The increase of monounsaturated 18-1 (omega-9) fatty acid was probably related to the absence of sporulation observed in hypertonic media, since its increase has been shown to inhibit the KinA sensor of sporulation. The significance of the other wall and membrane composition variations (and hydrophobic surface properties) in relation to the osmotic adaptation are discussed.

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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.

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Este trabajo se realizó con el objetivo de estudiar las posibles ventajas de utilizar cepas de Bacillus megaterium como huésped en la producción de bio-insecticidas, siendo las cepas naturales de Bacillus thuringiensis poco estables en campo, especialmente en las aguas donde son necesarias en la lucha contra larvas de mosquitos. Gracias a la producción de inclusiones lipídicas de poli-Beta-hidroxi-butirato o PHB los cultivos B. megaterium aumentan la flotabilidad, razón por la cual se eligió estudiar la viabilidad de cepas tanto PHB+ como PHB-. Cultivos de distintos estadios fueron sometidos a dilución 1/100 en aguas y después de incubar 24 a 48h a 32ºC, se determinó viabilidad y formación de esporas. La dilución fue en agua (W) o en aguas iso-tónicas como solución fisiológica (S) o agua dulce artificial (ADA) a fin de comparar los efectos de un doble estrés, nutricional e hipo-osmótico, al de un simpl

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Este trabajo se realizó con el objetivo de estudiar las posibles ventajas de utilizar cepas de Bacillus megaterium como huésped en la producción de bio-insecticidas, siendo las cepas naturales de Bacillus thuringiensis poco estables en campo, especialmente en las aguas donde son necesarias en la lucha contra larvas de mosquitos. Gracias a la producción de inclusiones lipídicas de poli-Beta-hidroxi-butirato o PHB los cultivos B. megaterium aumentan la flotabilidad, razón por la cual se eligió estudiar la viabilidad de cepas tanto PHB+ como PHB-. Cultivos de distintos estadios fueron sometidos a dilución 1/100 en aguas y después de incubar 24 a 48h a 32§C, se determinó viabilidad y formación de esporas. La dilución fue en agua (W) o en aguas iso-tónicas como solución fisiológica (S) o agua dulce artificial (ADA) a fin de comparar los efectos de un doble estrés, nutricional e hipo-osmótico, al de un simple estrés nutricional. La mutante PHB mostró ser extremadamente sensible al ensayo en agua perdiendo viabilidad (<0,1 o/o), mientras que la cepa salvaje se duplica 5 veces y esporula. Sin embargo la cepa mutante no mostró tal sensibilidad cuando el estrés es sólo de nutrientes (diluciones en S o ADA). Se discuten las ventajas de utilizar B. megaterium como húésped para la producción de bioinsecticidas destinados a ambientes acuáticos, en lugar de las cepas naturales

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Cultures of Bacillus megaterium strains, producers or not of poly-beta-hydroxy-butyrate (PHB+ and PHB-) were submitted to several shift-downs: nutritional (one hundred fold dilution in saline water S or artificial fresh water ADA) or nutritional and osmotic (one hundred fold dilution in water or W). In all conditions tested, the wild type strain survived, duplicated five times and sporulated. However, the PHB- mutant strain showed a drastic loss of viability in water (< 0.1%) not observed when the shift was only nutritional (S or ADA). Discussion was focused on the advantages of the potential use of Bacillus megaterium as host for delivering bio-insecticides in waters instead of natural hosts such as B. thuringiensis strains.

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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).

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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.

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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.

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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.

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Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.

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