Biocontrol of biofilm forming Burkholderia cepacia using a quorum quenching crude lactonase enzyme extract from a marine Chromohalobacter sp. strain D23.

Biofilm plays advantageous role in Burkholderia cepacia by exerting multi-drug resistance. As quorum sensing (QS) system regulates biofilm formation and pathogenicity in B. cepacia strains, quorum quenching (QQ) may be a novel strategy to control persistent B. cepacia infections. In these regards, 120 halophilic bacteria were isolated from marine sample and tested using Chromobacterium violaceum and C. violaceum CV026-based bioassays initially, showing reduced violacein synthesis by QQ enzyme by 6 isolates. Among them, Chromohalobacter sp. D23 significantly degraded both C6-homoserine lactone (C6-HSL) and C8-HSL due to potent lactonase activity, which was detected by C. violaceum CV026 biosensor. Further high-performance liquid chromatography (HPLC) study confirmed degradation of N-acyl homoserine lactones (N-AHLs) particularly C6-HSL and C8-HSL by crude lactonase enzyme. Chromohalobacter sp. D23 reduced biofilm formation in terms of decreased total biomass and viability in biofilm-embedded cells in B. cepacia significantly which was also evidenced by fluorescence microscopic images. An increase in antibiotic susceptibility of B. cepacia biofilm was achieved when crude lactonase enzyme of Chromohalobacter sp. strain D23 was combined with chloramphenicol (1-5 × MIC). Chromohalobacter sp. D23 also showed prominent decrease in QS-mediated synthesis of virulence factors such as extracellular polymeric substances (EPS), extracellular protease, and hemolysin in B. cepacia. Again crude lactonase enzyme of Chromohalobacter sp. strain D23 inhibited B. cepacia biofilm formation inside nasal oxygen catheters in vitro. Finally, antibiotic susceptibility test and virulence tests revealed sensitivity of Chromohalobacter sp. strain D23 against a wide range of conventional antibiotics as well as absence of gelatinolytic, hemolytic, and serum coagulating activities. Therefore, the current study shows potential quorum quenching as well as anti-biofilm activity of Chromohalobacter sp. D23 against B. cepacia.

Whole-genome sequencing and genome-scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use.

Salt tolerant organisms are increasingly being used for the industrial production of high-value biomolecules due to their better adaptability compared to mesophiles. Chromohalobacter canadensis is one of the early halophiles to show promising biotechnology potential, which has not been explored to date. Advanced high throughput technologies such as whole-genome sequencing allow in-depth insight into the potential of organisms while at the frontiers of systems biology. At the same time, genome-scale metabolic models (GEMs) enable phenotype predictions through a mechanistic representation of metabolism. Here, we sequence and analyze the genome of C. canadensis 85B, and we use it to reconstruct a GEM. We then analyze the GEM using flux balance analysis and validate it against literature data on C. canadensis. We show that C. canadensis 85B is a metabolically versatile organism with many features for stress and osmotic adaptation. Pathways to produce ectoine and polyhydroxybutyrates were also predicted. The GEM reveals the ability to grow on several carbon sources in a minimal medium and reproduce osmoadaptation phenotypes. Overall, this study reveals insights from the genome of C. canadensis 85B, providing genomic data and a draft GEM that will serve as the first steps towards a better understanding of its metabolism, for novel applications in industrial biotechnology.

A new carbonic anhydrase identified in the Gram-negative bacterium (Chromohalobacter sp.) and the interaction of anions with the enzyme.

In this study, the characterization and inhibition characteristic of α-class carbonic anhydrase from Chromohalobacter (ChCA) was documented for the first time. The carbonic anhydrase enzyme had 47.77% yield and 54.45-fold purity. The specific activity of the enzyme was determined as 318.52 U/mg proteins. Alternative substrate (4-nitrophenyl trifluoroacetate, 4-nitrophenyl phosphate, 4-nitrophenyl sulphate and 4-nitrophenyl acetate) were tested for the enzyme. KM and Vmax values for 4-nitrophenyl acetate were 4.57 mM and 4.29 EU/mL and for 4-nitrophenyl trifluoroacetate were 2.39 mM and 2.41 EU/mL. The anions, Cl-, NO2-, NO3-, Br-, ClO3-, ClO4-, I-, CO32- and SO42-, inhibited the ChCA hydratase activity. Among nine anions, the strongest inhibitor activities were obtained with micro molar concentrations of NO2-, NO3-, Br-, I-, CO32- (KI values of 160-255 µM). Other four anions tested (Cl-, ClO3-, ClO4- and SO42-) showed moderate inhibitory activities (KI values of 680-813.5 µM). The results obtained demonstrate that the anions we tested inhibit the Chromohalobacter CA (ChCA) enzyme as in other α-CAs in mammals; however, the susceptibility of ChCA resulted from anions differed significantly from that of other organism CAs.

