Moderate Halophile Research Articles

Use of halophilic aquaporin for preparation of biomimetic thin film composite membrane

A new aquaporin from a moderate halophile, Halomonas elongata, for fabrication of desalination membrane is introduced in this study. For activity comparison, H. elongata aqp gene and E. coli aqpZ gene were cloned into pGEX-4T-2 vector and expressed in E. coli BL21 cells. E. coli AqpZ (EcAqpZ) appeared as tetrameric form, whereas H. elongata aquaporin (HeAqp) was found mostly as monomers in SDS-PAGE analysis. Light scattering measurements showed that incorporation of aquaporins into the liposomes vesicles resulted in increase in water permeability. HeAqp and EcAqpZ were embedded in 200 nm DOPC liposomes and immobilized with m-phenylene-diamine (MPD) and trimesoyl chloride (TMC) salt rejection layer on polysulfone membrane (PSf). Water with 800 mM NaCl concentration was used for salt rejection experiments. Increasing aquaporin concentration resulted in increase in water permeability of Halomonas aquaporin embedded membranes. % salt rejection of HeAqp and EcAqp incorporated PSf membranes were increased by three fold as compared to control membrane. Salt rejection and water permeability of HeAqp embedded PSf membrane was three and 25 fold higher than that of control membrane, respectively. H. elongata aquaporin, a halophilic aquaporin, was shown to be a suitable candidate for preparation of desalination membranes.

Aminopeptidase Activity of Haloalkalophilic Bacteria of the Genus Halomonas Isolated from the Soda-Saline Lakes in the Badain Jaran Desert

Three strains of haloalkaliphilic bacteria were isolated from microbial mats of soda-saline lakes of the Badain Jaran Desert, Inner Mongolia (China). Based on the data on ribosomal phylogeny, they were identified as members of the genus Halomonas. These bacteria were moderate alkaliphiles and extreme halophiles. The peptidases secreted by these bacteria were shown to have narrow substrate specificity. They hydrolyzed proteins and para-nitroanilide substrates and showed maximal activity in the hydrolysis of L-leucyl-p-nitroanilides (LpNA). The maximum activity of the peptidases occurred at alkaline pH values (8–10) and elevated salinity (50–100 g/L); the enzymes were thermostable (up to 50°С). The results of inhibitor analysis and substrate specificity examination of extracellular enzymes indicated them to belong to the class of aminopeptidase-like metallopeptidases.

Bacteria from the “Saline di Tarquinia” marine salterns reveal very atypical growth profiles with regards to salinity and temperature

Several thousand bacterial colonies were isolated from “Saline di Tarquinia” (Italy) marine salterns; after dereplication, thirty-one strains were obtained, confirming the low culturable bacterial diversity attributed to similar hypersaline environments. Strains were identified by 16S ribosomal DNA sequencing and phylogenetic analysis; most strains belonged to the class Gammaproteobacteria, with minorities from Actinobacteria, Bacilli and Alphaproteobacteria. Halomonas and Salinivibrio were predominant genera. To profile growth preferences, strains were cultivated at different salinities and temperatures. Non-metric multi-dimensional scaling (nMDS) analysis on growth data at different salinities identified four groups that were significantly correlated with the ecological categories conventionally used to describe halophiles. The majority were moderate halophiles with optimal growth at 40-80‰ salinity. Some were markedly euryhaline and grew from 40-80‰ to 240-280‰.Very uncommon behaviour was observed for some strains, with a clear optimum growth in the slight to moderate halophilic range, but also the ability to grow without salt. These bacteria could barely be included in the mentioned traditional definitions and we suggest to consider them as “norm-tolerant halophilic”, being somehow in-between the slight halophiles and the halotolerants. As for temperature, nMDS groups were less defined and uncorrelated to ecological categories. Most strains were mesophilic-psychrotolerant or mesophilic, and some psychrotolerants were also recorded. Generally, diffused eurythermism was observed and various strains displayed unusual “flat growth profiles” that showed a broad range of thermal optima. Overall, the majority of strains appeared to be well adapted to the “Saline di Tarquinia” environment and showed great temperature and salinity variations compared to species observed in other salterns.

