Application value of EV/EV71/CA16-SAT detection in children with hand-foot-mouth disease.

The objective of this work was to explore the application value of a new type of fluorescent nucleic acid isothermal amplification (SAT) to detect EV/EV71/CA16-SAT in children with hand-foot-mouth disease (HFMD). For this purpose, from March 2017 to September 2019, Chengdu Children's Specialized Hospital collected throat swabs from children with clinical manifestations of hand, foot and mouth disease, and used SAT technology to screen and detect universal enterovirus (EV) nucleic acid (There were 1860 children with EV-RNA) positive. Patients who are EV-RNA positive at any time: first use the same throat swab specimen to detect EV71/CA16-RNA; secondly, collect venous blood and use the colloidal gold method to detect IgM antibodies in EV71/CA16 serum. The patients with positive EV71/CA16-RNA or EV71/CA16-IgM (or both) were repeated the above two methods 2 weeks and 4 weeks after standard treatment for review and comprehensive analysis. Results showed that 763 cases were enrolled for the first time: 59.76% were male and 40.24% were female; the age ranged from 1 month to 13 years, of which 69.06% were from 1 to 4 years old; CA16-RNA positive 56.23%, EV71-RNA positive 21.89%, CA16/EV71 -RNA were all positive in 1.57%; CA16-IgM was positive in 64.48%, EV71-IgM was positive in 54.26%, and CA16/EV71-IgM were both positive in 18.74%. After 2 weeks, 722 cases were reexamined: 26.73% were positive for CA16-RNA, 7.89% were positive for EV71-RNA, 0.28% were both positive for CA16/EV71-RNA; 66.21% were positive for CA16-IgM, 51.52% were positive for EV71-IgM, and IgM were all positive in 17.73%. Four weeks later, 489 cases were reexamined: among them, CA16-RNA positive 5.73% of which were positive for EV71 color RNA (0.005%), and 12.68% of them were all positive for EV71lym. The strategy of combining SAT technology and colloidal gold method to detect EV/EV71/CA16 nucleic acid (RNA) and serum IgM antibody in children HFMD can improve the early detection rate and accuracy of HFMD; According to the comprehensive analysis of the detection results of children with HFMD at the early stage, 2 weeks and 4 weeks of the present study, it is suggested that EV/EV71/CA16-SAT nucleic acid detection can be used to judge the prognosis, follow-up treatment, set isolation time, return students to school, and community management in children with HFMD. and prevention and control have more clinical application value.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

HbWRKY82, a novel IIc WRKY transcription factor from Hevea brasiliensis associated with abiotic stress tolerance and leaf senescence in Arabidopsis.

WRKY group transcription factors of model plants and major crops are confirmed to play essential roles in stress responses, senescence, secondary metabolism processes and hormone signal transduction. Previous studies have identified 81 HbWRKY genes from Hevea brasiliensis (the Pará rubber tree), but the functions of HbWRKYs in response to abiotic stresses and leaf senescence are unclear. In this study, one novel group IIc WRKY transcription factor named HbWRKY82 was identified and characterized as a stress-associated WRKY in rubber tree. Transient expression and transcriptional activation analyses indicated that HbWRKY82 encoded a nuclear protein and functioned as a transcription activator. The transcription levels of HbWRKY82 were induced by exogenous Ethrel (ET) (ethylene releaser) and abscisic acid (ABA) stimulations, down-regulated in tapping panel dryness rubber trees, and also exhibits significant decrease during the progression of leaf senescence. Overexpression of HbWRKY82 in Arabidopsis improved the tolerance to dehydration and salinity, and decreased the sensitivity to exogenous ABA. Moreover, real-time quantitative PCR analysis demonstrated that HbWRKY82 regulated the transcriptional expression of several stress-responsive genes (DREB1A, ERD10, HKT1, P5CS, RD22, RD29B, SKOR), leaf senescence marker genes (EIN3, WRKY53, NAP), ROS-related genes (RbohD, CSD1, CSD2, FSD3) and hormone signaling genes (EIN3, ABF3, ABF4). Collectively, our findings suggested that HbWRKY82 might function as an important transcriptional regulator in ET- and ABA-mediated leaf senescence and abiotic stress responses, and also be involved in tapping panel dryness, latex flow and regeneration processes of rubber trees via participating in the ET and reactive oxygen species signaling pathways.

Molecular characterization and functional analysis of a novel WRKY transcription factor HbWRKY83 possibly involved in rubber production of Hevea brasiliensis.

