Anaerobic methane oxidation linked to Fe(III) reduction in a Candidatus Methanoperedens-enriched consortium from the cold Zoige wetland at Tibetan Plateau

Anaerobic oxidation of methane (AOM) is a microbial process degrading ample methane in anoxic environments, and Ca. Methanoperedens mediated nitrate- or metal-reduction linked AOM is believed important in freshwater systems. This work, via 16S rRNA gene diversity survey and 16S rRNA quantification, found abundant Ca. Methanoperedens along with iron in the cold Zoige wetland at Tibetan Plateau. The wetland soil microcosm performed Fe(III) reduction, rather than nitrate- nor sulphate-reduction, coupled methane oxidation (3.87 μmol d⁻¹) with 32.33 μmol Fe(II) accumulation per day at 18°C, but not at 30°C. A metagenome-assembled genome (MAG) recovered from the microcosm exhibits ~74% average nucleotide identity with the reported Ca. Methanoperedens spp. that perform Fe(III) reduction linked AOM, thus a novel species Ca. Methanoperedens psychrophilus was proposed. Ca. M. psychrophilus contains the whole suite of CO₂ reductive methanogenic genes presumably involving in AOM via a reverse direction, and comparative genome analysis revealed its unique gene categories: the multi-heme clusters (MHCs) cytochromes, the S-layer proteins highly homologous to those recovered from lower temperature environments and type IV pili, those could confer Ca. M. psychrophilus of cold adaptability. Therefore, this work reports the first methanotroph implementing AOM in an alpine wetland.     

Thermotolerance,cell filamentation,and induced protein synthesis in psychrophilic and psychrotrophic bacteria

Both the psychrophile Aquaspirillum arcticum and the psychrotroph Bacillus psychrophilus were found to acquire thermotolerance when either heat shocked or treated with nalidixic acid; two conditions which also resulted in the induction of heat shock proteins and/or stress proteins and also cell filamentation. The possible relatedness of acquisition of thermotolerance and cell filamentation was examined by inhibiting cell filamentation with 1.5% KCl. A. arcticum cells which were heat shocked in the presence of KCl did not become filamentous nor acquire thermotolerance suggesting that these two responses may be related. On the other hand, when cells of B. psychrophilus were treated in a similar fashion, they also were prevented from cell filamentation but their ability to become thermotolerant was unaffected. When A. arcticum cells were heat shocked in the presence of chloramphenicol, heat shock protein synthesis was inhibited but not the acquistion of thermotolerance. Similar experiments with B. psychrophilus revealed that partial induction of heat shock proteins still occurred; however, no thermotolerance was exhibited.Abbreviations hsp(s) heat shock proteins(s) - SEM standard error of the mean     

Molecular Cloning of the Genes for Pyruvate Kinase of Two Bacilli,Bacillus psychrophilus and Bacillus licheniformis,and Comparison of the Properties of the Enzymes Produced in Escherichia coli

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by A TP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.     

ACandida transformant produced by mycobacterial nucleic acids

A variant ofCandida albicans, believed to be a transformant, was obtained in the presence of a preparation containing nucleic acids and proteins ofMycobacterium tuberculosis. The transformant produced small, oval cells and small colonies and differed biochemically and serologically from the parental strain. The transformant proved to be non-virulent but able to confer anti-mycobacterial immunity in guinea pigs.     

Diversity of Aerobic Methanotrophic Bacteria in a Permafrost Active Layer Soil of the Lena Delta,Siberia

With this study, we present first data on the diversity of aerobic methanotrophic bacteria (MOB) in an Arctic permafrost active layer soil of the Lena Delta, Siberia. Applying denaturing gradient gel electrophoresis and cloning of 16S ribosomal ribonucleic acid (rRNA) and pmoA gene fragments of active layer samples, we found a general restriction of the methanotrophic diversity to sequences closely related to the genera Methylobacter and Methylosarcina, both type I MOB. In contrast, we revealed a distinct species-level diversity. Based on phylogenetic analysis of the 16S rRNA gene, two new clusters of MOB specific for the permafrost active layer soil of this study were found. In total, 8 out of 13 operational taxonomic units detected belong to these clusters. Members of these clusters were closely related to Methylobacter psychrophilus and Methylobacter tundripaludum, both isolated from Arctic environments. A dominance of MOB closely related to M. psychrophilus and M. tundripaludum was confirmed by an additional pmoA gene analysis. We used diversity indices such as the Shannon diversity index or the Chao1 richness estimator in order to compare the MOB community near the surface and near the permafrost table. We determined a similar diversity of the MOB community in both depths and suggest that it is not influenced by the extreme physical and geochemical gradients in the active layer.     

Membrane phase behavior of a psychrotrophic and mesophilic bacillus

The phase properties of membranes isolated from the psychrotrophBacillus psychrophilus and the mesophileB. megaterium were examined using wide-angle X-ray diffraction. The temperature at which the transition from liquid-crystalline to crystalline (gel) phase occurred was below −30°C for both microorganisms, regardless of the temperature at which the microbial cells were grown. Thus the membranes for both microorganisms were exclusively liquid-crystalline over the entire growth temperature range. Indeed, the membrane was completely fluid at temperatures where growth of the psychrotroph ceases, thus indicating that the phase transition temperature is not the determinant of the minimum growth temperature.     