Characterization of a Functionally Unknown Arginine-Aspartate-Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues.

Arginine–aspartate–aspartate (RDD) family, representing a category of transmembrane proteins containing one highly conserved arginine and two highly conserved aspartates, has been functionally uncharacterized as yet. Here we present the characterization of a member of this family designated RDD from the moderate halophile Halobacillus andaensis NEAU-ST10-40T and report for the first time that RDD should function as a novel Na+(Li+, K+)/H+ antiporter. It’s more interesting whether the highly conserved arginine/aspartate residues among the whole family or between RDD and its selected homologs are related to the protein function. Therefore, we analyzed their roles in the cation-transporting activity through site-directed mutagenesis and found that D154, R124, R129, and D158 are indispensable for Na+(Li+, K+)/H+ antiport activity whereas neither R35 nor D42 is involved in Na+(Li+, K+)/H+ antiport activity. As a dual representative of Na+(Li+, K+)/H+ antiporters and RDD family proteins, the characterization of RDD and the analysis of its important residues will positively contribute to the knowledge of the cation-transporting mechanisms of this novel antiporter and the roles of highly conserved arginine/aspartate residues in the functions of RDD family proteins.

Possible correlation among osmophilic bacteria, levan yield, and the probiotic activity of three bacterial honey isolates

Abstract Three bacterial strains were isolated from different honey sources. They were identified based on 16S rRNA as Bacillus endophyticus SH, Bacillus subtilis WA and Bacillus subtilis MO. All the isolates had the ability to yield levan in the presence of 80 g/L sucrose with a degree of variation. The osmotic tolerance capacity of the three honey isolates was examined on sucrose, NaCl, sea water, NaCl/ sucrose. The results categorized the isolates as moderate halophiles where they reported their maximum growth between (4–8%) NaCl or (20–50%) sea water, also their highest growth was obtained at 4% sucrose and they were tolerant to sucrose stress till 20%. The probiotic studies revealed that the vegetative cells of the three isolates were highly resistant to severe acidic and alkaline pHs and to different concentrations of bile salt and pancreatic enzymes with variant degrees. Isolates safety was proved by negative blood hemolysis, an absence of hemolytic cytotoxin K (cytK) and non-hemolytic enterotoxin (nheA, nheB, and nheC) genes. In addition, the isolates were sensitive to the tested antibiotics including streptomycin, ampicillin, novobiocin, nalidixic acid, vancomycin, kanamycin, ciprofloxacin, and oxytetracyclin. Finally, they reported varying antimicrobial activities against different pathogens causing human problems. This study suggested B. endophyticus SH for the first time as a safe probiotic bacterium, also recommended the three isolates for using in different aspects such as food supplement.

Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics.

Members of the genus Spiribacter are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. Spiribacter salinus M19-40 is the type species of this genus and its first cultivated representative. In the habitats of S. salinus M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the in silico mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. S. salinus M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical ectABC operon. Instead, they are scrambled (ectAC; ectB) and are physically separated from each other on the S. salinus M19-40 genome. Genomes of many phylogenetically related bacteria also exhibit a non-canonical organization of the ect genes. S. salinus M19-40 also synthesizes trehalose, but this compatible solute seems to make only a minor contribution to the cytoplasmic solute pool under osmotic stress conditions. However, its cellular levels increase substantially in stationary phase cells grown under optimal salt concentrations. In silico genome mining revealed that S. salinus M19-40 possesses different types of uptake systems for compatible solutes. Among the set of compatible solutes tested in an osmostress protection growth assay, glycine betaine and arsenobetaine were the most effective. Transport studies with radiolabeled glycine betaine showed that S. salinus M19-40 increases the pool size of this osmolyte in a fashion that is sensitively tied to the prevalent salinity of the growth medium. It was amassed in salt-stressed cells in unmodified form and suppressed the synthesis of ectoine. In conclusion, the data presented here allow us to derive a genome-scale picture of the cellular adjustment strategy of a species that represents an environmentally abundant group of ecophysiologically important halophilic microorganisms.

