Mei-Chin Lai

Methanofollis aquaemaris sp. nov., a methanogen isolated from an aquaculture fish pond.

A novel methanogen, designated strain N2F9704T, was isolated from an aquaculture fish pond near Wang-gong, Taiwan. The cells were irregular cocci, non-motile, 1.2-2.0 microm in diameter and stained gram-negative. Cells of strain N2F9704T lysed easily by SDS treatment (0.1 g l(-1)) and the S-layer protein had an Mr of 137000. The catabolic substrates used included formate and H2+CO2, but not acetate, methanol, trimethylamine or secondary alcohols. The optimal growth parameters for strain N2F9704T were pH 6.5, 37 degrees C with 0.5% NaCl. Trace amounts of tungstate not only promoted growth but also extended the range of growth conditions. Analysis of the 16S rDNA sequence revealed a phylogenetic relationship to Methanofollis species and the name Methanofollis aquaemaris sp. nov. is therefore proposed for strain N2F9704T (= OCM 746T = CCRC 16166T). Additionally, the strain was infected with a novel coccus-shaped, enveloped virus with a diameter of 200 nm.

Glycine Betaine Transport in the Obligate Halophilic Archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.

Characterization and Regulation of the Osmolyte Betaine Synthesizing Enzymes GSMT and SDMT from Halophilic Methanogen Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize the osmolyte betaine de novo in response to extracellular salt stress. Betaine is generated by the stepwise methylation of glycine to form sarcosine, N, N-dimethylglycine and betaine by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. The complete gene cluster of Mpgsmt-sdmt was cloned from Southern hybridization and heterologous expressed in E. coli respectively. The recombinant MpGSMT and MpSDMT both retained their in vivo functional activities in E. coli BL21(DE3)RIL to synthesize and accumulate betaine and conferred elevated survival ability in betaine transport deficient mutant E. coli MKH13 under high salt stress. The dramatic activating effects of sodium and potassium ions on the in vitro methyltransferase activities of MpGSMT, but not MpSDMT or bacterial GSMT and SDMT, revealed that GSMT from halophilic methanoarchaeon possesses novel regulate mechanism in betaine biosynthesis pathway. The circular dichroism spectra showed the fluctuated peaks at 206 nm were detected in the MpGSMT under various concentrations of potassium or sodium ions. This fluctuated difference may cause by a change in the β-turn structure located at the conserved glycine- and sarcosine-binding residue Arg167 of MpGSMT. The analytical ultracentrifugation analysis indicated that the monomer MpGSMT switched to dimeric form increased from 7.6% to 70% with KCl concentration increased from 0 to 2.0 M. The level of potassium and sodium ions may modulate the substrate binding activity of MpGSMT through the conformational change. Additionally, MpGSMT showed a strong end product, betaine, inhibitory effect and was more sensitive to the inhibitor AdoHcy. The above results indicated that the first enzymatic step involved in synthesizing the osmolyte betaine in halophilic archaea, namely, GSMT, may also play a major role in coupling the salt-in and compatible solute (osmolyte) osmoadaptative strategies in halophilic methanogens for adapting to high salt environments.

Methanogenic Archaea Isolated from Taiwan's Chelungpu Fault

ABSTRACT Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification, and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, St545MbT of newly proposed species Methanolobus chelungpuianus and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 m below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod-shaped organism capable of using H2-CO2 and formate as substrates for methanogenesis. The morphological, biochemical, and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545MbT, isolated from a sandstone sample at 545 mbls, is a nonmotile, irregular, coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545MbT was 99.0% similar to that of Methanolobus psychrophilus strain R15 and was 96 to 97.5% similar to the those of other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545MbT were different from those of M. psychrophilus strain R15, and whole-genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545MbT (DSM 19953T; BCRC AR10030; JCM 15159) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.

Methanosarcina mazei strain O1M9704, methanogen with novel tubule isolated from estuarine environment

Abstract. A new methanogenic isolate, designated as strain O1M9704 (=OCM 667), was isolated from the sediment of the estuarine environment in Eriln Shi, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H2-CO2, acetate, or formate. Cells grew optimally at 37°C with 0.5% NaCl in neutral pH. The cells were stained Gram-negative, nonmotile, irregular coccus 0.3–0.6 μm in diameter. A comparison of 16S rDNA sequences phylogenetically related strain O1M9704 to Methanosarcina mazei. Gas vacuoles were observed both under phase contrast microscope and in thin sections in the electron microscope. Negative stain of electron micrographs showed a novel character of strain O1M9704 with tubule structure extended out of the cells. The tubule structure and gas vacuoles may benefit the adaptation of methanoarchaea in estuarine environment.

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

Abstract. A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H2-CO2, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond.

