Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis

Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, l-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the l-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus.     

Analysis of Protein Expression Profiles of <Emphasis Type="Italic">Halobacillus dabanensis</Emphasis> D-8<Superscript>T</Superscript> Under Optimal and High Salinity Conditions

Two-dimensional (2-D) gel electrophoresis was employed to display the expression profiles of proteins of Halobacillus dabanensis D-8^T under 1%, 10%, and 20% salinities. Approximately 700 protein spots could be detected in the 2-D gels by Imagemaster™ 2D Platinum software. The molecular masses of the majority of intracellular proteins were distributed in the range of 17.5 kDa–66 kDa and isoelectric points of 4.0–5.9. In total 133 protein spots were observed with a changed expression level under different salinity conditions. Sixty-two protein spots showed upregulation and 26 new protein spots were found under high salinity conditions, while 25 protein spots were downregulated and 20 spots disappeared. Twenty-seven proteins with a markedly changed expression in hypersaline environments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and MASCOT. A changed expression pattern was observed for proteins related to energy-producing pathways, stress regulators, and proteins involved in the survival of strain D-8^T under high salt challenges. Many proteins play necessary roles in the adaptation to high salt or as a general stress protein, and some proteins are salt-stressed specific proteins that improve the capability of salt-tolerance of strain D-8^T growth under extremely hypersaline condition.     

Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z”

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles.     

转录组学分析盐单胞菌四氢嘧啶合成代谢相关的表达差异基因与qRT-PCR验证

[目的]探究盐适应条件下坎帕尼亚盐单胞菌(Halomonas campaniensis)的差异基因表达水平,挖掘四氢嘧啶(ectoine)合成代谢相关联的差异基因.[方法]设置无盐组NS(0 mol/LNaCl)、中盐组 MS(1.5 mol/L NaCl)和高盐组 HS(2.5 mol/L NaCl),培养H.cam...     

Taxonomic description and genome sequence of <Emphasis Type="Italic">Halobacillus marinus</Emphasis> sp. nov., a novel strain isolated from Chilika Lake,India

moderately halophilic spore forming, motile, Gram-positive, rod-shaped bacterial strain designated as KGW1^T was isolated from water sample of Chilika Lake and characterized taxonomically using polyphasic approach. The strain grew in the presence of 0–25% (w/v) NaCl in marine salt agar media, hydrolyzes casein, and gelatin and shows presence of alkaline proteases. The major cell wall menaquinone was MK7 and major cellular fatty acids were anteiso-C_15:0 (44.89%), anteiso-C_17:0 (6.18%), isoC_15:0 (19.38%), and iso-C_16:0 (7.39%). Several chemotaxonomic features conform the isolate be a member of genus Halobacillus. The isolate KGW1^T contained A1γ meso-Dpm-direct type of peptidoglycan which is different from its phylogenetically closest neighbours. The 16S rRNA gene sequence based phylogenetic analysis also revealed the strain KGW1^T was affiliated to the genus Halobacillus and sequence similarity between the isolated strain and the type strains of Halobacillus species were found closest to, H. dabanensis D-8 DSM 18199^T (99.08%) and H. faecis IGA7-4 DSM 21559^T (99.01%), H. trueperi SL-5 DSM 10404^T (98.94%). The in silico DDH showed that the values in a range of 14.2–17.5% with the most closest strain H. dabanensis D-8 DSM 18199^T and other type strains of the genus Halobacillus for which whole genome sequence is reported. DNA-DNA relatedness between strain KGW1^T and the closest type strain Halobacillus trueperi DSM 10404^T was 11.75% (± 1.15). The draft genome sequence includes 3,683,819 bases and comprises of 3898 predicted coding sequences with a G + C content of 46.98%. Thus, the significant distinctiveness supported by phenotypic and genotypic data with its closest neighbors and other closely related species confirm the strain KGW1^T to be classified as a novel species within the genus Halobacillus, for which the name Halobacillus marinus sp. nov. is proposed. The type strain is KGW1^T (= DSM 29522 = JCM 30443).     

Functional characterization,secondary structure prediction and analysis of ectoine biosynthesis genes from <Emphasis Type="Italic">Bacillus halodurans</Emphasis>: an osmolyte involved in stress tolerance

An open reading frame encoding the ectoine biosynthesis genes was cloned from the Bacillus halodurans genome. An expression plasmid containing the operon was introduced into Escherichia coli cells, and the recombinant ectoine was quantified. The secondary structure of ectoine biosynthesis proteins were predicted and was quite similar to that of reported proteins from eubacteria.     

Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter⁻¹ ectoine with a space-time yield of 40 mg liter⁻¹ h⁻¹, with the vast majority of the ectoine being excreted.     

Characterization and phylogenetic analysis of ectoine biosynthesis genes from <Emphasis Type="Italic">Bacillus halodurans</Emphasis>

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved.     

