Identification of ColR binding consensus and prediction of regulon of ColRS two-component system

<p>Abstract</p> <p>Background</p> <p>Conserved two-component system ColRS of <it>Pseudomonas </it>genus has been implicated in several unrelated phenotypes. For instance, deficiency of <it>P. putida </it>ColRS system results in lowered phenol tolerance, hindrance of transposition of Tn<it>4652 </it>and lysis of a subpopulation of glucose-grown bacteria. In order to discover molecular mechanisms behind these phenotypes, we focused here on identification of downstream components of ColRS signal transduction pathway.</p> <p>Results</p> <p>First, highly similar ColR binding sites were mapped upstream of outer membrane protein-encoding <it>oprQ </it>and a putative methyltransferase-encoding PP0903. These two ColR binding sequences were used as an input in computational genome-wide screening for new potential ColR recognition boxes upstream of different genes in <it>P. putida</it>. Biological relevance of a set of <it>in silico </it>predicted ColR-binding sites was analysed <it>in vivo </it>by studying the effect of ColR on transcription from promoters carrying these sites. This analysis disclosed seven novel genes of which six were positively and one negatively regulated by ColR. Interestingly, all promoters tested responded more significantly to the over-expression than to the absence of ColR suggesting that either ColR is limiting or ColS-activating signal is low under the conditions applied. The binding sites of ColR in the promoters analysed were validated by gel mobility shift and/or DNase I footprinting assays. ColR binding consensus was defined according to seven ColR binding motifs mapped by DNase I protection assay and this consensus was used to predict minimal regulon of ColRS system.</p> <p>Conclusion</p> <p>Combined usage of experimental and computational approach enabled us to define the binding consensus for response regulator ColR and to discover several new ColR-regulated genes. For instance, genes of outer membrane lipid A 3-O-deacylase PagL and cytoplasmic membrane diacylglycerol kinase DgkA are the members of ColR regulon. Furthermore, over 40 genes were predicted to be putatively controlled by ColRS two-component system in <it>P. putida</it>. It is notable that many of ColR-regulated genes encode membrane-related products thus confirming the previously proposed role of ColRS system in regulation of membrane functionality.</p

The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

<p>Abstract</p> <p>Background</p> <p>We have recently found that <it>Pseudomonas putida </it>deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the <it>colR</it>-deficient <it>P. putida</it>.</p> <p>Results</p> <p>By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of <it>colR</it>-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the <it>ttgABC </it>operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the <it>colR </it>mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the <it>colR</it>-deficient <it>P. putida </it>are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type <it>P. putida </it>as well. Comparison of phenol tolerance of growing <it>versus </it>starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the <it>ttgC- </it>and <it>colRttgC-</it>deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the <it>colR </it>mutant, respectively. However, cell division of <it>P. putida </it>with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the <it>colR</it>-mutant strain than in the wild-type <it>P. putida</it>.</p> <p>Conclusions</p> <p>ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of <it>P. putida</it>. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.</p

Unchaining miniBacillus PG10:Relief of FlgM-mediated repression of autolysin genes

Cell chaining in Bacillus subtilis is naturally observed in a subset of cells during exponential growth and during biofilm formation. However, the recently constructed large-scale genome-minimized B. subtilis strain PG10 displays a severe and permanent defect in cell separation, as it exclusively grows in the form of long filaments of nonseparated cells. In this study, we investigated the underlying mechanisms responsible for the incomplete cell division of PG10 by genomic and transcriptomic analyses. Repression of the SigD regulon, including the major autolysin gene lytF, was identified as the cause for the cell separation problem of PG10. It appeared that SigD-regulated genes are downregulated in PG10 due to the absence of the flagellar export apparatus, which normally is responsible for secretion of FlgM, the anti-sigma factor of SigD. Although mild negative effects on growth and cell morphology were observed, deletion of flgM could revert the aberrant cell-chaining phenotype and increased transformation efficiency. Interestingly, our work also demonstrates the occurrence of increased antisense transcription of slrR, a transcriptional repressor of autolysin genes, in PG10 and provides further understanding for this observation. In addition to revealing the molecular basis of the cell separation defect in PG10, our work provides novel targets for subsequent genome reduction efforts and future directions for further optimization of miniBacillus PG10. IMPORTANCE Reduction of the size of bacterial genomes is relevant for understanding the minimal requirements for cellular life as well as from a biotechnological point of view. Although the genome-minimized Bacillus subtilis strain PG10 displays several beneficial traits as a microbial cell factory compared to its parental strain, a defect at the final stage of cell division was introduced during the genome reduction process. By genetic and transcriptomic analyses, we identified the underlying reasons for the cell separation problem of PG10. In addition to enabling PG10 to grow in a way similar to that of B. subtilis wild-type strains, our work points toward subsequent targets for fine-tuning and further reduction of the genome of PG10. Moreover, solving the cell separation defect facilitates laboratory handling of PG10 by increasing the transformation efficiency, among other means. Overall, our work contributes to understanding and improving biotechnologically attractive minimal bacterial cell factories

Quorum sensing in Pseudomonas savastanoi pv. savastanoi and Erwinia toletana: role in virulence and interspecies interactions in the olive knot