WRKY transcription factors play important roles in plant growth and developmental processes and various stress responses, and are also associated with jasmonic acid (JA) signaling in the regulation of secondary metabolite biosynthesis in plants. The regulatory networks mediated by WRKY proteins in the latex production of Hevea brasiliensis (the Pará rubber tree) are poorly understood. In this study, one novel WRKY gene (designated HbWRKY83) was identified from the latex of H. brasiliensis, and its functions were characterized via gene expression analysis in both the latex and HbWRKY83-overexpressing transgenic Arabidopsis. HbWRKY83 gene contains an open reading frame (ORF) of 921 bp encoding a 306-amino-acid protein which is clustered with group IIc WRKY TF. HbWRKY83 is a nuclear-localized protein with transcriptional activity. Real-time quantitative PCR analysis demonstrated that the transcription level of HbWRKY83 was up-regulated by exogenous methyl jasmonate, Ethrel (ethylene releaser) stimulation, and bark tapping (mechanical wounding). Compared with the wild-type plants, overexpression of HbWRKY83 improved the tolerance of transgenic Arabidopsis lines to drought and salt stresses by enhancing the expression levels of ethylene-insensitive3 transcription factors (EIN3s) and several stress-responsive genes, including Cu/Zn superoxide dismutases CSD1 (Cu/Zn-SOD1) and CSD2 (Cu/Zn-SOD2), related to reactive oxygen species scavenging. Additionally, these genes were also significantly up-regulated by bark tapping. In combination, these results suggest that HbWRKY83 might act as a positive regulator of rubber production by activating the expression of JA-, ethylene-, and wound-responsive genes in the laticiferous cells of rubber trees.

Grafted copolymerization of N-phenylmaleimide and styrene in porous polyvinyl chloride particles suspended in aqueous solution.

N-phenylmaleimide (N-PMI) and its precursor, (Z)-4-oxo-4-(phenylamino)but-2-enoic acid, were synthesized from aniline and maleic anhydride. Copolymerization between N-phenylmaleimide and styrene was initiated in micropores and outside surface of porous polyvinyl chloride (PVC) resin suspended in aqueous phase. The modified PVC was characterized with Gel Permeation Chromatography, thermal gravimetric analysis and scanning electron microscopy. The result of high performance liquid chromatography shows that the purity of N-PMI reached 97.3%. Thermal gravimetric analysis indicated that the introduction of N-PMI could clearly affect the thermal degradation behavior of PVC, and when PMI was at 6.25% of the PVC mass, the decomposition temperature T0.5 of modified polymer was increased to 314.2°C. The glass transition temperature of modified polymer was increased from 70°C to 80°C.

Genome-wide comparative analysis of papain-like cysteine protease family genes in castor bean and physic nut.

Papain-like cysteine proteases (PLCPs) are a class of proteolytic enzymes involved in many plant processes. Compared with the extensive research in Arabidopsis thaliana, little is known in castor bean (Ricinus communis) and physic nut (Jatropha curcas), two Euphorbiaceous plants without any recent whole-genome duplication. In this study, a total of 26 or 23 PLCP genes were identified from the genomes of castor bean and physic nut respectively, which can be divided into nine subfamilies based on the phylogenetic analysis: RD21, CEP, XCP, XBCP3, THI, SAG12, RD19, ALP and CTB. Although most of them harbor orthologs in Arabidopsis, several members in subfamilies RD21, CEP, XBCP3 and SAG12 form new groups or subgroups as observed in other species, suggesting specific gene loss occurred in Arabidopsis. Recent gene duplicates were also identified in these two species, but they are limited to the SAG12 subfamily and were all derived from local duplication. Expression profiling revealed diverse patterns of different family members over various tissues. Furthermore, the evolution characteristics of PLCP genes were also compared and discussed. Our findings provide a useful reference to characterize PLCP genes and investigate the family evolution in Euphorbiaceae and species beyond.

Papain-like cysteine protease encoding genes in rubber (Hevea brasiliensis): comparative genomics, phylogenetic, and transcriptional profiling analysis.