Pseudoruegeria limi sp. nov. isolated from mud flats in the Yellow Sea in Korea

A Gram-negative, aerobic, non-motile and rod-shaped bacterial strain, D-17^T, was isolated from mud flats in the Yellow Sea in Korea. Phylogenetic trees based on the 16S rRNA gene sequence showed that strain D-17^T belongs to the genus Pseudoruegeria and it shared 97.5 % similarity with the type strain of Pseudoruegeria haliotis WM67^T. The sequence similarities with Pseudoruegeria litimaris HD-43^T and Pseudoruegeria aquimaris SW-255^T were 96.9 and 96.1 %, respectively. Strain D-17^T was found to grow with 0.5–6 % (w/v) NaCl, at 20–30 °C, and at pH 6.5–8.0. Strain D-17^T was determined to contain Q-10 as the predominant ubiquinone and summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c, as defined by the MIDI system) as the major fatty acids. The major polar lipids were identified as phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid, an unidentified glycolipid, an unidentified lipid and four unidentified phospholipids. The DNA G+C content was determined to be 63.6 mol%. The DNA–DNA relatedness with P. haliotis WM67^T was 32.5 %. The differential phenotypic properties revealed that strain D-17^T can be separated from other Pseudoruegeria species. Based on the data presented in this study, strain D-17^T represents a novel species, for which the name Pseudoruegeria limi sp. nov. is proposed. The type strain is D-17^T (=KCTC 32460^T =JCM 19487^T).     

A process for the production of ectoine and poly(3-hydroxybutyrate) by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH₄⁺, and PO₄³⁻. In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.     

Production of ectoine through a combined process that uses both growing and resting cells of <Emphasis Type="Italic">Halomonas salina</Emphasis> DSM 5928<Superscript>T</Superscript>

Using ectoine-excreting strain Halomonas salina DSM 5928^T, we developed a new process for high-efficiency production of ectoine, which involved a combined process of batch fermentation by growing cells and production by resting cells. In the first stage, batch fermentation was carried out using growing cells under optimal fermentation conditions. The second stage was the production phase, in which ectoine was synthesized and excreted by phosphate-limited resting cells. Optimal conditions for synthesis and excretion of ectoine during batch fermentation in a 10 l fermentor were 0.5 mol l⁻¹ NaCl and an initial monosodium glutamate concentration of 80 g l⁻¹ respectively. The pH was adjusted to 7.0 and the temperature was maintained at 33°C. In phosphate-limited resting cells medium, monosodium glutamate and NaCl concentration was 200 g l⁻¹ and 0.5 mol l⁻¹, respectively, as well as pH was 7.0. The total concentration of ectoine produced was 14.86 g l⁻¹, the productivity and yield of ectoine was 7.75 g l⁻¹ day⁻¹ and 0.14 g g⁻¹, respectively, and the percentage of ectoine excreted was 79%. These levels of ectoine production and excretion are the highest reported to date.     

Comparative genomics analysis of Nitriliruptoria reveals the genomic differences and salt adaptation strategies

The group Nitriliruptoria, recently classified as a separate class of phylum Actinobacteria, has five members at present, which belong to halophilic or halotolerant Actinobacteria. Here, we sequenced the genomes of Egicoccus halophilus EGI 80432^T and Egibacter rhizosphaerae EGI 80759^T, and performed a comparative genomics approach to analyze the genomic differences and salt adaptation mechanisms in Nitriliruptoria. Phylogenetic analysis suggested that Euzebya tangerina F10^T has a closer phylogenetic relationship to Euzebya rosea DSW09^T, while genomic analysis revealed highest genomic similarity with Nitriliruptor alkaliphilus ANL-iso2^T and E. halophilus EGI 80432^T. Genomic differences of Nitriliruptoria were mainly observed in genome size, gene contents, and the amounts of gene in per functional categories. Furthermore, our analysis also revealed that Nitriliruptoria possess similar synthesis systems of solutes, such as trehalose, glutamine, glutamate, and proline. On the other hand, each member of Nitriliruptoria species possesses specific mechanisms, K⁺ influx and efflux, betaine and ectoine synthesis, and compatible solutes transport to survive in various high-salt environments.

     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

Marinobacter alkaliphilus sp. nov., a novel alkaliphilic bacterium isolated from subseafloor alkaline serpentine mud from Ocean Drilling Program Site 1200 at South Chamorro Seamount, Mariana Forearc

Novel alkaliphilic, mesophilic bacteria were isolated from subseafloor alkaline serpentine mud from the Ocean Drilling Program (ODP) Hole 1200D at a serpentine mud volcano, South Chamorro Seamount in the Mariana Forearc. The cells of type strain ODP1200D-1.5^T were motile rods with a single polar flagellum. Growth was observed between 10 and 45–50°C (optimum temperature: 30–35°C, 45-min doubling time), between pH 6.5 and 10.8–11.4 (optimum: pH 8.5–9.0), and between NaCl concentrations of 0 and 21% (w/v) (optimum NaCl concentration: 2.5–3.5%). The isolate was a facultatively anaerobic heterotroph utilizing various complex substrates, hydrocarbons, carbohydrates, organic acids, and amino acids. Nitrate or fumarate could serve as an electron acceptor to support growth under anaerobic conditions. The G+C content of the genomic DNA was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Marinobacter and was the most closely related to M. aquaeolei strain VT8^T and M. hydrocarbonoclasticus strain SP.17^T, while DNA–DNA hybridization demonstrated that the new isolate could be genetically differentiated from the previously described species of Marinobacter. Based on the physiological and molecular properties of the new isolate, we propose the name Marinobacter alkaliphilus sp. nov., type strain: ODP1200D-1.5^T (JCM12291^T and ATCC BAA-889^T).