Genomic and physiological characterization and description of Marinobacter gelidimuriae sp. nov., a psychrophilic, moderate halophile from Blood Falls, an antarctic subglacial brine

Antarctic subice environments are diverse, underexplored microbial habitats. Here, we describe the ecophysiology and annotated genome of a Marinobacter strain isolated from a cold, saline, iron-rich subglacial outflow of the Taylor Glacier, Antarctica. This strain (BF04_CF4) grows fastest at neutral pH (range 6-10), is psychrophilic (range: 0°C-20°C), moderately halophilic (range: 0.8%-15% NaCl) and hosts genes encoding potential low temperature and high salt adaptations. The predicted proteome suggests it utilizes fewer charged amino acids than a mesophilic Marinobacter strain. BF04_CF4 has increased concentrations of membrane unsaturated fatty acids including palmitoleic (33%) and oleic (27.5%) acids that may help maintain cell membrane fluidity at low temperatures. The genome encodes proteins for compatible solute biosynthesis and transport, which are known to be important for growth in saline environments. Physiological verification of predicted metabolic functions demonstrate BF04_CF4 is capable of denitrification and may facilitate iron oxidation. Our data indicate that strain BF04_CF4 represents a new Marinobacter species, Marinobacter gelidimuriae sp. nov., that appears well suited for the subglacial environment it was isolated from. Marinobacter species have been isolated from other cold, saline environments in the McMurdo Dry Valleys and permanently cold environments globally suggesting that this lineage is cosmopolitan and ecologically relevant in icy brines.

Microbial community structure of a low sulfate oil producing facility indicate dominance of oil degrading/nitrate reducing bacteria and Methanogens

ABSTRACTAnalysis of microbial community structure of a low sulfate oil producing facility in Nigeria using 16S rRNA gene sequencing technique revealed dominance of oil degrading and nitrate reducing bacteria and methanogenic archaea in produced waters and oil samples namely, Marinobacter (37%), Azovibrio (21%), Thauera (10–28%), and Methanolobus (22%). On the contrary, the associated oil pipeline samples revealed massive dominance of potentially corrosive Methanolobus (60%) and Methanobacterium (25-27%). Further experimentation shows that the methanogens implicated in oil pipelines are corrosive moderate halophile that utilizes H2/CO2 and methanol as substrates. More emphasis should therefore be on methanogenic archaea as opposed to sulfate reducing bacteria (SRBs) during mitigation plans for microbially induced corrosion (MIC) in a low sulfate oil producing facility.

Biodegradation of phenol by a moderately halophilic bacterial consortium

Bacterial degradation of phenols in the environment is influenced by number of factors such as salinity, pH, temperature, and nutrient availability. Halophiles are the group of microorganisms which could grow at saline conditions and thus renders a variety of environmental applications. Moderate halophiles can grow at 3–5% NaCl concentration and are able to utilize a diverse class of toxic pollutants persists in the environment as a result of industrial activities. The moderately halophilic bacterial consortia enriched from wastewater collected from Industrial Effluent Treatment Plant of Yanbu, Saudi Arabia utilized phenol as sole carbon source for its growth. The halophilic consortia could survive and utilize phenol at higher salt concentrations (8% and 12% NaCl). Yeast extract supplied as additional substrate significantly enhanced the phenol biodegradation at higher salt concentrations. The consortia effectively degraded 100% of added phenol within 4 days at haloalkaline conditions (4% NaCl, pH 10). Addition of urea as alternate nitrogen substrate greatly reduced the lag time of the bacterial consortia for the phenol biodegradation. PCR‐DGGE analysis of the moderately halophilic bacterial consortia revealed the presence of three dominant strains which are identified as Ochrobactrum sp. SAR1, Pseudomonas sp. SAR2, and Ochrobactrum sp. SAR3. Through this study, the moderately halophilic bacterial consortia can serve as versatile candidate for the treatment of phenol present in saline and alkaline industrial wastewaters. © 2017 American Institute of Chemical Engineers Environ Prog, 37: 1587–1593, 2018

