RESUMO

Inorganic phosphate (Pi) concentration modulates polyphosphate (polyP) levels in diverse bacteria, affecting their physiology and survival. Lactiplantibacillus paraplantarum CRL 1905 is a lactic acid bacterium isolated from quinoa sourdough with biotechnological potential as starter, for initiating fermentation processes in food, and as antimicrobial-producing organism. The aim of this work was to evaluate the influence of the environmental Pi concentration on different physiological and molecular aspects of the CRL 1905 strain. Cells grown in a chemically defined medium containing high Pi (CDM + P) maintained elevated polyP levels up to late stationary phase and showed an enhanced bacterial survival and tolerance to oxidative stress. In Pi sufficiency condition (CDM-P), cells were ~ 25% longer than those grown in CDM + P, presented membrane vesicles and a ~ 3-fold higher capacity to form biofilm. Proteomic analysis indicated that proteins involved in the "carbohydrate transport and metabolism" and "energy production and conversion" categories were up-regulated in high Pi stationary phase cells, implying an active metabolism in this condition. On the other hand, stress-related chaperones and enzymes involved in cell surface modification were up-regulated in the CDM-P medium. Our results provide new insights to understand the CRL 1905 adaptations in response to differential Pi conditions. The adjustment of environmental Pi concentration constitutes a simple strategy to improve the cellular fitness of L. paraplantarum CRL 1905, which would benefit its potential as a microbial cell factory.

RESUMO

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes.

RESUMO

In this work, the thermotolerance of Lactobacillus rhamnosus CRL1505, an immunobiotic strain, was studied as a way to improve the tolerance of the strain to industrial processes involving heat stress. The strain displayed a high intrinsic thermotolerance (55°C, 20 min); however, after 5 min at 60°C in phosphate buffer a two log units decrease in cell viability was observed. Different heat shock media were tested to improve the cell survival. Best results were obtained in the mediumcontaining inorganic salts (KH2PO4, Na2HPO4, MnSO4, and MgSO4) likely as using 10% skim milk. Flow cytometry analysis evinced 25.0% live cells and a large number of injured cells (59.7%) in the inorganic salts medium after heat stress. The morphological changes caused by temperature were visualized by transmission electronic microscopy (TEM). In addition, TEM observations revealed the presence of polyphosphate (polyP) granules in the cells under no-stress conditions. A DAPI-based fluorescence technique, adjusted to Gram-positive bacteria for the first time, was used to determine intracellular polyP levels. Results obtained suggest that the high initial polyP content in L. rhamnosus CRL 1505 together with the presence of inorganic salts in the heat shock medium improve the tolerance of the cells to heat shock. To our knowledge, this is the first report giving evidence of the relationship between polyP and inorganic salts in thermotolerance of lactic acid bacteria.

Assuntos

RESUMO

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk(-)ppx(-) or ppk(-)ppx(-)/ppk(+) strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.

Assuntos