Distribution of genes for synthesis of trehalose and Mannosylglycerate in Thermus spp. and direct correlation of these genes with halotolerance

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.     

Mannosylglycerate is essential for osmotic adjustment in Thermus thermophilus strains HB27 and RQ-1

We disrupted the mpgS encoding mannosyl-3-phosphoglycerate synthase (MpgS) of Thermus thermophilus strains HB27 and RQ-1, by homologous recombination, to assess the role of the compatible solute mannosylglycerate (MG) in osmoadaptation of the mutants, to examine their ability to grow in NaCl-containing medium and to identify the intracellular organic solutes. Strain HB27 accumulated only MG when grown in defined medium containing 2% NaCl; mutant HB27M9 did not grow in the same medium containing more than 1% NaCl. When trehalose or MG was added, the mutant was able to grow up to 2% of NaCl and accumulated trehalose or MG, respectively, plus amino acids. T. thermophilus RQ-1 grew in medium containing up to 5% NaCl, accumulated trehalose and lower amounts of MG. Mutant RQ-1M1 lost the ability to grow in medium containing more than 3% NaCl and accumulated trehalose and moderate levels of amino acids. Exogenous MG did not improve the ability of the organism to grow above 3% NaCl, but caused a decrease in the levels of amino acids. Our results show that MG serves as a compatible solute primarily during osmoadaptation at low levels of NaCl while trehalose is primarily involved in osmoadaptation during growth at higher NaCl levels.     

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/ OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.     

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.Rhizobia induce the formation of nodules on the roots of legume plants, in which atmospheric nitrogen is fixed and supplied to the host plant, thereby enhancing growth under nitrogen-limiting conditions. The symbiotic interaction between rhizobia and their cognate leguminous plants is important for agricultural productivity, especially in less developed countries. However, physiological stresses, such as desiccation and salinity, negatively affect these symbiotic interactions by limiting nitrogen fixation (44). The osmotic environment within the rhizosphere may affect root colonization, infection thread development, nodule development, and the formation of effective N₂-fixing nodules (21). Moreover, when legume seeds are inoculated with appropriate rhizobial strains prior to planting in the field, the vast majority of nodules produced are often not formed by the inoculant bacteria but rather by indigenous strains in the soil (36). This is in part due to the death of inoculant strains from rapid seed coat-mediated desiccation. Therefore, improvement of the survival of rhizobia under conditions of physiological stresses may promote biological nitrogen fixation and enhance plant growth.Rhizobia synthesize and accumulate compatible solutes, including trehalose, in response to desiccation and solute-mediated physiological stresses (5, 21, 42). Trehalose, a nonreducing disaccharide with an α,α-1,1 linkage between the two glucose molecules, has been shown to protect cell membranes and proteins from stress-induced inactivation and denaturation (8, 23, 24). The relationship between trehalose accumulation and symbiotic phenotype is dependent on rhizobial species and host genotype. Suarez et al. (39) reported an increase in root nodule number and nitrogen fixation by Phaseolus vulgaris inoculated with a trehalose-6-phosphate synthase-overexpressing strain of Rhizobium etli. In contrast, trehalose accumulation in Rhizobium leguminosarum and Sinorhizobium meliloti cells did not result in an increase in nitrogen-fixing nodules but led to enhancement of competitiveness on clover and on certain alfalfa genotypes, respectively (1, 16, 20).Four trehalose biosynthetic pathways, mediated by OtsAB, TreS, TreYZ, and TreT, have been reported thus far for prokaryotes (8, 25). The OtsAB pathway results in the condensation of glucose-6-phosphate with UDP-glucose by trehalose-6-phosphate synthase (OtsA) to form trehalose-6-phosphate. Trehalose is subsequently formed from trehalose-6-phosphate by the action of trehalose-6-phosphate phosphatase (OtsB). The TreS pathway involves a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with α,α-1,4 linkage between the two glucose molecules, to trehalose. The third pathway, mediated by TreYZ, involves the conversion of maltodextrins into trehalose. The terminal α-1,1-glycosylic bond at the end of the maltodextrin polymer is hydrolyzed by maltooligosyltrehalose synthase (TreY), and trehalose is subsequently released from the end of the polymer via hydrolysis by maltooligosyltrehalose trehalohydrolase (TreZ). More recently, a trehalose glycosyltransferring synthase (TreT) was shown to catalyze the reversible formation of trehalose from ADP-glucose and glucose (25).In addition to biosynthesis, Gram-negative bacteria have also been reported to have trehalose degradation systems. Typically, trehalose is hydrolyzed into two glucose moieties by periplasmic and cytoplasmic trehalase enzymes, TreA and TreF, respectively (13, 15). However, Sinorhizobium meliloti also uses ThuA and ThuB for trehalose utilization (16).Bradyrhizobium japonicum, the root nodule symbiont of soybeans, accumulates trehalose in cultured cells and bacteroids (34, 35). Biochemical studies indicated that B. japonicum has three independent trehalose biosynthetic pathways involving trehalose synthase (TreS), maltooligosyltrehalose synthase (TreYZ), and trehalose-6-phosphate synthetase (OtsAB) (38). Sequence analysis of the B. japonicum USDA 110 genome identified the genes that encode these biosynthetic pathways: otsAB (bll0322 to bll0323), two homologs of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771), but not treT (17). Orthologous gene sequences to the trehalose degradation genes treA, treF, and thuAB have not been found in the genome of B. japonicum USDA 110. Cytryn et al. (6) reported that expression of otsA, treS (blr6767), and treY genes were highly induced by desiccation stress. Moreover, the concentrations of these three enzymes increased when B. japonicum was cultured in the presence of salt (38). Trehalose concentration in B. japonicum has been reported to increase due to desiccation stress (6), and this sugar is purported to act as an osmoprotectant. The addition of exogenously supplied trehalose has been reported to enhance the survival of B. japonicum in response to desiccation and salinity stresses (9, 37). Despite this information, little is known about how the various trehalose biosynthetic pathways modulate stress tolerance and symbiotic performance in B. japonicum.The purpose of this study was to examine the functional role(s) of the B. japonicum trehalose biosynthetic pathways on stress survival by constructing single, double, and triple mutants and by producing strains that overexpress the trehalose biosynthesis enzymes. Here we report on the relationship between trehalose accumulation and physiological responses to salinity and desiccation stresses in mutant and overexpression strains and that mutations in the trehalose biosynthesis pathways altered the symbiotic performance of B. japonicum USDA 110 on soybeans. Results of these studies indicate that trehalose accumulation in B. japonicum plays a prominent role in the saprophytic and symbiotic competence of this agriculturally important soil bacterium.     

