Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway

Abstract

A recent study indicated that Bacillus subtilis catabolizes acetoin by enzymes encoded by the acu gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259-271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum, genomic fragments from B. subtilis harboring acoA, acoB, acoC, acoL, and acoR homologous genes were identified, and some of them were functionally expressed in E. coli. Furthermore, acoA was inactivated in B. subtilis by disruptive mutagenesis; these mutants were impaired to express PPi-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in B. subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure.

FIG. 1

Molecular organization of the B. subtilis aco gene cluster. (A) Scale in kilobase pairs. (B) Structural genes of the aco gene cluster. The positions of putative promoters (P) and hairpin-like structures (loops) are indicated. (C) Relevant restriction sites of the sequenced region. (D) Relevant fragments of the analyzed region.

FIG. 2

Disruptive mutagenesis of acoA. By PCR a 700-bp DNA fragment of acoA, covering the inner region of acoA, was synthesized, and this fragment was inserted into pLGW200 (1). The ligation mixture was transformed into E. coli Top10 (Invitrogen, San Diego, Calif.) for amplification. The isolated recombinant plasmid pLGSA700 was transformed into cells of B. subtilis 168, which were selected for chloramphenicol resistance.

FIG. 3

Localization of pLGSA700 integrations. PstI and EcoRI digests of genomic DNA from B. subtilis 168 (wt) and from pLGSA700-disrupted acoA mutants (SA35, SA36, SA37, SA41, SA42, and SA43) were separated in 0.8% (wt/vol) agarose gels, blotted onto a nylon membrane, and hybridized with biotin-labeled vector pLGW200 (22). The positions of PstI and EcoRI fragments which gave signals with the probe are indicated by arrows. The sizes of standard fragments (Std) are indicated at the left. (A) Agarose gel stained with ethidium bromide; (B) blot hybridized with probe pLGW200.

FIG. 4

Acetoin utilization by B. subtilis 168 and the acoA-defective mutant SA35. Cells of B. subtilis 168 and of SA35 were cultivated in NSM at 37°C. At the time point indicated by an arrow, 10 mM acetoin was added to the culture of SA35 (▵, ▴) and to one culture of B. subtilis 168 (○, ●), whereas it was omitted from the other culture of B. subtilis 168 (□, ■). Growth (○, □, ▵) was monitored photometrically, and acetoin in the culture supernatants (●, ■, ▴) was measured gas chromatographically.