Microbiota dynamics and volatile compounds in lupine based Moromi fermented at different salt concentrations.

Fermented soy sauces are used as food seasonings in Eastern countries and all over the world. Depending on their cultural origins, their production differs in parameters such as wheat addition, temperature, and salt concentration. The fermentation of lupine seeds presents an alternative to the use of soybeans; however, the microbiota and influencing factors are currently unknown. In this study, we analyse the microbiota of lupine Moromi (mash) fermentations for a period of six months and determine the influence of different salt concentrations on the microbiota dynamics and the volatile compound composition. Cultured microorganisms were identified by protein profiling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene amplicon sequencing provided an overview of the microbiota including non-cultured bacteria. The volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS). At all salt concentrations, we found that Tetragenococcus halophilus (up to 1.4 × 109 colony forming units (CFU)/mL on day 21) and Chromohalobacter japonicus (1.9 × 109 CFU/mL, day 28) were the dominating bacteria during Moromi fermentation. Debaryomyces hansenii (3.6 × 108 CFU/mL, day 42) and Candida guilliermondii (2.2 × 108 CFU/mL, day 2) were found to be the most prevalent yeast species. Interestingly, Zygosaccharomyces rouxii and other yeasts described as typical for soy Moromi were not found. With increasing salinity, we found lower diversity in the microbiota, the prevalence-gain of typical species was delayed, and ratios differed depending on their halo- or acid tolerance. GC-MS analysis revealed aroma-active compounds, such as pyrazines, acids, and some furanones, which were mostly different from the aroma compounds found in soy sauce. The absence of wheat may have caused a change in yeast microbiota, and the use of lupine seeds may have led to the differing aromatic composition. Salt reduction resulted in a more complex microbiome, higher cell counts, and did not show any spoiling organisms. With these findings, we show that seasoning sauce that uses lupine seeds as the sole substrate is a suitable gluten-free, soy-free and salt reduced alternative to common soy sauces with a unique flavour.

Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+ The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG- mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants.IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.

Potential for biocolloid transport of cesium at high ionic strength.

In subsurface repositories, active bacterial populations may directly influence the fate and transport of radionuclides including in salt repository systems like the Waste Isolation Pilot Plant in Carlsbad, NM. This research quantified the potential for transport and interaction between Chromohalobacter sp. and Cs in a high ionic strength system (2.6 M NaCl) containing natural minerals. Mini-column experiments showed that Chromohalobacter moved nearly un-retarded under these conditions and that there was neither association of Cs with microbes nor dolomite despite changes in bacterial metabolic phases. Growth batch experiments that monitored the potential uptake of Cs into the microbes confirmed results in column experiments where intracellular uptake of Cs by Chromohalobacter was not observed. These results show that Cs may be highly mobile if released in high ionic strength systems and/or carbonate minerals with negligible inhibition by these microbes.

Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. RESULTS: Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. CONCLUSIONS: Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.

Extracellular polymer substance synthesized by a halophilic bacterium Chromohalobacter canadensis 28.

Halophilic microorganisms are producers of a lot of new compounds whose properties suggest promising perspectives for their biotechnological exploration. Moderate halophilic bacterium Chromohalobacter canadensis 28 was isolated from Pomorie salterns as an extracellular polymer substance (EP) producer. The best carbon source for extracellular polymer production was found to be lactose, a sugar received as a by-product from the dairy industry. After optimization of the culture medium and physicochemical conditions for cultivation, polymer biosynthesis increased more than 2-fold. The highest level of extracellular polymer synthesis by C. canadensis 28 was observed in an unusually high NaCl concentration (15% w/v). Chemical analysis of the purified polymer revealed the presence of an exopolysaccharide (EPS) fraction (14.3% w/w) and protein fraction (72% w/w). HPLC analysis of the protein fraction showed the main presence of polyglutamic acid (PGA) (75.7% w/w). EPS fraction analysis revealed the following sugar composition (% w/w): glucosamine 36.7, glucose 32.3, rhamnose 25.4, xylose 1.7, and not identified sugar 3.9. The hydrogel formed by PGA and EPS fractions showed high swelling behavior, very good emulsifying and stabilizing properties, and good foaming ability. This is the first report for halophilic bacterium able to synthesize a polymer containing PGA fraction. The synthesized biopolymer shows an extremely high hydrophilicity, due to the simultaneous presence of PGA and EPS. The analysis of its functional properties and the presence of glucosamine in the highest proportion in EPS fraction clearly determine the potential of EP synthesized by C. canadensis 28 for application in the cosmetics industry.

