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Characterization of an alkaliphilic thermophilic bacillus isolate TA2.A1

A novel aerobic, alkaliphilic, thermophilic Bacillus species was isolated from a thermal bore in Te Aroha, New Zealand with a source temperature and pH of 77°C and pH 8.28. The strain designated TA2.A1 was a Gram positive spore-forming rod with a filamentous morphology that grew optimally at pH 9.2 and 70°C. The organism grew over a temperature range of 45°C to at least 75°C and a pH range of 7.7-10.5. The substrate range that supported growth was unusual and included glycerol, sodium pyruvate, trehalose and sucrose. The monosaccharide components of sucrose, glucose and fructose, did not support growth. Membrane lipid analysis was typical of that of many thermophilic bacteria with a very high proportion of saturated branched chain fatty acids. The DNA base composition of isolate TA2.A1 was 47.3 mol% guanine plus cytosine. When the 16S rDNA sequences of Bacillus isolate TA2.A1 and other Bacillus strains are aligned, six sequences including Bacillus isolate TA2.A1 contained a five base pair insert positioned at bases 36-40 from the 5′ end of the isolate TA2.A1 sequence. The five base pair insert was the same for isolate TA2.A1 and fragment ab009 (complete 16S rDNA sequence not currently available) from a clone library from mesophilic sewage sludge. A phylogenetic dendogram indicated that isolate TA2.A1 was a deeply branching member of the Bacillus genus. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth and sodium chloride (> 5mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (∆pNa) across the cell membrane. N,N- dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1 suggesting that an F₁F₀-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (∆Ψ), but only when sodium was present. In the absence of sodium, the rate of ∆Ψ-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of ∆pNa alone (i.e., no ∆Ψ). Glutamate uptake was specifically inhibited by monensin, and the Kₘ for sodium was 5.6mM. The Hill plot had a slope of approximately 1 suggesting that sodium binding was non-cooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Kₘ for glutamate was 2.90 μM, and the Vₘₐₓ was 0.7 nmol.min⁻¹ mg of protein. Glutamate transport was specifically inhibited by glutamate analogues. Sucrose uptake by isolate TA2.A1 was also evaluated and was actively transported by the presence of sodium ions, probably by a sucrose/Na⁺ symport. A number of thermophilic microorganisms use sodium-coupled uptake for amino acid utilization. However, the fact that both sucrose and glutamate uptake in isolate TA2.A1 were sodium dependent was unusual. [¹⁴C]sucrose uptake was inhibited by cold trehalose which suggested that both carbohydrates may be competing for the same uptake system. Sucrose grown cells could transport [¹⁴C]glucose at low concentrations but the rate was 10 fold lower than for [¹⁴C]sucrose transport. No transport of [¹⁴C]fructose by sucrose grown cells could be demonstrated.
Type of thesis
The University of Waikato
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