Influence of host cultivars and Rhizobium species on the growth and symbiotic performance of Phaseolus vulgaris under salt stress

In order to study the effect of salt stress on the Rhizobium-common bean symbiosis, we investigated the response of both partners, separately and in symbiosis. The comparison of the behaviour of five cultivars of Phaseolus vulgaris differing in seed colour, growing on nitrates and different concentrations of NaCl, showed genotypic variation with respect to salt tolerance. Coco Blanc was the most sensitive cultivar, whereas SMV 29-21 was the most tolerant one. At the Rhizobium level, two strains previously selected for their salt tolerance were used: Rhizobium tropici strain RP163 and Rhizobium giardinii strain RP161. Their relative growth was moderately decreased at 250mM NaCl, but they were able to grow at a low rate in the presence of 342 mM NaCl. Their viability at the minimal inhibitory concentration was slightly affected. The effect of salinity on Rhizobium-plant association was studied by using the tolerant variety SMV 29-21 and the sensitive one Coco Blanc inoculated separately with both strains. In the absence of salinity, the strains induced a significantly higher number of nodules on the roots of the cultivar SMV 29-21 compared to those of Coco Blanc. Concerning effectiveness, both strains were similarly effective with SMV 29-21, but not with Coco Blanc. In the presence of salinity, Coco Blanc was more severely affected when associated with RP163 than with RP161. Salinity affected the nodulation development more than it affected the infection steps. Neither of the two strains was able to nodulate SMV 29-21 under saline conditions, in spite of the fact that this was considered the most salt-tolerant variety. The unsuccessful nodulation of SMV 29-21 could be related to the inhibition by salt of one or more steps of the early events of the infection process. In conclusion, N-fixing plants were found to be more sensitive to salt stress than those depending on mineral nitrogen. Evidence presented here suggests that a best symbiotic N2 fixation under salinity conditions could be achieved if both symbiotic partners, as well as the different steps of their interaction (early events, nodule formation, activity, etc.), are all tolerant to this stress.     

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco

AIMS: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. METHODS AND RESULTS: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified. CONCLUSIONS: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.     

The life of root nodule bacteria in the acidic underground

Abstract The effects of low pH on the growth and survival of root nodule bacteria are important in determining the success of legume inoculation in practical agriculture. The mechanisms for maintenance of intracellular pH (pH_i) are crucial, but little is yet known about them in root nodule bacteria. Like other Gram-negative bacteria, root nodule bacteria show an adaptive acid tolerance response, with growth at moderately acidic conditions protecting against an extreme acid shock. Variations in acid tolerance within species of root nodule bacteria imply a genetic basis to low pH tolerance and studies of acid-sensitive mutants suggest that as many as 20 genes could be involved. A general model for acid tolerance in root nodule bacteria is presented.     

Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments

Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.     

Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.     

Tolerance of Bradyrhizobium sp. (Lupini) strains to salinity, pH, CaCO3 and antibiotics

AIMS: Ten rhizobial isolates obtained from different locations in Egypt were examined for their ability to survive under stress conditions and their growth response to increasing levels of NaCl (1-8% w/v), pH (4-10), CaCO3 (1-10% w/v) and 12 antibiotics. METHODS AND RESULTS: All the rhizobial isolates tolerated a NaCl concentration up to 5% and were divided into two groups with respect to NaCl tolerance. The rhizobial isolates from group two showed significantly (P < 0.05) better survival under high NaCl concentration. All the tested isolates survived acidic (pH 4-5) and alkaline conditions (pH 9-10) and CaCO3 (up to 10% w/v) in liqued YEM medium. CONCLUSION: Antibiotic resistance patterns did not correlate to NaCl, pH or CaCO3 tolerance. Variations among different strains showed that there is potential to improve strain performance under stress conditions. Significance and Impact of the Study: The results suggest that selection of adapted strains under stress conditions is possible and can be used as inoculants for successful lupin growth.     