MAIN CONCLUSION: 43 HbPLCPs representing nine subfamilies or 20 orthologous groups were found in rubber, where paralogs were resulted from the recent WGD and local duplication. Several senescence-associated genes were also identified. Papain-like cysteine proteases (PLCPs) comprise a large family of proteolytic enzymes involved in plant growth and development, seed germination, organ senescence, immunity, and stress response. Despite their importance and the extensive research in the model plant Arabidopsis thaliana, little information is available on rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study performed a genome-wide identification of PLCP family genes in rubber, resulting in a relatively high number of 43 members. The phylogenetic analysis assigned these genes into nine subfamilies, i.e., RD21 (6), CEP (4), XCP (4), XBCP3 (2), THI (1), SAG12 (18), RD19 (4), ALP (2), and CTB (2). Most of them were shown to have orthologs in Arabidopsis; however, several members in SAG12, CEP and XBCP3 subfamilies form new groups as observed in other core eudicots such as Manihot esculenta, Ricinus communis, Populus trichocarpa, and Vitis vinifera. Based on an expert sequence comparison, 20 orthologous groups (OGs) were proposed for core eudicots, and rubber paralogs were shown to be resulted from the recent whole-genome duplication (WGD) as well as local duplication. Transcriptional profiling showed distinct expression pattern of different members across various tissues, e.g., root, leaf, bark, laticifer, flower, and seed. By using the senescence-specific HbSAG12H1 as the indicator, the transcriptome of senescent rubber leaves was deeply sequenced and several senescence-associated PLCP genes were identified. Results obtained from this study provide valuable information for future functional analysis and utilization of PLCP genes in Hevea and other species.

Survey of the rubber tree genome reveals a high number of cysteine protease-encoding genes homologous to Arabidopsis SAG12.

Arabidopsis thaliana SAG12, a senescence-specific gene encoding a cysteine protease, is widely used as a molecular marker for the study of leaf senescence. To date, its potential orthologues have been isolated from several plant species such as Brassica napus and Nicotiana tabacum. However, little information is available in rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study presents the identification of SAG12-like genes from the rubber tree genome. Results showed that an unexpected high number of 17 rubber orthologues with a single intron were found, contrasting the single copy with two introns in Arabidopsis. The gene expansion was also observed in another two Euphorbiaceae plants, castor bean (Ricinus communis) and physic nut (Jatropha curcas), both of which contain 8 orthologues. In accordance with no occurrence of recent whole-genome duplication (WGD) events, most duplicates in castor and physic nut were resulted from tandem duplications. In contrast, the duplicated HbSAG12H genes were derived from tandem duplications as well as the recent WGD. Expression analysis showed that most HbSAG12H genes were lowly expressed in examined tissues except for root and male flower. Furthermore, HbSAG12H1 exhibits a strictly senescence-associated expression pattern in rubber tree leaves, and thus can be used as a marker gene for the study of senescence mechanism in Hevea.

Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis.

Aquaporins (AQPs) are channel-forming integral membrane proteins that transport water and other small solutes across biological membranes. Despite the vital role of AQPs, to date, little is known in physic nut (Jatropha curcas L., Euphorbiaceae), an important non-edible oilseed crop with great potential for the production of biodiesel. In this study, 32 AQP genes were identified from the physic nut genome and the family number is relatively small in comparison to 51 in another Euphorbiaceae plant, rubber tree (Hevea brasiliensis Muell. Arg.). Based on the phylogenetic analysis, the JcAQPs were assigned to five subfamilies, i.e., nine plasma membrane intrinsic proteins (PIPs), nine tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), two X intrinsic proteins (XIPs), and four small basic intrinsic proteins (SIPs). Like rubber tree and other plant species, functional prediction based on the aromatic/arginine selectivity filter, Froger's positions, and specificity-determining positions showed a remarkable difference in substrate specificity among subfamilies of JcAQPs. Genome-wide comparative analysis revealed the specific expansion of PIP and TIP subfamilies in rubber tree and the specific gene loss of the XIP subfamily in physic nut. Furthermore, by analyzing deep transcriptome sequencing data, the expression evolution especially the expression divergence of duplicated HbAQP genes was also investigated and discussed. Results obtained from this study not only provide valuable information for future functional analysis and utilization of Jc/HbAQP genes, but also provide a useful reference to survey the gene family expansion and evolution in Euphorbiaceae plants and other plant species.

Gene Structures, Evolution and Transcriptional Profiling of the WRKY Gene Family in Castor Bean (Ricinus communis L.).

WRKY proteins comprise one of the largest transcription factor families in plants and form key regulators of many plant processes. This study presents the characterization of 58 WRKY genes from the castor bean (Ricinus communis L., Euphorbiaceae) genome. Compared with the automatic genome annotation, one more WRKY-encoding locus was identified and 20 out of the 57 predicted gene models were manually corrected. All RcWRKY genes were shown to contain at least one intron in their coding sequences. According to the structural features of the present WRKY domains, the identified RcWRKY genes were assigned to three previously defined groups (I-III). Although castor bean underwent no recent whole-genome duplication event like physic nut (Jatropha curcas L., Euphorbiaceae), comparative genomics analysis indicated that one gene loss, one intron loss and one recent proximal duplication occurred in the RcWRKY gene family. The expression of all 58 RcWRKY genes was supported by ESTs and/or RNA sequencing reads derived from roots, leaves, flowers, seeds and endosperms. Further global expression profiles with RNA sequencing data revealed diverse expression patterns among various tissues. Results obtained from this study not only provide valuable information for future functional analysis and utilization of the castor bean WRKY genes, but also provide a useful reference to investigate the gene family expansion and evolution in Euphorbiaceus plants.

Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue.

BACKGROUND: Natural rubber, an important industrial raw material, is specifically synthesized in laticifers located inside the rubber tree (Hevea brasiliensis Muell. Arg.) trunk. Due to the absence of plasmodesmata, the laticifer water balance is mediated by aquaporins (AQPs). However, to date, the characterization of H. brasiliensis AQPs (HbAQPs) is still in its infancy. RESULTS: In this study, 51 full-length AQP genes were identified from the rubber tree genome. The phylogenetic analysis assigned these AQPs to five subfamilies, including 15 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 9 NOD26-like intrinsic proteins (NIPs), 4 small basic intrinsic proteins (SIPs) and 6 X intrinsic proteins (XIPs). Functional prediction based on the analysis of the aromatic/arginine (ar/R) selectivity filter, Froger's positions and specificity-determining positions (SDPs) showed a remarkable difference in substrate specificity among subfamilies. Homology analysis supported the expression of 44 HbAQP genes in at least one of the examined tissues. Furthermore, deep sequencing of the laticifer transcriptome in the form of latex revealed a key role of several PIP subfamily members in the laticifer water balance, and qRT-PCR analysis showed diverse expression patterns of laticifer-expressed HbAQP genes upon ethephon treatment, a widely-used practice for the stimulation of latex yield. CONCLUSIONS: This study provides an important genetic resource of HbAQP genes, which will be useful to improve the water use efficiency and latex yield of Hevea.

Gene Structures, Evolution, Classification and Expression Profiles of the Aquaporin Gene Family in Castor Bean (Ricinus communis L.).

Aquaporins (AQPs) are a class of integral membrane proteins that facilitate the passive transport of water and other small solutes across biological membranes. Castor bean (Ricinus communis L., Euphobiaceae), an important non-edible oilseed crop, is widely cultivated for industrial, medicinal and cosmetic purposes. Its recently available genome provides an opportunity to analyze specific gene families. In this study, a total of 37 full-length AQP genes were identified from the castor bean genome, which were assigned to five subfamilies, including 10 plasma membrane intrinsic proteins (PIPs), 9 tonoplast intrinsic proteins (TIPs), 8 NOD26-like intrinsic proteins (NIPs), 6 X intrinsic proteins (XIPs) and 4 small basic intrinsic proteins (SIPs) on the basis of sequence similarities. Functional prediction based on the analysis of the aromatic/arginine (ar/R) selectivity filter, Froger's positions and specificity-determining positions (SDPs) showed a remarkable difference in substrate specificity among subfamilies. Homology analysis supported the expression of all 37 RcAQP genes in at least one of examined tissues, e.g., root, leaf, flower, seed and endosperm. Furthermore, global expression profiles with deep transcriptome sequencing data revealed diverse expression patterns among various tissues. The current study presents the first genome-wide analysis of the AQP gene family in castor bean. Results obtained from this study provide valuable information for future functional analysis and utilization.

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake.

In this study, metagenomic DNA was screened for the Na(+)/H(+) antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na(+)/H(+) antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na(+)/H(+) antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6%) with a putative NhaD-type Na(+)/H(+) antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na(+)/H(+) antiporters from Halomonas elongata (20.8%), Alkalimonas amylolytica (19.0%), Vibrio parahaemolyticus (18.9%) and Vibrio cholerae (18.7 %). pH-dependent Na(+)(Li(+))/H(+) antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na(+)(Li(+))/H(+) antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na(+)/H(+) antiporters. Also, the apparent K m values of Hb_NhaD for Na(+) and Li(+) at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na(+)/H(+) antiporter.

Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na(+)/H(+) antiporter of Halobacillus dabanensis.

NhaH is a novel Na(+)/H(+) antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+) binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.

Putative paired small multidrug resistance family proteins PsmrAB, the homolog of YvdSR, actually function as a novel two-component Na(+)/H(+) antiporter.