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Insights into metabolic osmoadaptation of the ectoines-producer bacterium Chromohalobacter salexigens through a high-quality genome scale metabolic model.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore and use C. salexigens as cell factory for ectoine(s) production, a comprehensive knowledge at the systems level of its metabolism is essential. For this purpose, the construction of a robust and high-quality genome-based metabolic model of C. salexigens was approached. RESULTS: We generated and validated a high quality genome-based C. salexigens metabolic model (iFP764). This comprised an exhaustive reconstruction process based on experimental information, analysis of genome sequence, manual re-annotation of metabolic genes, and in-depth refinement. The model included three compartments (periplasmic, cytoplasmic and external medium), and two salinity-specific biomass compositions, partially based on experimental results from C. salexigens. Using previous metabolic data as constraints, the metabolic model allowed us to simulate and analyse the metabolic osmoadaptation of C. salexigens under conditions for low and high production of ectoines. The iFP764 model was able to reproduce the major metabolic features of C. salexigens. Flux Balance Analysis (FBA) and Monte Carlo Random sampling analysis showed salinity-specific essential metabolic genes and different distribution of fluxes and variation in the patterns of correlation of reaction sets belonging to central C and N metabolism, in response to salinity. Some of them were related to bioenergetics or production of reducing equivalents, and probably related to demand for ectoines. Ectoines metabolic reactions were distributed according to its correlation in four modules. Interestingly, the four modules were independent both at low and high salinity conditions, as they did not correlate to each other, and they were not correlated with other subsystems. CONCLUSIONS: Our validated model is one of the most complete curated networks of halophilic bacteria. It is a powerful tool to simulate and explore C. salexigens metabolism at low and high salinity conditions, driving to low and high production of ectoines. In addition, it can be useful to optimize the metabolism of other halophilic bacteria for metabolite production.

Improved production and characterization of a highly stable laccase from the halophilic bacterium Chromohalobacter salexigens for the efficient delignification of almond shell bio-waste.

Extremozymes have gained importance for their ability to efficiently develop the processes in rigorous industrial conditions with incidence in the recycling of especially robust natural wastes. The production of an extracellular laccase from the halophilic bacterium Chromohalobacter salexigens aided for the bio-delignification of almond shell was optimized using response surface methodology followed by one-factor-at-a-time, resulting in an 80-fold increase in the enzyme yield. Out of 10 different medium components, CuSO4, ZnSO4, glucose, and urea were shown to have the greatest effects on the laccase production. The crude laccase was surprisingly stable against the various solvents, salts, chemicals, pH ranges, and temperatures, and it exhibited a high catalytic efficiency to a wide range of phenolic and non-phenolic substrates. Laccase reduced the kappa number of the lignin of almond shell by approximately 27% without the aid of a mediator, and the delignification efficiency strengthened by up to 58% reduction in kappa number in the used harsh conditions. Due to the high potential of the enzyme in delignification, specifically under extreme conditions, laccase from C. salexigens can be considered as an ideal alternative for chemical treatment methods in cellulose fibres extraction of lignocellulosic bio-wastes or delignification of the lignin and lignin-derived industrial wastes.

Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of Chromohalobacter salexigens.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. RESULTS: Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L-1) and volumetric ectoine yields (up to 4.21 g L-1) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. CONCLUSIONS: Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.

Chromohalobacter is a Causing Agent for the Production of Organic Acids and Putrescine during Fermentation of Ganjang, a Korean Traditional Soy Sauce.

