Role of carnitine in adaptation of Chromohalobacter salexigens DSM 3043 and its mutants to osmotic and temperature stress in defined medium.

L-Carnitine is widespread in nature, but little information is available on its metabolism and physiological functions in moderate halophiles. In this study, we found that Chromohalobacter salexigens DSM 3043 could utilize carnitine not only as a nutrient, but also as an osmolyte. When grown at 37 °C under salt-stress conditions, the strain utilized carnitine as an osmoprotectant by enzymatically converting it into GB. When grown at low and high temperature, both carnitine and its metabolic intermediate GB were simultaneously accumulated intracellularly, serving as cryoprotectants and thermoprotectants. The genes (csal_3172, csal_3173, and csal_3174) which were predicted to participate in L-carnitine degradation to GB were deleted to construct the corresponding mutants. The effects of salinity and temperature on the growth rates and cytoplasmic solute pools of the C. salexigens wild-type and mutant strains were investigated. 13C-NMR analysis revealed that GB was still detected in the Δcsal_3172Δcsal_3173Δcsal_3174 mutant grown in a defined medium with added DL-carnitine, but not with L-carnitine, indicating that an unidentified D-carnitine degradation pathway exists in C. salexigens. Taken together, the data presented in this study expand our knowledge on carnitine metabolism and its physiological functions in C. salexigens exposed to single or multiple environmental abiotic stress.

First report of Colletotrichum gloeosporioides causing leaf spot on Cyclobalanopsis glauca in China.

Cyclobalanopsis glauca (Thunb.) Oerst. is one of the most widely distributed species of evergreen broad-leaved tree in subtropical areas of China. It is also grown in Korea, Japan, and India. Because of its beautiful shape, C. glauca is commonly used for greening gardens and walkways. In July 2018, leaf spots on C. glauca were observed in Zhejiang province (Lishui, N: 28°26' 6.75";E: 119°54'11.22), China. About 70% of the trees were found to be diseased, with approximately 50% of leaves showing symptoms. The symptoms on C. glauca leaves initially appeared as small brown-yellow spots which gradually expanded, developing a light brown central and dark brown to black margin. The spots ranged from 4 to 15 mm in diameter. Ten symptomatic fragments measuring approximately 5×5 mm from each leaf were surface disinfested with 70% ethanol for 30 s, and then they were rinsed in sterile distilled water and placed on potato dextrose agar (PDA) medium at 25 °C in the dark for five days. Segments of colony perimeters were then transferred to new plates. The colonies initially produced white mycelia that later turned gray-white with pink and occasionally black dots scattered on the surface of the mycelium. Spores were aseptate, cylindrical, 8 to 15 µm in length, and 3 to 5 µm wide, most with rounded ends, a few with one apex round and the other fusiform, as described for Colletotrichum gloeosporioides (Penz.) Sacc. (Agostini et al. 1993). The internal transcribed spacer rDNA (ITS: MK758005) and two nuclear protein-coding genes (CHS: MK784770, ACT: MK784769) were amplified with ITS1/ITS4, CHS-79F/CHS-345, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequence had 99.61% identity to GQ485605 for ITS, 99.56% to GQ856782 for ACT, and 100% to GQ856733 for CHS of C. gloeosporioides CBS 953.97 in GenBank, respectively. To fulfill Koch's postulates, spores (1×108) of the isolate were sprayed onto leaves of twelve 2-year-old C. glauca plants (at least six leaves per plant). The fungus was inoculated on one side of each leaf, and distilled water was used as a mock inoculated control on the other side. The plants were cultivated in the greenhouse to maintain high humidity and a temperature near 25 °C. After 9 days, 100% of the leaf halves that had been inoculated had symptoms identical to those observed on affected C. glauca leaves in the field, while no symptoms were observed on the mock inoculated half of each leaf. The fungus was reisolated from the symptoms and identified as C. gloeosporioides using techniques previously described. To our knowledge, this is the first report of C. gloeosporioides infecting C. glauca in China. This study will establish a foundation for the further study of C. gloeosporioides to address the disease effectively. References: Agostini, J. P., et al. 1993. Phytopathology. 82:1177. Weir, B. S., et al. 2012. Stud. Mycol. 73:115. This work was supported by the National Science Foundation for Young Scientists of China (31800035).

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.

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.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25 °C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37 °C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Molecular basis of P450 OleTJE: an investigation of substrate binding mechanism and major pathways.