Comparative genomics of Vibrio campbellii strains and core species of the Vibrio Harveyi clade

The core of the Vibrio Harveyi clade contains V. harveyi, V. campbellii, V. owensii, V. jasicida, and V. rotiferianus. They are well recognized aquatic animal pathogens, but misclassification has been common due to similarities in their rDNA sequences and phenotypes. To better understand their evolutionary relationships and functional features, we sequenced a shrimp pathogen strain V. harveyi 1114GL, reclassified it as V. campbellii and compared this and 47 other sequenced Vibrio genomes in the Harveryi clade. A phylogeny based on 1,775 genes revealed that both V. owensii and V. jasicida were closer to V. campbellii than to V. harveyi and that V. campbellii strains can be divided into two distinct groups. Species-specific genes such as intimin and iron acquisition genes were identified in V. campbellii. In particular, the 1114GL strain contains two bacterial immunoglobulin-like genes for cell adhesion with 22 Big_2 domains that have been extensively reshuffled and are by far the most expanded among all species surveyed in this study. The 1114GL strain differed from ATCC BAA-1116 by ~9% at the synonymous sites, indicating high diversity within V. campbellii. Our study revealed the characteristics of V. campbellii in the Harveyi clade and the genetic basis for their wide-spread pathogenicity.

High prevalence of Streptococcus agalactiae from vaginas of women in Taiwan and its mechanisms of macrolide and quinolone resistance

BACKGROUND/PURPOSE Streptococcus agalactiae (GBS), is the most common pathogen causing infections among perinatal women and neonatal babies. Nonetheless, there are few studies on the occurrence of GBS among the pregnant women and the mechanisms of GBS resistance to quinolones and macrolides in Taiwan. METHODS GBS were isolated from vaginas of the pregnant and non-pregnant symptomatic women in Taiwan. The prevalence, antimicrobial susceptibility, and mechanisms of resistance against erythromycin and quinolone of total 188 isolates were studied. RESULTS The isolation rate of GBS from pregnant women was significantly higher at 21.8% compare with the non-pregnant women of 13.2%. Antibiotic susceptibility test of the 188 GBS isolates revealed a high non-susceptible rate for erythromycin (50.0%) while the rate for levofloxacin was only 4.8%. Among 94 erythromycin non-susceptible GBS isolates, ermB gene was detected 83.1% (59/71) for those GBS that were non-susceptible to both clindamycin and tetracycline, which was significantly higher than GBS that are susceptible to clindamycin but resistant to tetracycline at 43.8% (7/16). No ermA or mef gene was detected in any isolate. Mutations were detected in the parC and gyrA genes in 14 out of 18 levofloxacin non-susceptible isolates. The predominant mutation type was the combination of Ser79Tyr in parC and Ser81Leu mutations in gyrA. CONCLUSION GBS is the most common isolated pathogens in vaginal infections in Taiwan, resistance to tetracycline and erythromycin is higher than the rate observed for other regions of the world, while the resistance rate for levofloxacin was relatively lower in Taiwan.

Characterization of Methanococcus voltaei Strain P2F9701a: A New Methanogen Isolated from Estuarine Environment

A new methanogen, designated as strain P2F9701a (= OCM 745), was isolated from a water sample of estuarine environment in Elrin Shi, Taiwan. Cells of strain P2F9701a were motile coccus (0.7∼1.1 μm) with tufts of flagella. Gas vacuoles were observed, and the protein cell wall was composed of S-layer protein subunit with Mr of 74,700. Cells catabolized formate and H2+CO2 to produce methane, but not acetate, methanol, and trimethylamine. Strain P2F9701a grew in the range of 30–42°C, with optimal growth temperature at 37°C, but did not grow below 28°C or above 42°C. This estuarine isolate P2F9701a tolerated well the NaCl concentration between 0.02 and 1.03 m, and the optimal salt for growth was 0.17 m. Although phylogenetic analytic results indicated that P2F9701a belongs to the mesophilic, hydrogenotrophic marine methanogen of Methanococcus voltaei, the occurrence of gas vacuoles, tufts of flagella, eury-halotolerant and steno-thermotolerant characters of strain P2F9701a are different from mesophilic Methanococcus spp. that had been reported.

The biotechnological potential of the extreme halophilic archaea Haloterrigena sp. H13 in xenobiotic metabolism using a comparative genomics approach.

Extreme halophilic archaea are thriving and dominant populations within hypersaline environments. Because of the extreme properties of the enzymes of halophilic archaea and similar metabolic abilities to their bacterial counterparts, our interests focus on their potential biotechnological applications. In this study, the partial genome of a newly isolated extreme halophilic archaeon, Haloterrigena sp. H13, was investigated. The genome size was estimated to be about 3.9 MB, and a genomic shotgun library was constructed. A total of 1479 clones from the library were sequenced once, and 1186 contigs were obtained. From these contigs, 580 open reading frames (ORFs) were identified, and 394 ORFs were annotated. From the partial genome of strain H13, we identified genes that may be involved in 1,2‐dichloroethane degradation, naphthalene/anthracene degradation, γ‐hexachlorocyclohexane degradation, 1‐/2‐methylnaphthalene degradation and benzoate degradation via CoA ligation. Among the identified ORFs, gene homologs of (S)‐2‐haloacid dehalogenase (EC 3.8.1.2) and salicylate hydroxylase (EC 1.14.13.1), which might be involved in the degradation of dichloroethane, γ‐hexachlorocyclohexane and naphthalene, were found in the partial genome sequence of strain H13. According to the current genome annotation of peripheral metabolic pathways and the putative xenobiotic‐degrading enzymes, the potential of extreme haloarchaea in bioremediation applications is proposed.