The olive-knot disease (Olea europea L.) is caused by the bacterium Pseudomonas savastanoi pv. savastanoi (PSV). PSV in the olive-knot undergoes interspecies interactions with the harmless endophyte Erwina toletana (ET); PSV and ET co-localize and form a stable community resulting in a more aggressive disease. PSV and ET produce the same type of the N-acylhomoserine lactone (AHL) quorum sensing (QS) signal and they share AHLs in planta. In this work we have further studied the AHL QS systems of PSV and ET in order to determine possible molecular mechanism(s) involved in this bacterial inter-species interaction/cooperation. The AHL QS regulons of PSV and ET were determined allowing the identification of several QS-regulated genes. Surprisingly, the PSV QS regulon consisted of only a few loci whereas in ET many putative metabolic genes were regulated by QS among which several involved in carbohydrate metabolism. One of these loci was the aldolase-encoding gene garL, which resulted to be essential for both co-localization of PSV and ET cells inside olive knots as well as knot development. This study further highlighted that pathogens can cooperate with commensal members of the plant microbiome. SIGNIFICANCE OF THIS STUDY: This is a report on studies of the quorum sensing (QS) systems of olive knot pathogen Pseudomonas savastanoi pv. savastanoi and olive-knot cooperator Erwinia toletana. These two bacterial species form a stable community in the olive knot, share QS signals and cooperate resulting in a more aggressive disease. In this work we further studied the QS systems by determining their regulons as well studying QS-regulated genes which might play a role in this cooperation. This represents a unique in vivo interspecies bacterial virulence model and highlights the importance of bacterial interspecies interaction in disease

The Effect of Cellular Redox Status on the Evolvability of New Catabolic Pathways

Oxidation of aromatic compounds can be mutagenic due to the accumulation of reactive oxygen species (ROS) in bacterial cells and thereby facilitate evolution of corresponding catabolic pathways. To examine the effect of the background biochemical network on the evolvability of environmental bacteria hosting a new catabolic pathway, Akkaya and colleagues (mBio 9:e01512-18, 2018, https://doi.org/10.1128/mBio.01512-18) introduced the still-evolving 2,4-dinitrotoluene (2,4-DNT) pathway genes from the original environmental Burkholderia sp.Oxidation of aromatic compounds can be mutagenic due to the accumulation of reactive oxygen species (ROS) in bacterial cells and thereby facilitate evolution of corresponding catabolic pathways. To examine the effect of the background biochemical network on the evolvability of environmental bacteria hosting a new catabolic pathway, Akkaya and colleagues (mBio 9:e01512-18, 2018, https://doi.org/10.1128/mBio.01512-18) introduced the still-evolving 2,4-dinitrotoluene (2,4-DNT) pathway genes from the original environmental Burkholderia sp. isolate into the genome of Pseudomonas putida KT2440. They show that the mutagenic effect of 2,4-DNT oxidation, which is associated with the accumulation of ROS and oxidative damage on DNA, can be avoided by preserving high NADPH levels in P. putida. The observations of this study highlight the impact of the cellular redox status of bacteria on the evolvability of new metabolic pathways

Fluorescent Lead(IV) sulfide nanoparticles synthesized by Idiomarina sp. strain PR58-8 for bioimaging applications

The fabrication of nanoparticles by microorganisms presents a “green” method for generating biocompatible nanomaterials. We discovered the intracellular biosynthesis of fluorescent lead(IV) sulfide nanoparticles by the moderate halophile, Idiomarina sp. strain PR58-8. The bacterium tolerated up to 8 mM Pb(NO3)2 during growth. Non-protein thiols dose-dependently increased in response to metal exposure, which suggests they are involved in the growth of PbS2 crystals and lead detoxification. Using X-ray diffraction, transmission electron microscopy (TEM), high-resolution TEM, and energy dispersive analysis of X-rays, the nanoparticles were characterized as spherical β-PbS2 nanoparticles (PbS2NPs) with a tetragonal crystal lattice, a crystallite domain size of 2.38 nm, and an interplanar distance of 0.318 nm. A narrow symmetric emission spectrum with a Gaussian distribution and an emission maximum at 386 nm was obtained when the particles were excited at 570 nm. The PbS2NPs exhibited a large Stokes' shift (8,362 cm−1) and a relatively high quantum yield (67%). These properties, along with fluorescence that was maintained in various microenvironments and their biocompatibility, make these nanoparticles excellent candidates for bioimaging. The particles were internalized by HeLa cells and evenly distributed within the cytoplasm, exhibiting their potential for in situ bioimaging applications. The “as-synthesized” lead(IV) sulfide nanoparticles may provide expanded opportunities for targeted bioimaging via modifying the surface of the particles. IMPORTANCE This article reports the intracellular synthesis of fluorescent lead(IV) sulfide nanoparticles (PbS2NPs) by a microorganism. All previous reports on the microbial synthesis of lead-based nanoparticles are on lead(II) sulfide that exhibits near-infrared fluorescence, requiring expensive instrumentation for bioimaging. Bioimaging using PbS2NPs can be achieved using routine epifluorescence microscopes, as it fluoresces in the visible range. The research on PbS2 nanoparticles to date is on their chemical synthesis employing toxic precursors, extreme pH, pressure, and temperature, resulting in cytotoxic products. In this context, the synthesis of PbS2 nanoparticles by Idiomarina sp. strain PR58-8, described in this work, occurs at ambient temperature and pressure and results in the generation of biocompatible nanoparticles with no hazardous by-products. The excellent fluorescence properties that these particles exhibit, as well as their abilities to easily penetrate the cells and evenly distribute within the cytoplasm, make them exceptional candidates for bioimaging applications. This study demonstrated the synthesis and fluorescence bioimaging application of microbially synthesized PbS2 nanoparticles