Cytochrome P450 OleTJE has attracted much attention for its ability to catalyze the decarboxylation of long chain fatty acids to generate alkenes, which are not only biofuel molecule, but also can be used broadly for making lubricants, polymers and detergents. In this study, the molecular basis of the binding mechanism of P450 OleTJE for arachidic acid, myristic acid, and caprylic acid was investigated by utilizing conventional molecular dynamics simulation and binding free energy calculations. Moreover, random acceleration molecular dynamics (RAMD) simulations were performed to uncover the most probable access/egress channels for different fatty acids. The predicted binding free energy shows an order of arachidic acid < myristic acid < caprylic acid. Key residues interacting with three substrates and residues specifically binding to one of them were identified. The RAMD results suggest the most likely channel for arachidic acid, myristic acid, and caprylic acid are 2e/2b, 2a and 2f/2a, respectively. It is suggested that the reaction is easier to carry out in myristic acid bound system than those in arachidic acid and caprylic acid bound system based on the distance of Hß atom of substrate relative to P450 OleTJE Compound I states. This study provided novel insight to understand the substrate preference mechanism of P450 OleTJE and valuable information for rational enzyme design for short chain fatty acid decarboxylation.

Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS-A18 and inulin hydrolysis by the enzyme.

To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain, Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin, but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity, 14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH 7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report on alkaline inulinase production from microorganism.

[Effect of ultrasound-guided acupoint electrical stimulation on diaphragmatic dysfunction associated with mechanical ventilation].

OBJECTIVE: To observe the effect of ultrasound-guided acupoint electrical stimulation on the patients with diaphragmatic dysfunction associated with mechanical ventilation in ICU. METHODS: Fifty-two patients were randomly divided into an observation group (26 cases, 3 cases dropping) and a control group (26 cases). Conventional treatment was given to all patients. On the basis of conventional treatment, acupoint electrical stimulation therapy was applied at Zhangmen (LR 3), Dabao (SP 21), Pishu (BL 20), Shenshu (BL 23), etc. In the observation group, the treatment was given for 30 min each time, 3 times a day for 7 days. Diaphragm thickening fraction (TFdi) was used as an index to guide the individualized setting of stimulation intensity and judge the effect, and the difference of mechanical ventilation time, ICU time, total hospitalization time, hospital mortality and reintubation rate between the two groups were observed. RESULTS: The mechanical ventilation time in the observation group was shorter than that in the control group (P<0.05). There was no significant difference in the ICU time and total hospitalization time between the two groups (P>0.05). During hospitalization, 2 patients died in the observation group and 3 patients died in the control group, there was no significant difference in hospital mortality (P>0.05). One patient in the observation group was reintubated and 8 patients in the control group (P<0.05). The use of acupoint electrical stimulation was a factor in shortening the mechanical ventilation time and reducing the reintubation events (P<0.05). CONCLUSION: Ultrasound-guided acupoint electrical stimulation can relieve ventilator-induced diaphragmatic dysfunction, reduce ventilator support time and reintubation events.

Genome Sequence of the Fructan-Degrading Organism Marinimicrobium sp. Strain LS-A18, Isolated from a Marine Solar Saltern.

Marinimicrobium sp. strain LS-A18 is a fructan-degrading organism isolated from a brine sample from a marine solar saltern in Jiaozhou Bay, China. The draft genome sequence of this bacterium is 3,815,107 bp in length, with a G+C content of 59.03%. To our knowledge, this is the first genome announcement of a fructan-degrading strain of the genus Marinimicrobium.

Extracellular production of novel halotolerant, thermostable, and alkali-stable carboxymethyl cellulase by marine bacterium Marinimicrobium sp. LS-A18.

Cellulases which are active and stable under extreme conditions have attracted considerable attention because of their potential industrial applications. Marinimicrobium sp. LS-A18 showed high extracellular carboxymethylcellulase (CMCase) activity when grown on mineral salt medium containing carboxymethylcellulose as the sole carbon source. Maximum CMCase activity was obtained at 55°C and pH 7.0 in the absence of NaCl. Under the optimized fermentation conditions, the yield of CMCase was increased up to 2.5 U/ml, which was 3.1-fold higher than that before optimization. The enzyme retained 84 % of residual activity after incubation at 60°C for 1 h and more than 88 % of residual activity after incubation for 72 h in the presence of different pH (5-11) and NaCl concentrations (0-25 %, w/v), indicating it was halotolerant, thermostable and alkali-stable. These characteristics made the CMCase from Marinimicrobium sp. LS-A18 as a potentially novel biocatalyst in biotechnological and industrial applications.