The coexistence of symbiosis and pathogenicity-determining genes in Rhizobium rhizogenes strains enables them to induce nodules and tumors or hairy roots in plants

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

Identification of acid-stress-tolerant proteins from promising cyanobacterial isolates

Cyanobacterial cultures were isolated from acidic (pH 4.9–6.2) rice grown soils in Tamil Nadu, India. The predominant genera were Anabaena (50%), Westiellopsis (17.5%), Nostoc (15%), Oscillatoria (5%) and others that were unicellulars (12.5%) viz., Microcystis, Calothrix and Phormidium. The levels of tolerance to acidity varied among these strains, which were tested and authenticated for their acid tolerance capacity under both in vitro and pot culture conditions. Westiellopsis sp. was found to predominate from pH 4.9 to pH 6.2, indicating its adaptability. Cultures tolerant to acidic conditions were characterized for growth, biomass production and biochemical constituents. Under acidic conditions, Westiellopsis sp. showed pronounced chlorophyll a content, phycobilin pigment content, ammonia excretion and nitrogenase activity compared to normal conditions. Molecular characterization, particularly isozyme and random amplified polymorphic DNA (RAPD) analysis, were also carried out. Three strains of Westielliopsis sp. strains were selected, of which two were able to grow at an acidity level of pH 4.0, while one strain was able to sustain growth at an acidity level of 5.0. These three cultures, along with acid susceptible strains of Westielliopsis sp. and Anabaena sp. PCC 7120 (standard check) were subjected to acid shock for different time intervals. Protein profiling of both the acid-tolerant and acid-susceptible strains was carried out with samples collected at different time intervals. Based on the presence/absence of protein bands in the tolerant/susceptible strains, some low- and medium-molecular weight proteins can be linked to conferring acid tolerance. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Further studies on RpoS in enterobacteria: identification of rpoS in Enterobacter cloacae and Kluyvera cryocrescens

RpoS, the alternative sigma factor sigma(s), is important for bacterial survival under extreme conditions. Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence. To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species. A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri. Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E. coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers. The rpoS gene from E. cloacae and from K. cryocrescens was cloned and sequenced and a mutant allele was constructed in E. cloacae. Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential- and stationary-phase cells of both strains. E. cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.     

Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH

The glutamate decarboxylase (GAD) system is critical to the survival of Listeria monocytogenes LO28 at low-pH stress (相似文献   

Pleiotropic Menaquinone-Deficient Mutant of Bacillus subtilis

A multiple aromatic amino acid auxotroph of Bacillus subtilis 168 has been isolated which is unable to synthesize menaquinone-7 (MK-7) unless supplied with shikimic acid (SHK). The mutant, RB163, was isolated by selecting for resistance to low levels (1.5 mug/ml) of kanamycin. Enzymatic and genetic analyses show that the strain is an aroD mutant lacking 5-dehydroshikimate reductase. Under growth conditions in which its MK-7 deficiency is expressed, RB163 is deficient in cytochromes a, b, and c, exhibits low growth yields, and does not sporulate. Genetic analysis indicates that this pleiotropic phenotype is the result of a single genetic event. All phenotypic characteristics are reversible when the mutant is grown under conditions such that MK is synthesized. Comparison of strain RB163 with other aro mutants blocked before SHK ("early-aro" mutants) reveals interesting differences. Most early-aro mutants are cytochrome- and MK-sufficient, sporogenous, and sensitive to kanamycin when grown in the absence of SHK. However, in addition to strain RB163, two other aro mutants were found to show the pleiotropic phenotype. These three mutants have in common, and differ from other early-aro strains in, the inability to synthesize MK. It is suggested that the phenotypically wild-type aro mutants are bradytrophic, allowing enough substrate flow through the common aromatic pathway to satisfy the MK requirement. The pleiotropic mutants are thought to be completely blocked in the common pathway, thus accounting for their inability to synthesize MK.     

Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.     

Role of Partially Saturated Canthaxanthin and Ergosterol in the Survival of <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> Mutant at Extreme Acidic Condition

Unlike the wild type, the mutant Aspergillus carbonarius synthesized a yellow pigment, partially saturated canthaxanthin (PSC) when the growth medium acidified to low pH. Since the pigment found pharmaceutical applications, the possible mechanism involved in its ability to grow at extreme acidic conditions is described. To understand the mutation in the pathway, specific inhibitors affecting carotenoid biosynthesis were used in the medium and PSC synthesis and cell integrity were studied. Results suggested that the possible occurrence of mutation in the isoprenoid pathway for higher production of carotenoid as well as ergosterol caused the mutant to grow in extremely acidic conditions. The results also suggested that the flow of carbon for sterol biosynthesis and that of carotenoids are dependent. The deposition of carotenoids and ergosterol in the cell membrane causing the cells to maintain pH homeostasis under the acidic growth conditions is of significant importance. In A. carbonarius, understanding the cause of stress induced PSC accumulation is essential for efficient expression and production of the pharmaceutically significant carotenoid and this will further facilitate research into the role of carotenoids in stress tolerance of other filamentous fungi.     

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.