Tetronasin

Streptomyces longisporoflavus produces the polyketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DNA was cloned in Streptomyces lividans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb KpnI fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (ATP binding cassette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.

Concentrations of Na(+), K(+) and Ca(2+) in the growth medium were varied within limits normally found in vivo to determine how cation concentrations affect the sensitivity of ruminal bacteria to the ionophores, monensin (a Na(+)/H(+) and K(+)/H(+) exchanger) and tetronasin (Ca(2+)/H(+)). High [Na(+)] (172 mM cf. 137 mM in control medium) enhanced the efficacy of monensin towards Eubacterium ruminantium 2388, Streptococcus bovis C277, Lactobacillus casei LB17 and Prevotella albensis M384. High [K(+)] (35 mM cf. 19 mM) alone caused a decreased potency of both ionophores, except with L. casei. Added Ca(2+) (7.4 cf. 2.8 mM) increased the potency of tetronasin when [Na(+)] was low. High [Na(+)] alone also potentiated the efficacy of tetronasin. Monensin caused intracellular [Na(+)] and [K(+)] to be decreased in the most sensitive of these organisms, E. ruminantium, whereas only intracellular [Ca(2+)] fell with tetronasin. The changes were small; however, Δp fell by only 20 mV after 2 h when ionophores caused immediate cessation of growth. ATP concentrations fell by 77% and 75% with monensin and tetronasin, respectively. Thus, altering cation concentrations might be used to potentiate the efficacy of ionophores, by increasing the rate of energy expenditure to maintain ionic homoeostasis in sensitive bacteria.

Four ionophores differing in cation selectivity were compared for their effect on microbial fermentation and biohydrogenation by ruminal bacteria in continuous culture. Monensin and nigericin are monovalent antiporters with selective binding affinities for Na+ and K+, respectively. Tetronasin is a divalent antiporter that binds preferentially with Ca2+ or Mg2+. Valinomycin is a monovalent uniporter and does not exchange K+ for H+. Steady-state concentrations of 2 micrograms/ml of monensin, nigericin, tetronasin, or valinomycin were maintained by constant infusion into fermenters. Molar percentages of acetate were lower, and those of propionate were higher, in the presence of monensin, nigericin, and tetronasin; all three ionophores also decreased CH4 production. Concentrations of valinomycin as high as 8 micrograms/ml had no effect on volatile fatty acids or CH4 production. Monensin, nigericin, and tetronasin inhibited the rate of biohydrogenation of linoleic acid. Continuous infusion of C18:2n-6 at a steady-state concentration of 314 micrograms/ml into fermenters receiving monensin, nigericin, or tetronasin resulted in lower amounts of stearic acid and higher amounts of oleic acid. Ionophores increased total C18:2 conjugated acids mainly because of an increase in the cis-9, trans-11-C18:2 isomer. If reflected in milk fat, ionophore-induced changes in ruminal lipids could enhance the nutritional qualities of milk.