Ammonia-hyperproducing bacteria from New Zealand ruminants

Abstract

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria, Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genus Peptostreptococcus, that D4 and D5 belonged to the family Bacteroidaceae, where D4 was similar to Fusobacterium necrophorum; and that C2 was most similar to an unidentified species from the genus Eubacterium. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h-1, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h-1, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants.

FIG. 1

16S rDNA RFLPs of HAP bacteria and isolates from New Zealand ruminants. PCR-amplified 16S ribosomal genes were digested with the restriction endonucleases MspI, CfoI, and HaeIII and separated in a 1.2% (wt/vol) agarose gel. The marker lane contains a 1-kb ladder (GIBCO BRL).

FIG. 2

Dot blots of DNA extracted from deer, cow, and sheep rumen samples hybridized with oligonucleotide probes F966, SR836, and C72, which are specific for C. aminophilum, C. sticklandii, and P. anaerobius, respectively. All rumen DNA samples were applied at 1 μg per dot. Each membrane also included control target DNA spotted at the amounts shown to the right of the figure, except for the P. anaerobius membrane, in which control DNA was applied only up to 5 μg.

FIG. 3

Nitrogen source utilization by HAP isolates. Fast (a)- and slow (b)-growing isolates were grown on media containing either tryptone (Tryp), Casamino Acids (Cas), casein, gelatin, tryptone plus Casamino Acids (HAP), or NH₄Cl (Basal) as the sole nitrogen source. OD₆₀₀ readings were taken throughout the growth of each isolate and were used to calculate the maximum specific growth rate and maximum OD₆₀₀ for each nitrogen source. Results are the means of duplicate cultures.

FIG. 4

Ammonia production by HAP isolates. Cultures of HAP isolates grown on HAP medium were sampled and assayed for ammonia (8). Results are the means of duplicate cultures.

FIG. 5

Unrooted phylogenetic trees showing the relationship of isolate D4 (a) and isolates C2, D1, and S1 (b) to closely related bacteria. The trees were derived from similarity matrices (18) by the method of Fitch and Margoliash with the PHYLIP package (12). The scale bar represents a 10% difference in nucleotide sequences.