Bioremediation potential of a halophilic bacterium Chromohalobacter salexigens EG1QL3: exopolysaccharide production, crude oil degradation, and heavy metal tolerance

Based on biochemical and phylogenetic analyses, isolated from a salt sample from Lake Qarun (Egypt) a halophilic strain EG1QL3 was identified as Chromohalobacter salexigens. The abilities of EG1QL3 to produce an extracellular polysaccharide, degrade oil, and resist to heavy metals were revealed.

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

ABSTRACT 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.

The Potential Use of Ectoine Produced by a Moderately Halophilic Bacteria Chromohalobacter salexigens KT989776 for Enhancing Germination and Primary Seedling of Flax “Linum usitatissimum L.” under Salinity Conditions

The similarity between plant and microbial cells encourage the use of microbial metabolites of halophilic bacteria for the alleviation of salt stress in plants. In the current research work, a compatible solute ectoine extracted from a moderately halophilic bacteria Chromohalobacter salexigens KT989776 was used to enhance flax germination and primary seedling under different levels of salinity. Two successive experiments including germination in Petri plates under six levels of salinity (0, 3, 5, 7, 9 and 11 dS.m-1) and a pot experiment under three irrigating water salinity levels (2, 3 and 4) with two types of ectoine application (spray and soil addition) were conducted. Germination parameters were recorded for the first experiment while a fresh and dry weight of plants and peroxidase activity in addition to sodium-potassium ratio were estimated in the pot experiment. Also, ectoine accumulation in plants was detected using HPLC. Results of LC-MS proved the production of ectoine by C. salexigens KT989776 and ectoine enhanced significantly all germination parameters of flax seeds, decreased sodium accumulation in the plant, increased potassium content, and lowered peroxidase and phenoloxidase activity. Also, HPLC analysis proved that ectoine was detected in all treated samples while not detected in non-treated control.

Draft Genome Sequence of Halotolerant Bacterium Chromohalobacter salexigens ANJ207, Isolated from Salt Crystal Deposits in Pipelines

Chromohalobacter salexigens ANJ207 was isolated from a salt crystal and is known to tolerate up to 30% NaCl concentration. Here, we report the de novo draft assembly of C. salexigens ANJ207.

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

ABSTRACT 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 13 C 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 Fe 2+ . 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.