Salt-tolerance aerobic granular sludge: Formation and microbial community characteristics

The salt-tolerance aerobic granular sludge (SAGS) dominated by moderately halophilic bacteria was successfully cultured in a 9% (w/v) salty, lab-scale sequence batch reactor (SBR) system. Influence of high salinity (0–9% w/v NaCl) on the formation, performance and microbial succession of the SAGS were explored. Crystal nucleus hypothesis, selection pressure hypothesis and compressed double electric layers hypothesis were used to discuss the formation mechanism of SAGS. Notably, salinity could be seen as a kind of selection pressure contributed to the formation of SAGS, while salinity also declined the performance of SAGS system. High throughput 16S rRNA gene analysis showed that the salinity had great influence on the species succession and community structure of SAGS. Moreover, Salinicola and Halomonas were dominant at 9% salt concentration, therefore moderate halophiles were identified as functional groups for the tolerance of hypersaline stress.

Recovery of metallo-tolerant and antibiotic resistant psychrophilic bacteria from Siachen glacier, Pakistan.

Cultureable bacterial diversity of previously unexplored Siachen glacier, Pakistan, was studied. Out of 50 isolates 33 (66%) were Gram negative and 17 (34%) Gram positive. About half of the isolates were pigment producers and were able to grow at 4–37°C. 16S rRNA gene sequences revealed Gram negative bacteria dominated by Proteobacteria (especially γ-proteobacteria and β-proteobacteria) and Flavobacteria. The genus Pseudomonas (51.51%, 17) was dominant among γ- proteobacteria. β-proteobacteria constituted 4 (12.12%) Alcaligenes and 4 (12.12%) Janthinobacterium strains. Among Gram positive bacteria, phylum Actinobacteria, Rhodococcus (23.52%, 4) and Arthrobacter (23.52%, 4) were the dominating genra. Other bacteria belonged to Phylum Firmicutes with representative genus Carnobacterium (11.76%, 2) and 4 isolates represented 4 genera Bacillus, Lysinibacillus, Staphylococcus and Planomicrobium. Most of the Gram negative bacteria were moderate halophiles, while most of the Gram positives were extreme halophiles and were able to grow up to 6.12 M of NaCl. More than 2/3 of the isolates showed antimicrobial activity against multidrug resistant S. aureus, E. coli, Klebsiella pneumonia, Enterococcus faecium, Candida albicans, Aspergillus flavus and Aspergillus fumigatus and ATCC strains. Gram positive bacteria (94.11%) were more resistant to heavy metals as compared to Gram negative (78.79%) and showed maximum tolerance against iron and least tolerance against mercury.

A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter

In this study, genomic DNA was screened from Halobacillus andaensis NEAU-ST10-40T by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated upf0118 exhibiting Na+(Li+)/H+ antiport activity was finally cloned. Protein alignment showed that UPF0118 shares the highest identity of 81.5% with an unannotated gene encoding a protein with uncharacterized protein function belonging to UPF0118 family from H. kuroshimensis, but shares no identity with all known specific Na+(Li+)/H+ antiporter genes or genes with Na+(Li+)/H+ antiport activity. Growth test, western blot and Na+(Li+)/H+ antiport assay revealed that UPF0118 as a transmembrane protein exhibits pH-dependent Na+(Li+)/H+ antiport activity. Phylogenetic analysis indicated that UPF0118 clustered with all its homologs belonging to UPF0118 family at a wide range of 22–82% identities with the bootstrap value of 92%, which was significantly distant with all known specific single-gene Na+(Li+)/H+ antiporters and single-gene proteins with the Na+(Li+)/H+ antiport activity. Taken together, we propose that UPF0118 should represent a novel class of Na+(Li+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of a protein with uncharacterized protein function as a representative of UPF0118 family containing the domain of unknown function, DUF20.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500mM NaCl at pH 6.0, fourfold by 250mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila.

