Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

BACKGROUND: The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. RESULTS: A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE) and the type II NADH dehydrogenases (NDH-2). In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II)-reductase activity is detected in a specific subset of NDH-2. CONCLUSION: The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2

Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration

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

Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level▿

We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in <em>Escherichia coli</em>

<div><p>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 <em>Escherichia coli</em> 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 <em>E. coli</em>. 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 <em>ppk⁻ppx⁻</em> or <em>ppk⁻ppx⁻</em>/<em>ppk⁺</em> strains, deficient in polyP synthesis and hydrolysis, respectively. <em>luxS</em> and <em>lsrK</em> 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.</p> </div

Lemon maturation causes anatomical and biochemical changes at the flavedo tissue level

Plants mobilize the photosynthates by three transport pathways: apoplastic, symplastic through plasmodesmata (PD), and transcellular. In flavedo of postharvest mature lemons, a high activity of cell wall-bound invertase (WI), an enzyme associated with transcellular transport of monosaccharides, has been detected. In order to elucidate whether this high enzymatic activity is related to restricted transport in the symplastic pathway with fruit maturation, the aim of the present work was to compare anatomical and biochemical parameters in peel tissues of immature and mature lemons. Anatomical structure focusing on cell walls, callose deposition, WI activity, and sucrose content were analyzed in peel tissues of immature and mature lemons. The parenchyma of flavedo tissue of immature lemons presented an elevated number of primary pit fields (PPF). These PPF, associated to PD or cell wall interruptions, had the appearance of a string of beads. However, in mature lemons, the number of PPF was scarce due to callose deposition. WI activity and apoplastic sucrose content increased significantly in flavedo of mature lemons in comparison to immature lemons. Present findings lay structural and functional bases relevant to understand differences between immature and mature lemons, which would help to design agricultural practices in pre- and post-harvest management.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Inorganic salts and intracellular polyphosphate inclusions play a role in the thermotolerance of the immunobiotic Lactobacillus rhamnosus CRL 1505.

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

Biofilm formation after reinoculation in CM.

*<p>Biofilm formation was determined 24 h after the cells transference to CM. 48 h MT cultures were used as control. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Biofilm formation by <i>luxS</i> mutant in conditioned media.

<p><i>luxS⁻</i> cells growing in MT medium during 24 h were reinoculated in wild type MT CM or MT+P CM and in <i>ppk⁻ppx⁻</i> MT CM from different growth times, as indicated. Biofilm formation was determined 24 h after the shift to CM. 48 h MT cultures were used as control (–). Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

Biofilm formation after changes of phosphate concentration in stationary phase.

<p>Biofilm formation by MC4100 was measured at indicated times of growth in MT and in MT with the addition of 40 mM phosphate buffer pH 7 at 24 h (MT+24P) or at 48 h (MT+48P) (<b>left panel</b>), or in MT+P and in MT+P culture switched to fresh MT at 24 h (MT+P→24MT) (<b>right panel</b>). Result represents the mean ± SD of at least three independent experiments performed in triplicate. Different letters indicate significant differences according to Tukey's test with a <i>p</i>-value of 0.05.</p

PolyP levels and biofilm formation in different Pi concentrations media.

<p>The biofilm amount (white bars) and DAPI-polyP fluorescence (black dots) were determined at 48 h in the indicated <i>E. coli</i> strains grown in static conditions in MT medium modified with the indicated Pi concentrations. Data represent the mean ± SD of at least three independent experiments.</p