Neomycin resistance as a selectable marker in Methanococcus maripaludis.

We cloned the aminoglycoside phosphotransferase genes APH3'I and APH3'II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis.

Plasmid transfer by conjugation in Desulfovibrio desulfuricans.

Conjugational transfer of several IncQ plasmids from Escherichia coli to the strictly anaerobic, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G100A was demonstrated. Plasmid DNA from exconjugants was visualized on agarose gels and was used to transform E. coli to the appropriate antibiotic resistances. Neither transfer of IncW and IncP plasmids to strain G100A, nor transfer of any plasmid to D. desulfuricans strain ATCC 27774 was observed. Conjugation of suicide plasmids containing either Tn5 or Tn9 into D. desulfuricans did not result in detectable transposition. Optimal conditions for conjugational transfer and antibiotic resistance levels of strain G100A were examined.

Effects of amino acids and peptides on rumen microbial growth yields.

Experiments were conducted using mixed rumen bacterial cultures to determine which amino acids limited growth. Complete amino acid mixtures stimulated microbial growth alone and when added to casein. Amino acid subgroups did not stimulate growth alone or when added to casein or casein hydrolysates. Results were similar whether growth was limited by periodic addition of low amounts of carbohydrate or when higher amounts were added to batch cultures. Little growth occurred with ammonia as sole N source. Addition of 100 mg/L of amino acids and peptides quadrupled growth, peptides at 10 mg/L resulted in higher growth than the corresponding amount of free amino acids. Apparent saturation of growth occurred when 10 mg/L of a complete amino acid mixture or trypticase was added to cultures. The Michaelis constant values for amino acids and trypticase were determined to be .5 and 1.0 mg/L, respectively. Growth was a linear function of amount of carbohydrate fermented with the coefficient of slope increasing with increasing amino acid concentrations. These experiments demonstrate that growth stimulation from amino acids and proteins is due to the number of amino acids provided in a given mixture rather than specific growth limiting amino acids. Rumen bacterial growth is greatly stimulated by amino acids and peptides, with low affinity constant values, allowing good growth in the concentrations of amino acids and peptides found in vivo

Modeling of rumen water kinetics and effects of rumen pH changes.

Mechanistic elements for determining water kinetics and effects of pH on VFA production and cellulose hydrolysis were incorporated into a model of rumen digestion and metabolism. Elements necessary for water kinetics were estimates of salivary flow during eating, resting, and ruminating, time course and amount of drinking water, osmotic flux across the rumen wall based on changes in rumen fluid osmolality, and liquid turnover rate. Osmotic flux equations predicted a significant net absorption of water from rumens of dairy cattle. Water kinetic equations predicted experimental changes in rumen volume during twice daily feeding, and produced marker dilution data qualitatively resembling published data. Increased marker dilution during feeding was due to increased rumen volume. An empirical equation predicting rumen pH from VFA concentration was validated against literature data. Changes in molar percentages of acetate and propionate as pH declined below 6.2 were produced by decreasing cellulose hydrolysis and fermentation and by altering stoichiometric coefficients for fermentation of soluble sugars and starch. Insufficient data prevented an adequate challenge of equations predicting decreases in cellulose hydrolysis and changes in stoichiometric coefficients as pH decreased.