In this study, metagenomic DNA was screened for the Na(+)/H(+) antiporter gene from the halophilic bacteria from Daban Salt Lake using Escherichia coli KNabc lacking three major Na(+)/H(+) antiporters, and two genes psmrAB predicted to encode paired small multidrug resistance (PSMR) family proteins, the homolog of YvdSR, were finally cloned. Only the simultaneous presence of psmrAB, but not the single gene alone, conferred the tolerance of E. coli KNabc to up to 0.6 M NaCl and at alkaline pH. pH-dependent Na(+)(Li(+))/H(+) antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc cells carrying psmrAB, which had the highest activity at pH 9.0. However, a detailed test for antimicrobial drugs showed that E. coli DH5α with psmrAB only exhibited slight resistance to chloramphenicol, but not other representative antimicrobial drugs especially ethidium bromide. Protein sequence alignment showed that neither PsmrA nor PsmrB has homology with known single-gene or multiple-gene Na(+)/H(+) antiporters, or such proteins as TetA(L) and MdfA with Na(+)/H(+) antiport activity. Taken together, PsmrAB should function mainly as a novel two-component Na(+)/H(+) antiporter. This is the first example of a PSMR family member that exhibits Na(+)/H(+) antiporter activity.

[Soil moisture content and fine root biomass of rubber tree (Hevea brasiliensis) plantations at different ages].

By using soil core sampling method, this paper studied the soil moisture regime of rubber plantations and the fine root biomass of Hevea brasiliensis in immature period (5 a), early yielding period (9 a), and peak yielding period (16 a). With the increasing age of rubber trees, the soil moisture content of rubber plantations increased but the fine root biomass decreased. The soil moisture content at the depth of 0-60 cm in test rubber plantations increased with soil depth, and presented a double-peak pattern over the period of one year. The fine root biomass of rubber trees at different ages had the maximum value in the top 10 cm soil layers and decreased with soil depth, its seasonal variation also showed a double-peak pattern, but the peak values appeared at different time. Soil moisture content and soil depth were the main factors affecting the fine root biomass of H. brasiliensis.

[Study progress on compatible solutes in moderately halophilic bacteria].

Moderately halophilic bacteria which grow best in media with 3% to 15% salt constitute a heterogenous group of microorganisms which belong to different genera. These bacteria can inhabit the salt or soda lakes, coastal lagoons or man-made salterns. Moderately halophilc bacteria living in higher saline environments can not only cope with high osmotic stress but also adapt osmotic shock in short time. To adapt to these environments, all the species make a osmoprotection by the accumulation a restricted range of low molecular mass molecules, small, organic compatible solutes, such as sugars, amino acids, betaines and ectoines. Therefore, the osmoadaptation of moderately halophilc bacteria is regulated by the so-called "compatible solute" strategy. Compatible solutes are operationally defined as organic osmolytes that can be amassed by the cell in exceedingly high concentrations without disturbing vital cellular functions and the correct folding of proteins. As a result, compatible solutes can make important contributions to the restoration of the turgor under conditions of low water activity by counteracting the efflux of water from the cell. In addition, they have a stabilizing, both in vivo and vitro, on the native structure of proteins and cell components. This mechanism has a minimal requirement for genetic change and a high degree of flexibility in allowing moderate halophiles to adapt to saline environment. In this review, the adaptation to saline environments, the variety and characteristic of compatible solutes, and the functional mechanism of moderately halophilic bacteria are reviewed and discussed.

Cloning and characterization of the genes encoding a glycine betaine ABC-type transporter in Halobacillus trueperi DSM10404T.

By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404(T). Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the sigma(A)-dependent promoter of B. subtilis. Reverse transcription (RT)-PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter.

[Sodium ion transportation system and its possible mechanisms in bacteria].

Sodium ion with high concentration is toxic to living cells, and microorganisms adapt to the environment containing high concentration of salt by the strategies of salt-in-cytoplasm and compatible solutes. The Na+ extrusion system plays important roles in maintaining cytoplasmic Na+ homeostasis and pH level in microbial cells. Two possible mechanisms of Na+ circulation across the cytoplasmic membrane have been proposed, namely primary Na+ pump and secondary Na+/H+ antiporter. Primary sodium pumps coupled the extrusion of Na+ to respiration, and the activity of which was insensitive to uncoupler CCCP ( carbonyl-cyanide m-chlorophenylhydrazone). There were two types of secondary Na+/H+ antiporters-encoding genes designated single gene and multiple subunits, respectively. The types of transportation systems for Na+, possible mechanisms of Na+ extrusion, and projects for further study in bacteria are reviewed.

The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter.

Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.