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Log Analyzer 7.0b serial key or number

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A consolidated analysis of the physiologic and molecular responses induced under acid stress in the legume-symbiont model-soil bacterium Sinorhizobium meliloti.

Rhizobia are Gram-negative α- and β-proteobacteria that have the ability to fix atmospheric nitrogen in symbiotic association with legumes1,2 as well as with nonleguminous plants in a few known examples3. That capacity gives these associations a central role in the N cycle, thus making rhizobia also a subject of interest in agricultural-production systems4,5,6,7. The establishment of symbiosis in natural soils, however, is frequently limited by abiotic-stress conditions which affect that mutualistic interaction in different ways8. Thus, the acidity of soils limits nodulation and N2 fixation in many legume-rhizobia symbioses of agronomic interest9. Soil acidity is an extended edaphic condition in culturable lands over the entire globe10, and is known to be a major limiting circumstance for legume productivity. The increased concentration of hydrogen ions negatively affects at once the host plant11,12, the rhizobia13, and the symbiotic relationship14,15,16. Different stages in the interaction between rhizobia and the host roots have been reported to be affected by acidity, including the production of nodulation factors14,17,18,19,20, the attachment of rhizobia to roots14, the number of root nodules15, and the nitrogenase activity18.

Sinorhizobium meliloti and Sinorhizobium medicae are rhizobia with the ability to establish symbiosis with legumes of the genera Medicago, Melilotus, and Trigonella. These rhizobia are particularly sensitive to low pH21, growing either very slowly or not at all at pHs between 6.0–5.5 depending on the medium and the cultivation condition12,22,23. For this reason, acid tolerance has been considered a positive trait to perform well—in both survival and symbiosis—under acid conditions24. The screening for acid-tolerant alfalfa-nodulating isolates that could colonize and/or persist in acidic media and/or soils thus resulted in a selection of novel strains with enhanced saprophytic competence11 and/or improved symbiosis under moderately-acid conditions23,25,26,27,28,29. On the basis of these and other previous results, the genetic analysis of acid tolerance in rhizobia in general, and in alfalfa symbionts in particular, emerged as a potential powerful approach for a rational improvement of the rhizobial performance at low pH. As a result of the screening of Tn5-mutant libraries, several genes for acid tolerance (namely act, among others) could be identified in S. medicae—including actA30 and actR/S31,32, actP33, exoH34, and exoR9 along with some other genes that were induced at low pH, such as phrR35 and lpiA36,37. The collected genetic evidence indicates that tolerance to acidity in these rhizobia is a multigenic phenotype. Such a view is consistent with the failure to convert acid-sensitive rhizobia into acid-tolerant variants simply by the transfer of single cosmid clones generated from a more tolerant genotype (results from our laboratory). A more extensive understanding at the molecular level of the metabolism and responses of rhizobia to low pH remain essential for making an effective use of genetic manipulations in order to enhance bacterial acid tolerance.

Aiming at exploring other alternatives for improving acid tolerance in rhizobia, we attempted to induce transient phenotypes of acid tolerance. As previously demonstrated in E. coli and other bacteria38,39, an acid-tolerance response (ATR) could be induced in different rhizobia, including S. medicae40 and S. meliloti41 among others42. An increase in the tolerance to severe acid shocks was observed when rhizobia had been previously cultivated in batch under sublethal acidic conditions41,42. Tiwari et al.31 had demonstrated that the genes actSR were necessary for the induction of an ATR in S. medicae. Later on, Draghi et al.41 demonstrated that the ATR in S. meliloti was coupled to an increased symbiotic competitiveness for nodule occupancy, opening the possibility of exploring the stabilization of the ATR in rhizobia to be inoculated into acid soils. Unfortunately, no data were available on the molecular changes associated with that acid adaptation.

The subsequent approaches to improve the basic understanding of the rhizobial responses to extracellular acidity were based on different experimental configurations, all directed at characterizing the genetic expression and protein profile under either acid growth or acid shock. Transcriptional fusions generated by Tn5 transposition demonstrated that acid-induced promoters were associated with cytochrome synthesis, potassium-ion cycling, and lipid biosynthesis and transport among other functions43. After performing a complementary study using proteomic tools, Reeve et al.43 suggested that the folding, proteolysis, and transport processes were key activities in S. medicae growing in acidity. While all these studies were performed on low-pH batch cultures, Hellweg et al.44 undertook an in-depth time-course analysis of the transcriptomic response in S. meliloti after shifting the bacterial cells from neutral to acid pH. The work revealed that a short-term exposure of the bacteria to low pH was sufficient to induce significant transcriptional changes in diverse rhizobial genes. All these studies provided useful—albeit fragmented—information on the phenotypes, and in certain examples, on the molecular components displayed by the rhizobia after challenge with a high concentration of extracellular hydrogen ions.

