The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a fermentable growth substrate (i.e. sucrose) from pH 7.5 to 10.0 with no significant change in specific growth rate, suggesting that this bacterium is a facultative alkaliphile. However, when strain TA2.A1 was grown on non-fermentable carbon sources like succinate, no growth was observed until the external pH was > 9.0, suggesting this bacterium is an obligate alkaliphile. Growth on succinate at pH 9.5 was sensitive to both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin revealing that both the proton and sodium motive force ([Delta][mu][H⁺] and [Delta][mu][Na⁺], respectively) were obligate requirements for growth at alkaline pH values. Transport of succinate was driven by a chemical gradient of Na⁺ ([Delta]pNa⁺) that was strictly coupled to [Delta][Psi]. A single transport system was detected for the uptake of succinate, with an apparent K[m] of 19 [mu]M and V[max] of 0.45 nmol succinate/min/mg protein. Succinate transport was pH-dependent, and showed optimal activity at pH values greater than 8.5. Other C₄-dicarboxylates (e.g. malate, fumarate) inhibited the uptake of succinate suggesting that the permease was general for other C₄-dicarboxylates.
Cytochrome content, succinate dehydrogenase oxidoreductase, and F₁F₀-ATPase activities were lower in membranes from strain TA2.A1 cells grown at pH 7.5 compared to those cultured at 9.5. These data suggest that oxidative phosphorylation-linked processes are down-regulated at neutral pH values, an observation that mirrored oxygen consumption profiles of strain TA2.A1 in whole cells. To study this phenomenon at a molecular level, we measured ATP synthesis by the F₁F₀-ATP synthase from strain TA2.A1 as a function of pH. The strain TA2.A1 F₁F₀-ATP synthase had a pH optimum for ATP synthesis of 9.0-9.5, and significantly lower rates of ATP synthesis observed below pH 9.0. Analysis of the atp operon from the thermoalkaliphilic Bacillus sp. strain TA2.A1 and comparison with other atp operons from alkaliphilic bacteria reveals the presence of a conserved lysine residue at position 180 (Bacillus sp. strain TA2.A1 numbering) within the a subunit of these F₁F₀-ATP synthases. We hypothesize that the basic nature of this residue is ideally suited to capture protons from the bulk phase at high pH. To test this hypothesis, a heterologous expression system for the ATP synthase from Bacillus sp. TA2.A1 (TA2F₁F₀) was developed in Escherichia coli DK8 ([Delta]atp). Amino acid substitutions were made in the a subunit of TA2F₁F₀ at position 180. Lysine (aK180) was substituted for the basic residues histidine (aK180H) or arginine (aK180R), and the uncharged residue glycine (aK180G). ATP synthesis experiments were performed in ADP plus P[i]-loaded right-side out membrane vesicles energized by ascorbate-phenazine methosulfate. When these enzyme complexes were examined for their ability to perform ATP synthesis over the pH range from 7.0 to 10.0, TA2F₁F₀ and aK180R showed a similar pH profile having optimum ATP synthesis rates at pH 9.0 to 9.5 with no measurable ATP synthesis at pH 7.5. Conversely, aK180H and aK180G showed maximal ATP synthesis at pH�s 8.0 and 7.5, respectively. ATP synthesis under these conditions for all enzyme forms was sensitive to DCCD. These data strongly imply that amino acid residue K180 is a specific adaptation within the a subunit of TA2F₁F₀ to facilitate proton capture at high pH. At pH values near the pK[a] of K180, the trapped protons readily dissociate to reach the subunit c binding sites but this dissociation is impeded at neutral pH values causing either a blocking of the proposed H⁺ channel and/or mechanism of proton translocation, and hence ATP synthesis is inhibited.
The mechanisms where by alkaliphilic bacteria obtain iron remains unknown. Growth of strain TA2.A1 at pH 9.5 in the presence of the artificial iron chelators ethylenediamine O-hydroxyphenylacetic acid (EDDHA) and 2�2� dipyridal revealed that iron is an important requirement for aerobic growth at alkaline pH values. Furthermore, biochemical analysis showed that Bacillus alcalophilus and Bacillus pseudofirmus both synthesized orange catecholate siderophores, whilst Bacillus halodurans synthesized a hydroxamate siderophore. These tests showed that strain TA2.A1 synthesized both orange catecholate and hydroxamate siderophore/s. Attempts to purify the catecholate were unsuccessful. No homologues of previously identified non-ribosomal peptide synthase (NRPS) genes in Bacillus subtilis and B.halodurans were detected in the genome of strain TA2.A1 using both PCR and Southern hybridization using known non-ribosomal peptide synthase genes.
| Identifer | oai:union.ndltd.org:ADTP/202528 |
| Date | January 2008 |
| Creators | McMillan, Duncan George Glenn, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Duncan George Glenn McMillan |
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