Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.     

A Novel DNA-Binding Protein,PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.     

Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei

The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.     

Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei

The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC(Hme)) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE(Hme) and phaC(Hme) genes were constitutively expressed, and both the PhaE(Hme) and PhaC(Hme) proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC(Hme) genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC(Hme) genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC(Hme) was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE(Hme)/PhaC(Hme) (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC(Hme) and PhaE(Hme), accounted for the PHBV synthesis in H. mediterranei.     

Analysis of acidic surface of Haloferax mediterranei glucose dehydrogenase by site-directed mutagenesis

Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of glucose dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.     

Global Gene Expression Analysis of Long-Term Stationary Phase Effects in E. coli K12 MG1655

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.     

Evidence for salt-associated restriction pattern modifications in the archaeobacterium Haloferax mediterranei.

DNA restriction pattern modifications were detected when Haloferax mediterranei was grown in low (10%) salt concentrations. After cells were grown again in optimal (25%) salt concentrations, the original pattern was recovered. These salt-associated DNA modifications were revealed with 5% of the 160 DNA fragments cloned and used as probes in hybridization experiments. Patterns obtained when genomic DNA was digested with different restriction enzymes showed that these modifications are related not to insertions or deletions in genome but to modifications of some specific sequences.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Products of non-reductive CO2 assimilation in the halophilic archaebacterium Haloferax mediterranei.

The products of CO2 fixation by heterotrophically grown Haloferax mediterranei were analysed. The main 14C-labelled alpha-ketoacid detected following incubation with NaH14CO3 and pyruvate or propionate was pyruvate. In presence of these organic acids and NH4+, 14CO2 was incorporated into glutamic and aspartic acids and alanine.     

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5–3 M NaCl. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

Background

Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal Findings

Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion

The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.     

地中海富盐菌中phaB基因的鉴定及PHBV前体供应途径的分析

【目的】进一步揭示地中海富盐菌(Haloferax mediterranei)中聚羟基丁酸羟基戊酸酯[poly(3-hydroxybutyrate-co-3-hydroxyvalerate),PHBV]前体供应的途径并鉴定其中的关键基因。【方法】将细菌及其他古菌中已鉴定的与聚羟基脂肪酸酯前体(3-羟基脂酰-CoA)供应相关的酶与地中海富盐菌预测的全蛋白质组进行同源性比对,得到相似性比较高的5个基因,分别命名为:phaB1,phaB2,phaJ1,phaJ2和phaJ3。首先利用RT-PCR检测了5个基因在产PHBV的条件下的转录情况。然后利用同源重组双交换的方法,将5个基因分别或组合敲除,得到突变株:ΔphaB1,ΔphaB2,ΔphaJ1,ΔphaJ2,ΔphaJ3,ΔphaB1phaB2,ΔphaJ1phaJ2和ΔphaJ1phaJ2phaJ3。并在突变株ΔphaB1phaB2中分别互补phaB1和phaB2基因。【结果】无论是将3个phaJ基因单独敲除,还是组合敲除,对地中海富盐菌PHBV的积累都没有明显影响。单独敲除phaB1基因对PHBV的积累没有明显影响,单独敲除phaB2基因导致突变株PHBV产量明显下降,而且3-HV单体组分所占的摩尔比例也有所下降。将phaB1和phaB2基因同时敲除后,得到的突变株不再产生PHBV。【结论】在地中海富盐菌中可能主要存在由乙酰-CoA和丙酰-CoA提供PHBV前体的途径,其中编码乙酰乙酰-CoA还原酶的两个基因即为phaB1和phaB2。