Construction of a flocculating yeast for fuel ethanol production

The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Saccharomyces. cerevisiae ZWA46. The transformant, ZWA46-F2, showed strong and stable flocculation ability during 20 serial batch cultivations. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the culture medium. The flocculation ability of the transformant showed no difference with the initial pH ranging from 3.5 to 6.0. Furthermore, the ethanol concentration and other properties of the transformant strain ZWA46-F2 were similar to those of the wild-type strain ZWA46.     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

Expression of virulence and antibiotic resistance in anEscherichia coli transconjugant carrying a large plasmid pCAT120 ofShigella dysenteriae type I and its spontaneous fragmentations

The role of a 120-kb plasmid in relation to virulence and drug resistance factor inShigella dysenteriae was studied. For characterization of plasmids, the mating system is a useful and efficient means of transferring both large and small plasmids to a new host. The conjugative transfer of a 120-kb (pCAT120) ampicillin-resistant plasmid ofS. dysenteriae toE. coli K-12 was not successful. Introduction of anE. coli fertility factor plasmid F, did not help to mobilize the plasmid. Low transfer frequencies of antibiotic markers toE. coli were achieved by treatment of the donorS. dysenteriae with N-methyl-N'-nitro-N-nitrosoguanidine. The transconjugants showed resistance to ampicillin, chloramphenicol, tetracycline and cadmium. A transconjugant carrying the 120-kb plasmid ofS. dysenteriae produced keratoconjunctivitis in guinea pigs. Repeated subculture of Clm^r transconjugant (pCAT120) on tryptic soya agar plates became Clm^S and showed four distinct DNA bands ranging from 3 to 10 kb in size on agarose gel electrophoresis. Utilization of organic acids, metal resistance (Cd), dye-binding properties (Crb⁺, Ebr⁺) and drug resistance (Amp, Tet) were identified on 10, 7, 4 and 3-kb plasmid DNA fragment of pCAT120 respectively. Crb⁺ 4-kb DNA fragment of pCAT120 was isolated, purified and transferred to an avirulentE. coli K12 by trans-formation. However, transformant (pET4) showed poor growth on solid media and its growth in liquid culture was only possible after supplementation of the unknown low-molar-mass thermolabile factor(s) secreted by the recipient strain. A 130-kDa outer membrane protein was synthesized by the transformant (pET4) carrying a 4-kb Congo red binding plasmid DNA fragment of pCAT120. A highly reduced rate of synthesis of a few low-molar-mass outer membrane proteins was also observed among the transformant (pET4) in relation to the recipient strain. Transconjugant carrying four plasmid DNA fragments of pCAT120 and Crb⁺ transformant (pET4) failed to produce keratoconjunctivitis in guinea pigs. Presented in part at the57th Annual Meeting of Society of Biological Chemists (India), New Delhi, October, 1988 (Abstr. No. 269 & 272) andIndo-UK Workshop on Diarrhoeal Diseases, Calcutta, January 1989 (Abstr. Page No. 215-217).     

Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rap1

The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rapl gene is expressed both during growth and development in D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rapl transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rapl transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rapl transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rapl transformant. We propose that the Rapl protein functions in the regulation of cell morphology in D. discoideum. © 1993Wiley-Liss, Inc.     

Reduction of lactate production in Lactococcus lactis, a combined strategy: metabolic engineering by introducing foreign alanine dehydrogenase gene and hemin addition

An approach to decreasing the lactate production in Lactococcus lactis by metabolic engineering is presented. The inhibitory effects of a low pH due to the accumulation of lactate on cell growth and nisin production in L. lactis are well known. To avoid such inhibitory effects, a new strategy by rerouting carbon flow was considered. In an effort to suppress lactate production, a new gene was introduced into L. lactis to create a novel pathway for alanine synthesis to reroute the metabolic flow of lactate. Alanine dehydrogenase (E.C.1.4.1.1) encoded by alaD from Bacillus sphaericus was expressed in L. lactis. The enzyme was expressed to a specific activity of nearly 0.39 U/mg protein in the transformant. Hemin addition was also considered to decrease the lactate production in L. lactis. The effect of hemin on the alanine production in the transformant was investigated. This study showed that using the combined strategy, stronger effects on lactate and alanine productions were observed in the transformant.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Ethylene Glycol Utilization,Cold and Ethylene Glycol Shockand Acclimation Proteins in a Psychrotrophic Bacterium

A psychrotrophic Pseudomonas fluorescens was isolated that utilizes ethylene glycol as a sole carbon source, with removal efficiencies of 98% and 96% in 20 and 55 days at 25° and 5°C, respectively. The response of the psychrotroph to environmental shifts was investigated using two-dimensional SDS-PAGE and computing scanning laser densitometry. During a 25°C to 5°C cold shock, the microorganism induced ten cold shock proteins. Under conditions of constant growth at 5°C, five cold acclimation proteins were synthesized. Ethylene glycol shock induced 14 ethylene glycol shock proteins. Ten ethylene glycol acclimation proteins were found. Similarities between the shock proteins and acclimation proteins for cold shock and acclimation and the ethylene glycol shock and acclimation may suggest that these proteins are of significance to both shock recovery as well as constant growth in a new environment.     

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999