Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens

de Lucena DK, Pühler A, Weidner S. The role of sigma factor RpoH1 in the pH stress response of Sinorhizobium meliloti. BMC Microbiology. 2010;10(1): 256.Background: Environmental pH stress constitutes a limiting factor for S. meliloti survival and development. The response to acidic pH stress in S. meliloti is versatile and characterized by the differential expression of genes associated with various cellular functions. The purpose of this study was to gain detailed insight into the participation of sigma factors in the complex stress response system of S. meliloti 1021 using pH stress as an effector. Results: In vitro assessment of S meliloti wild type and sigma factor mutants provided first evidence that the sigma factor RpoH1 plays a major role in the pH stress response. Differential expression of genes related to rhizobactin biosynthesis was observed in microarray analyses performed with the rpoH1 mutant at pH 7.0. The involvement of the sigma factor RpoH1 in the regulation of S. meliloti genes upon pH stress was analyzed by comparing timecourse experiments of the wild type and the rpoH1 mutant. Three classes of S. meliloti genes could be identified, which were transcriptionally regulated in an RpoH1-independent, an RpoH1-dependent or in a complex manner. The first class of S. meliloti genes, regulated in an RpoH1-independent manner, comprises the group of the exopolysaccharide I biosynthesis genes and also the group of genes involved in motility and flagellar biosynthesis. The second class of S. meliloti genes, regulated in an RpoH1-dependent manner, is composed of genes known from heat shock studies, like ibpA, grpE and groEL5, as well as genes involved in translation like tufA and rplC. Finally, the third class of S. meliloti genes was regulated in a complex manner, which indicates that besides sigma factor RpoH1, further regulation takes place. This was found to be the case for the genes dctA, ndvA and smc01505. Conclusions: Clustering of time-course microarray data of S. meliloti wild type and sigma factor rpoH1 mutant allowed for the identification of gene clusters, each with a unique time-dependent expression pattern, as well as for the classification of genes according to their dependence on RpoH1 expression and regulation. This study provided clear evidence that the sigma factor RpoH1 plays a major role in pH stress response

Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance

Chromohalobacter salexigens, a Gammaproteobacterium belonging to the family Halomonadaceae, shows a broad salinity range for growth. Osmoprotection is achieved by the accumulation of compatible solutes either by transport (betaine, choline) or synthesis (mainly ectoine and hydroxyectoine). Ectoines can play additional roles as nutrients and, in the case of hydroxyectoine, in thermotolerance. A supplementary solute, trehalose, not present in cells grown at 37°C, is accumulated at higher temperatures, suggesting its involvement in the response to heat stress. Trehalose is also accumulated at 37°C in ectoine-deficient mutants, indicating that ectoines suppress trehalose synthesis in the wild-type strain. The genes for ectoine (ectABC) and hydroxyectoine (ectD, ectE) production are arranged in three different clusters within the C. salexigens chromosome. In order to cope with changing environment, C. salexigens regulates its cytoplasmic pool of ectoines by a number of mechanisms that we have started to elucidate. This is a highly complex process because (i) hydroxyectoine can be synthesized by other enzymes different to EctD (ii) ectoines can be catabolized to serve as nutrients, (iii) the involvement of several transcriptional regulators (σS, σ32, Fur, EctR) and hence different signal transduction pathways, and (iv) the existence of post-trancriptional control mechanisms. In this review we summarize our present knowledge on the physiology and genetics of the processes allowing C. salexigens to cope with osmotic stress and high temperature, with emphasis on the transcriptional regulation

Role of Trehalose in Salinity and Temperature Tolerance in the Model Halophilic Bacterium Chromohalobacter salexigens.