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10³ CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.     

Increased trehalose biosynthesis improves <Emphasis Type="Italic">Mesorhizobium ciceri</Emphasis> growth and symbiosis establishment in saline conditions

Cicer arietinum (chickpea) is a legume very sensitive to salinity, and so are most of its rhizobial symbionts belonging to the species Mesorhizobium ciceri. We observed that exogenous trehalose (i.e., added to the growth medium) can significantly improve growth of M. ciceri strain Rch125 under moderate salinity. In order to test if endogenous trehalose (i.e., synthesized by the cell) could also enhance salt tolerance, strain Rch125 was genetically modified with various trehalose biosynthesis genes from Sinorhizobium meliloti 1021 (otsA, treS, treY) and Mesorhizobium loti MAFF 303099 (otsAB). We found that overexpression of otsA or otsAB, but not treS or treY, significantly improved M. ciceri Rch125 growth in saline media. This growth improvement correlated with enhanced trehalose accumulation in otsA- and otsAB-modified cells, suggesting that increased trehalose synthesis via trehalose-6-phosphate can enhance bacterial salt tolerance. Chickpea plants inoculated with M. ciceri Rch125 derivatives carrying extra otsAB or otsA genes formed more nodules and accumulated more shoot biomass than wild type inoculated plants when grown in the presence of NaCl. These results support the notion that improved salt tolerance of the bacterial symbiont can alleviate the negative effects of salinity on chickpeas, and that such improvement in M. ciceri can be achieved by manipulating trehalose metabolism.     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.     

大肠杆菌海藻糖合成酶基因的克隆和表达

利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因,克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株,转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能,高渗透压诱导实验表明,转化株能够合成克隆基因 产物海藻糖,但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体,并在农杆菌的介导下 转入植物,赋予其抗高渗、耐干旱能力奠定了重要的研究基础。     

Transferable Denitrification Capability of Thermus thermophilus

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N₂O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.     

Truepera radiovictrix gen. nov., sp. nov., a new radiation resistant species and the proposal of Trueperaceae fam. nov

Two isolates, belonging to a new species of a novel genus of the Phylum “Deinococcus/Thermus ”, were recovered from hot spring runoffs on the Island of São Miguel in the Azores. Strains RQ-24^T and TU-8 are the first cultured representatives of a distinct phylogenetic lineage within this phylum. These strains form orange/red colonies, spherical-shaped cells, have an optimum growth temperature of about 50 °C, an optimum pH for growth between about 7.5 and 9.5, and do not grow at pH below 6.5 or above pH 11.2. These organisms grow in complex media without added NaCl, but have a maximum growth rate in media with 1.0% NaCl and grow in media containing up to 6.0% NaCl. The organisms are extremely ionizing radiation resistant; 60% of the cells survive 5.0 kGy. These strains are chemoorganotrophic and aerobic; do not grow in Thermus medium under anaerobic conditions with or without nitrate as electron acceptor and glucose as a source of carbon and energy, but ferment glucose to d-lactate without formation of gas. The organisms assimilate a large variety of sugars, organic acids and amino acids. Fatty acids are predominantly iso- and anteiso-branched; long chain 1,2 diols were also found in low relative proportions; menaquinone 8 (MK-8) is the primary respiratory quinone. Peptidoglycan was not detected. Based on 16S rRNA gene sequence analysis, physiological, biochemical and chemical analysis we describe a new species of one novel genus represented by strain RQ-24^T (CIP 108686^T = LMG 22925^T = DSM 17093^T) for which we propose the name Truepera radiovictrix. We also propose the family Trueperaceae fam. nov. to accommodate this new genus.     

Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Anaerobic Growth,a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles

Despite the fact that the extreme thermophilic bacteria belonging to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that this nar operon can be transferred by conjugation to an aerobic Thermus strain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives of Escherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative plasmid integrated into the chromosome. First, the nar operon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of the nar cluster. Finally, the mating time required for the transfer of the nar operon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream of nar.

Most extreme thermophiles that live in geothermal environments are strict anaerobes (3, 11) as a consequence of the adaptation to the low solubility of oxygen at these temperatures. However, members of the genus Thermus constitute an exception to this general rule, being described taxonomically as strictly aerobic chemorganotrophs (2).However, we recently showed that one of the most thermophilic isolates of this genus, Thermus thermophilus HB8, was able to grow anaerobically when nitrate was present in the medium. Biochemical and genetic evidence demonstrated that this ability was related to the synthesis of a membrane-bound respiratory nitrate reductase complex whose protein components, the α (NarG; 136 kDa), β (NarH; 57 kDa), and γ (NarI; 28 kDa) subunits, were homologous (about 48 to 50% sequence identity) to those from mesophilic facultative anaerobes (e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced under low oxygen concentrations when nitrate was present (21). In contrast to those described for most nitrate reducers, the product of nitrate respiration was secreted to the growth medium through an unknown transporter.We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains HB8 and HB27 of T. thermophilus is the absence of plasmids from the latter, the possibility that the nar operon could be encoded by a transferable genetic element, such as a plasmid, was considered.In this article, we analyze this possibility and demonstrate that the ability to grow by nitrate respiration can be transferred to the aerobic strain T. thermophilus HB27 by conjugation. We also relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal genes in a time-dependent way.     

基于比较基因组学分析嗜热链球菌的遗传多样性和防御系统

【目的】嗜热链球菌(Streptococcusthermophilus)是发酵乳制品的基础发酵菌种之一,全基因组水平解析嗜热链球菌的遗传多样性和工业发酵特性对于优良发酵菌株的筛选意义重大。【方法】本研究通过比较基因组学方法对27株嗜热链球菌的遗传多样性和防御系统进行分析。【结果】全基因组分析结果显示嗜热链球菌群体内具有较高的遗传多样性;基于核心基因集构建的系统发育树划分为2个分支,其中分支2菌株缺乏完整的组氨酸合成途径,经验证,分支2菌株在缺乏组氨酸的培养基中不能正常生长。通过对嗜热链球菌不同菌株的防御系统进行分析发现,同类型的CRISPR基因座和限制修饰系统在基因组中出现的位置相对固定。CRISPR-Cas系统(P0.05,r=0.43)和限制修饰系统(P0.01,r=–0.59)的数量与编码转座酶基因的数量均显著相关,表明嗜热链球菌为了阻止外源DNA入侵会进化出多种防御系统来保护自身遗传完整性。此外,分支1菌株的CRISPR-Cas系统数量极显著(P0.001)多于分支2,而限制修饰系统无显著差异,表明分支1菌株在噬菌体抗性方面可能更具优势。【结论】本研究基于核心基因构建的系统发育分析将27株嗜热链球菌分为2个分支,不同分支菌株在组氨酸代谢能力和防御系统方面有一定差异。该研究结果为今后快速筛选优良嗜热链球菌发酵剂提供了新思路。     

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342 mM NaCl as well as 127 or 99 mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44 °C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.     

Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.     

The ggpS Gene from Synechocystis sp. Strain PCC 6803 Encoding Glucosyl-Glycerol-Phosphate Synthase Is Involved in Osmolyte Synthesis

A salt-sensitive mutant of Synechocystis sp. strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis. Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11. This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (otsA homolog)- and glycerol-3-phosphate-synthesizing enzymes. After construction and characterization of mutants defective in these genes, it became obvious that an otsA homolog (sll1566) (T. Kaneko et al., DNA Res. 3:109–136, 1996) encodes GGPS, since only the mutant affected in sll1566 showed salt sensitivity combined with a complete absence of GG accumulation. Furthermore, the overexpression of sll1566 in Escherichia coli led to the appearance of GGPS activity in the heterologous host. The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of Synechocystis.     

Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97–3.72 μmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales.     

Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity

The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg²⁺ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.