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L-1 and to LiCl up to 0.2 mol·L-1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

Fluorescent Lead(IV) Sulfide Nanoparticles Synthesized by Idiomarina sp. Strain PR58-8 for Bioimaging Applications.

The fabrication of nanoparticles by microorganisms presents a "green" method for generating biocompatible nanomaterials. We discovered the intracellular biosynthesis of fluorescent lead(IV) sulfide nanoparticles by the moderate halophile, Idiomarina sp. strain PR58-8. The bacterium tolerated up to 8 mM Pb(NO3)2 during growth. Non-protein thiols dose-dependently increased in response to metal exposure, which suggests they are involved in the growth of PbS2 crystals and lead detoxification. Using X-ray diffraction, transmission electron microscopy (TEM), high-resolution TEM, and energy dispersive analysis of X-rays, the nanoparticles were characterized as spherical β-PbS2 nanoparticles (PbS2NPs) with a tetragonal crystal lattice, a crystallite domain size of 2.38 nm, and an interplanar distance of 0.318 nm. A narrow symmetric emission spectrum with a Gaussian distribution and an emission maximum at 386 nm was obtained when the particles were excited at 570 nm. The PbS2NPs exhibited a large Stokes' shift (8,362 cm-1) and a relatively high quantum yield (67%). These properties, along with fluorescence that was maintained in various microenvironments and their biocompatibility, make these nanoparticles excellent candidates for bioimaging. The particles were internalized by HeLa cells and evenly distributed within the cytoplasm, exhibiting their potential for in situ bioimaging applications. The "as-synthesized" lead(IV) sulfide nanoparticles may provide expanded opportunities for targeted bioimaging via modifying the surface of the particles.IMPORTANCE This article reports the intracellular synthesis of fluorescent lead(IV) sulfide nanoparticles (PbS2NPs) by a microorganism. All previous reports on the microbial synthesis of lead-based nanoparticles are on lead(II) sulfide that exhibits near-infrared fluorescence, requiring expensive instrumentation for bioimaging. Bioimaging using PbS2NPs can be achieved using routine epifluorescence microscopes, as it fluoresces in the visible range. The research on PbS2 nanoparticles to date is on their chemical synthesis employing toxic precursors, extreme pH, pressure, and temperature, resulting in cytotoxic products. In this context, the synthesis of PbS2 nanoparticles by Idiomarina sp. strain PR58-8, described in this work, occurs at ambient temperature and pressure and results in the generation of biocompatible nanoparticles with no hazardous by-products. The excellent fluorescence properties that these particles exhibit, as well as their abilities to easily penetrate the cells and evenly distribute within the cytoplasm, make them exceptional candidates for bioimaging applications. This study demonstrated the synthesis and fluorescence bioimaging application of microbially synthesized PbS2 nanoparticles.

Phylogenetic analysis of the bacterial community in a crystallizer pond, Pomorie salterns, Bulgaria

ABSTRACTThe aim of this study was to investigate the bacterial community habituating P18, the biggest crystallizer pond in Pomorie salterns (34% salinity). The obtained results showed that the bacterial community differs from many previous reports of low bacterial diversity in hypersaline environments and demonstrates unusually high diversity of presented taxa, some unusual domination of diverse genera not reported before as dominant and identification of previously unknown 16S rRNA sequences. The retrieved 23 bacterial operational taxonomic units (OTUs) affiliated with 15 bacterial genera from four phyla – Firmicutes, 47.5%; Proteobacteria, 23.1%; Bacteroidetes, 22%; Deinococcus–Thermus, 2.4%; and one-candidate division SR1, 4.8%. Representatives of the phylum Firmicutes predominated in the bacterial community with almost half of the retrieved sequences. Almost all clones branched together with cultured halophiles or uncultured clones retrieved from saline niches. Despite of the high salt concentration, some of the closest phylogenetic neighbours were moderate halophiles. New sequences represented 42.3% of bacterial OTUs. Some of them formed separate branches with similarity less than 85%.