Ganjang, a Korean traditional fermented soy sauce, is prepared by soaking doenjang-meju (fermented soybeans) in approximately 20% (w/v) solar salt solution. The metabolites and bacterial communities during ganjang fermentation were simultaneously investigated to gain a better understanding of the roles of the microbial population. The bacterial community analysis based on denaturing gradient gel electrophoresis of 16S rRNA gene sequences showed that initially, the genus Cobetia was predominant (0 to 10 d), followed by Bacillus (5 to 74 d), and eventually, Chromohalobacter became predominant until the end of the fermentation process (74 to 374 d). Metabolite analysis using (1)H-NMR showed that carbon compounds, such as fructose, galactose, glucose, and glycerol, probably released from doenjang-meju, increased rapidly during the early fermentation period (0 to 42 d). After removal of doenjang-meju from the ganjang solution (42 d), the initial carbon compounds remained nearly constant without the increase of fermentation products. At this point, Bacillus species, which probably originated from doenjang-meju, were predominant, suggesting that Bacillus is not mainly responsible for ganjang fermentation. Fermentation products including acetate, lactate, α-aminobutyrate, γ-aminobutyrate, and putrescine increased quickly with the rapid decrease of the initial carbon compounds, while Chromohalobacter, probably derived from the solar salts, was predominant. Multivariate redundancy analysis indicated that the Chromohalobacter population was closely correlated with the production of the organic acids and putrescine during the ganjang fermentation. These results may suggest that Chromohalobacter is a causing agent responsible for the production of organic acids and putrescine during ganjang fermentation and that the solar salts, not doenjang-meju, is an important microbial source for ganjang fermentation.

Contribution of RpoS to metabolic efficiency and ectoines synthesis during the osmo- and heat-stress response in the halophilic bacterium Chromohalobacter salexigens.

Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating ectoine and hydroxyectoine respectively. Evolution has optimized its metabolism to support high production of ectoines. We analysed the effect of an rpoS mutation in C. salexigens metabolism and ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered ectoines content, suggesting that RpoS regulates ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were deregulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.

The stimulatory effect of mannitol on levan biosynthesis: Lessons from metabolic systems analysis of Halomonas smyrnensis AAD6(T.).

Halomonas smyrnensis AAD(T) is a halophilic, gram-negative bacterium that can efficiently produce levan from sucrose as carbon source via levansucrase activity. However, systems-based approaches are required to further enhance its metabolic performance for industrial application. As an important step toward this goal, the genome-scale metabolic network of Chromohalobacter salexigens DSM3043, which is considered a model organism for halophilic bacteria, has been reconstructed based on its genome annotation, physiological information, and biochemical information. In the present work, the genome-scale metabolic network of C. salexigens was recruited, and refined via integration of the available biochemical, physiological, and phenotypic features of H. smyrnensis AAD6(T) . The generic metabolic model, which comprises 1,393 metabolites and 1,108 reactions, was then systematically analyzed in silico using constraints-based simulations. To elucidate the relationship between levan biosynthesis and other metabolic processes, an enzyme-graph representation of the metabolic network and a graph decomposition technique were employed. Using the concept of control effective fluxes, significant links between several metabolic processes and levan biosynthesis were estimated. The major finding was the elucidation of the stimulatory effect of mannitol on levan biosynthesis, which was further verified experimentally via supplementation of mannitol to the fermentation medium. The optimal concentration of 30 g/L mannitol supplemented to the 50 g/L sucrose-based medium resulted in a twofold increase in levan production in parallel with increased sucrose hydrolysis rate, accumulated extracellular glucose, and decreased fructose uptake rate.

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment.

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.

Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens.

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-(13)C]-, [2-(13)C]-, [6-(13)C]-, and [U-(13)C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.

Temperature- and salinity-decoupled overproduction of hydroxyectoine by Chromohalobacter salexigens.

Hydroxyectoine overproduction by the natural producer Chromohalobacter salexigens is presented in this study. Genetically engineered strains were constructed that at low salinity coexpressed, in a vector derived from a native plasmid, the ectoine (ectABC) and hydroxyectoine (ectD) genes under the control of the ectA promoter, in a temperature-independent manner. Hydroxyectoine production was further improved by increasing the copies of ectD and using a C. salexigens genetic background unable to synthesize ectoines.

Role of trehalose in salinity and temperature tolerance in the model halophilic bacterium Chromohalobacter salexigens.

The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established.

Halophilic properties of metal binding protein characterized by high histidine content from Chromohalobacter salexigens DSM3043.

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2-2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2-1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01-0.1 mM NiSO4 stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation.