Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Gene integration in the Escherichia coli chromosome mediated by Tn21 integrase (Int21).

A replication-thermosensitive, pSC101-derived plasmid containing the int gene and RHS-2 from the integron in Tn21 and a kanamycin resistance marker has been constructed and used to obtain Tn21 integrase (Int21)-mediated plasmid integration in the Escherichia coli chromosome. Colonies carrying an integrated plasmid were obtained after growth at 42 degrees C. Southern hybridization and PCR experiments indicated that they contained the plasmid specifically integrated through the RHS into different positions in the E. coli chromosome. Nucleotide sequence determination of the plasmid-chromosome junctions showed that integration sites in the chromosome were pentanucleotides with the sequence described for Int21 secondary sites.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Streptomycin-resistant, asporogenous mutant of Bacillus subtilis.

Summary Mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC 2.7.1.40) are described. These have less than 0.5% of the pyruvate kinase activity of the wild type. All the other glycolytic enymes are present in normal amounts in these mutants. The mutation is recessive and segregates in diploids as a single gene. Five alleles examined fail to complement one another. Tetrad analysis and mitotic recombination data place the mutation on the left arm of chromosome I distal to cys 1. The majority of single-step spontaneous revertants on glucose regain the enzyme activity fully and this activity appears, by a number of criteria, to be due to the same enzyme present in the wild type. Some of these revertants become nuclear petites. The mutants do neither grow on nor ferment sugars but do grow on ethyl alcohol or pyruvate. Glucose addition to cultures growing on alcohol arrests growth until glucose is exhausted. The steady state rate of glucose utilization is slower than in the wild type. This is associated with the accumulation of as much as 5 moles P-enolpyruvate per g wet weight of cells and proportional amounts of 2-P-glyceric and 3-P glyceric acids.The mutation is believed to involve some regulatory element in the synthesis of pyruvate kinase.     

Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.     

Water utilization of tropical hardwood hammocks of the Lower Florida Keys

Summary Predawn water potential of representative plant species, together with stable isotope composition of stem water and potential water sources were investigated in four low-elevation tropical hardwood hammocks in the Lower Florida Keys, during a one year period. Hammock species had the lowest water potentials when soil water content was low and/or soil salinity was high, but differences in groundwater salinity had no effect on the water potential. Comparison of D/H ratio of plant stem water with soil and ground water corroborates the conclusion that they are primarily utilizing soil water and not groundwater. Thus, tropical hardwood hammocks are buffered from saline groundwater, and are able to thrive in areas where groundwater salinity is as high as 25. The effect of sea level rise on these forests may depend more on changes in the frequency of tidal inundation of the soil surface than on changes in groundwater salinity.