The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli

Abstract Background The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. Results Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. Conclusions Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.This research was financially supported by grants from the CYTED (AGROSEQ; 107PIC0312), Spanish Ministerio de Ciencia e Innovación (BIO2011-22833), Spanish National Network on Extremophilic Microorganisms (BIO2011-12879-E), and Junta de Andalucía (P08-CVI-03724).Peer Reviewe

New insights into hydroxyectoine synthesis and its transcriptional regulation in the broad-salt growing halophilic bacterium Chromohalobacter salexigens

Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C. salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production.Red Nacional de Microorganismos Extremofílicos RED2018-102734-

The ectD Gene, Which Is Involved in the Synthesis of the Compatible Solute Hydroxyectoine, Is Essential for Thermoprotection of the Halophilic Bacterium Chromohalobacter salexigens

The halophilic bacterium Chromohalobacter salexigens synthesizes and accumulates compatible solutes in response to salt and temperature stress. (13)C-nuclear magnetic resonance analysis of cells grown in minimal medium at the limiting temperature of 45°C revealed the presence of hydroxyectoine, ectoine, glutamate, trehalose (not present in cells grown at 37°C), and the ectoine precursor, Nγ-acetyldiaminobutyric acid. High-performance liquid chromatography analyses showed that the levels of ectoine and hydroxyectoine were maximal during the stationary phase of growth. Accumulation of hydroxyectoine was up-regulated by salinity and temperature, whereas accumulation of ectoine was up-regulated by salinity and down-regulated by temperature. The ectD gene, which is involved in the conversion of ectoine to hydroxyectoine, was isolated as part of a DNA region that also contains a gene whose product belongs to the AraC-XylS family of transcriptional activators. Orthologs of ectD were found within the sequenced genomes of members of the proteobacteria, firmicutes, and actinobacteria, and their products were grouped into the ectoine hydroxylase subfamily, which was shown to belong to the superfamily of Fe(II)- and 2-oxoglutarate-dependent oxygenases. Analysis of the ectoine and hydroxyectoine contents of an ectABC ectD mutant strain fed with 1 mM ectoine or hydroxyectoine demonstrated that ectD is required for the main ectoine hydroxylase activity in C. salexigens. Although in minimal medium at 37°C the wild-type strain grew with 0.5 to 3.0 M NaCl, with optimal growth at 1.5 M NaCl, at 45°C it could not cope with the lowest (0.75 M NaCl) or the highest (3.0 M NaCl) salinity, and it grew optimally at 2.5 M NaCl. The ectD mutation caused a growth defect at 45°C in minimal medium with 1.5 to 2.5 M NaCl, but it did not affect growth at 37°C at any salinity tested. With 2.5 M NaCl, the ectD mutant synthesized 38% (at 37°C) and 15% (at 45°C) of the hydroxyectoine produced by the wild-type strain. All of these data reveal that hydroxyectoine synthesis mediated by the ectD gene is thermoregulated and essential for thermoprotection of C. salexigens

Biosynthesis of compatible solutes in rhizobial strains isolated from <it>Phaseolus vulgaris </it>nodules in Tunisian fields

<p>Abstract</p> <p>Background</p> <p>Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species <it>Sinorhizobium meliloti</it>, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of <it>Phaseolus vulgaris </it>in Tunisia, as well as by the reference strain <it>Rhizobium tropici </it>CIAT 899^T.</p> <p>Results</p> <p>The most NaCl-tolerant strain was <it>A. tumefaciens </it>10c2, followed (in decreasing order) by <it>R. tropici </it>CIAT 899, <it>R. leguminosarum </it>bv. <it>phaseoli </it>31c3, <it>R. etli </it>12a3 and <it>R. gallicum </it>bv. <it>phaseoli </it>8a3. ¹³C- and ¹H-NMR analyses showed that all <it>Rhizobium </it>strains synthesized trehalose whereas <it>A. tumefaciens </it>10c2 synthesized mannosucrose. Glutamate synthesis was also observed in <it>R. tropici </it>CIAT 899, <it>R. leguminosarum </it>bv. <it>phaseoli </it>31c3 and <it>A. tumefaciens </it>10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in <it>R. tropici </it>CIAT 899 and of mannosucrose in <it>A. tumefaciens </it>10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the <it>otsA </it>gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. <it>In vivo </it>¹³C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the β-1,2-cyclic glucan from <it>R. tropici </it>CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined.</p> <p>Conclusions</p> <p>The soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain <it>R. tropici </it>CIAT 899.</p