To date, most physiological and genetic studies on acid-stress in rhizobia have used batch cultures fraught with the consequent heterogeneity in cell physiology during cultivation, and also lacking an external control on the growth rate and other culture variables. The alternative use of continuous cultivation in a chemostat, already employed for the analysis of acid stress in rhizobia45, is an appropriate, and indeed preferable, choice for a more robust experimental design through the reduction of variables down to only the extracellular pH, thus leading to physiologically homogeneous bacterial populations and subsequent datasets46. Having used both classical and various omic tools, in this work we present a consolidated analysis of the responses of S. meliloti growing under a controlled acid stress. Changes in the transcriptome, proteome, and metabolome were integrated into a model to describe the cultural responses in the chemostat, together with the modifications in the enzymes and compounds of the central metabolic pathways.


Cultivation parameters of S. meliloti 2011 growing in continuous culture in chemostat either at pH 7.0 or at nearly growth-limiting acidity

A continuous culture of S. meliloti 2011 was established at pH 7.0 in Evans minimal medium with glucose and ammonium as the respective carbon and nitrogen sources. The cells were cultured under the conditions indicated in Materials and Methods (i.e., D=0.07.h−1, 28°C, dissolved oxygen at saturation). Because of the C:N ratio in Evans defined medium the bacterial culture became N-limited at pH 7.0. Under steady-state conditions at pH 7.0 the culture reached a cell density of ca. 4.7×109c.f.u.ml−1 (OD600nm=4.6) at a biomass of 1.66g.l−1 dry weight (Table 1). In order to determine the lowest extracellular pH at which S. meliloti was still able to grow in the chemostat, the pH of the extracellular medium was lowered stepwise at intervals of 0.2pH units to achieve discrete steady states at each new pH (between pHs of 7.0 and 6.0). When the extracellular pH reached the value 6.0 (the acidic pHlimit), the culture washed out, indicating that the rhizobia either stopped growing or duplicated at a rate that was lower than the one imposed by the chemostat-dilution rate. The continuous cultivation provided an optimal design for determining the lowest extracellular pH that was still compatible with the duplication rate imposed by the experimental setting. The acidic pHlimit in the chemostat proved to be comparable to those previously obtained in batch cultivation for S. meliloti and S. medicae (i.e., between 5.8–5.6)23,47. This information on the acidic pHlimit was used in a subsequent experiment to set up a continuous culture at pH 6.1 in order to have rhizobial cells growing just 0.1pH unit above the condition where they had reached their limit of acid tolerance. Table 1 lists the cultivation parameters that characterized the growth of S. meliloti in the chemostat at pH 7.0 and at pH 6.1, including the data on the biomass production, number of culturable cells, and respiration indices. Under acidity a clear change in the limiting substrate occurred (an excess of near 7mM ammonium was measured in the supernatant) accompanied by a 53% decrease in the biomass. The acid stress in the rhizobial cells at pH 6.1 was clearly reflected in that only 26% of the total bacteria counted in the Petroff-Hausser chamber were culturable (the number of living cells was estimated by plating). The more than 5-fold increase in the O2 consumption per living cell was observed in the culture at pH 6.1 compared to the same parameter in the culture at pH 7.0 indicated a higher aerobic respiration in the acid-stressed rhizobia. Finally, while cells produced significantly more EPS under acidity (YEPS/s; cf. Table 1), no major change in the amount of polyhydroxybutyrate per cell was detected. The connection between EPS production and acid tolerance in rhizobia has been suspected since long. In S. meliloti, however, there is no evidence that EPS could have a positive effect on the bacterial acid tolerance. It has been previously reported that an exoY S. meliloti mutant, which lacks EPS, did not display any significant difference in its death rate when exposed at low pH40.

Table 1

Cultivation parameters of S. meliloti 2011 grown in the chemostat at extracellular pH 7.0 and pH 6.1.
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, Log Analyzer 7.0b serial key or number

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry†

Author affiliations

* Corresponding authors

a School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Haidian Dist, Beijing, China

b Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijng, China


Established high-throughput proteomics methods provide limited information on the stereostructures of proteins. Traditional technologies for protein structure determination typically require laborious steps and cannot be performed in a high-throughput fashion. Here, we report a new medium throughput method by combining mobility capillary electrophoresis (MCE) and native mass spectrometry (MS) for the 3-dimensional (3D) shape determination of globular proteins in the liquid phase, which provides both the geometric structure and molecular mass information of proteins. A theory was established to correlate the ion hydrodynamic radius and charge state distribution in the native mass spectrum with protein geometrical parameters, through which a low-resolution structure (shape) of the protein could be determined. Our test data of 11 different globular proteins showed that this approach allows us to determine the shapes of individual proteins, protein complexes and proteins in a mixture, and to monitor protein conformational changes. Besides providing complementary protein structure information and having mixture analysis capability, this MCE and native MS based method is fast in speed and low in sample consumption, making it potentially applicable in top–down proteomics and structural biology for intact globular protein or protein complex analysis.

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This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry
Chem. Sci., 2020,11, 4758-4765

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry

H. Wu, R. Zhang, W. Zhang, J. Hong, Y. Xiang and W. Xu, Chem. Sci., 2020, 11, 4758

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