Isolation and identification of culturable halophilic bacteria with producing hydrolytic enzyme from Incheh Broun hypersaline wetland in Iran.

Incheh Broun hypersaline wetland is located near the border of Turkmenistan in thenorth of Iran. This wetland is notable because of salinity (280g/l) and alteration in pH range (2.8 to 6.8). Eastern part of wetland is affected by wastewater of iodine extraction factory. Samples were taken from soil, water and salt. Totally, 400 bacterial strains were isolated of which 194 strains were Gram-positive bacilli, 184 strains were Gram-negative rod and 22 strains were Gram-positive cocci. According to phylogenetic analysis of 16S rRNA, selected strains were placed in three taxonomic phyla including Firmicutes, Actinobacteria and Gammaproteobacteria. Optimum growth was evaluated for salt and 22 strains were found to be moderate halophile and 33 strains were halotolerant. Production of lipase, amylase, gelatinase and protease was examined. Gram-positive bacilli were the main producers of hydrolytic enzymes. Gelatinase and protease were the most frequent enzymes. Gram-positive cocci were the main producers of lipase but they didn't produce amylase.

Salinivibrio costicola GL6, a Novel Isolated Strain for Biotransformation of Caffeine to Theobromine Under Hypersaline Conditions

The present study has been conducted towards isolation of moderately halophilic bacteria capable of transforming caffeine into theobromine. A total of 45 caffeine-degrading moderate halophiles were enriched from hypersaline lakes and examined for the biotransformation of caffeine to theobromine by thin-layer chromatography (TLC) and high-performance liquid chromatography analyses. Strain GL6, giving the highest yield of theobromine, was isolated from the Hoz Soltan Lake, 20% w/v salinity, central Iran, and identified as Salinivibrio costicola based on morphological and biochemical features as well as its 16S rRNA gene sequence analysis (GeneBank Accession No. KT378066) and DNA-DNA relatedness. The biotransformation of caffeine with strain GL6 leads to the formation of two metabolites, identified as theobromine and paraxanthine, but the yield of paraxanthine was much lower. Further study on the production of theobromine from caffeine under resting cell experiment was carried out subsequently. The optimal yield of theobromine (56%) was obtained after a 32-h incubation using 5mM of caffeine and 15gl-1 (wet weight) of biomass in 0.1M saline phosphate buffer (pH 7.0 and 10% w/v NaCl) under agitation 180rpm at 30°C. The biotransformed theobromine was purified by preparative TLC and subjected to FTIR and mass spectroscopy for chemical identification. This is the first evidence for biotransformation of caffeine into theobromine by strains of the genus Salinivibrio.

Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli

Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called “bacterial milking” efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL−1h−1 respectively. The overall ectoine yield was 0.11g/g of glucose.

Integrated molecular, physiological and in silico characterization of two Halomonas isolates from industrial brine.

Two haloalkaliphilic bacteria isolated from industrial brine solutions were characterized via molecular, physiological, and in silico metabolic pathway analyses. Genomes from the organisms, designated Halomonas BC1 and BC2, were sequenced; 16S ribosomal subunit-based phylogenetic analysis revealed a high level of similarity to each other and to Halomonas meridiana. Both strains were moderate halophiles with near optimal specific growth rates (≥60% μ max) observed over <0.1-5% (w/v) NaCl and pH ranging from 7.4 to 10.2. Isolate BC1 was further characterized by measuring uptake or synthesis of compatible solutes under different growth conditions; in complex medium, uptake and accumulation of external glycine betaine was observed while ectoine was synthesized de novo in salts medium. Transcriptome analysis of isolate BC1 grown on glucose or citrate medium measured differences in glycolysis- and gluconeogenesis-based metabolisms, respectively. The annotated BC1 genome was used to build an in silico, genome-scale stoichiometric metabolic model to study catabolic energy strategies and compatible solute synthesis under gradients of oxygen and nutrient availability. The theoretical analysis identified energy metabolism challenges associated with acclimation to high salinity and high pH. The study documents central metabolism data for the industrially and scientifically important haloalkaliphile genus Halomonas.