Regulatory functions of the pyruvate decarboxylase β (E1β) and the dihydrolipoamide acetyltransferase (E2) subunits of the pyruvate dehydrogenase complex in Bacillus subtilis

Haichun Gao, Purdue University

Abstract

The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1α and E1β), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). Disruptions of the pdhB, pdhC and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful. The three mutants lacked PDH activity and were unable to grow on glucose. The pdhB and pdhC mutants sporulated to only 5% of the wild type whereas the pdhD deletion strain sporulated to 55% of the wild type. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to function(s) of these subunits independent of enzymatic activity. The sporulation defect could be enhanced by the addition of acetate, glutamate, succinate and divalent cations, suggesting that physiological conditions had no significant effect on improving the sporulation of these mutants. The Escherichia coli aceF gene failed to restore either PDH enzymatic activity or sporulation to the pdhC deletion strain. PDH enzymatic activity was fully recovered with either the B. subtilis or the Bacillus thuringiensis pdhC gene. With the B. thuringiensis pdhC gene, however, the sporulation efficiency was elevated to only 70% of wild type, whereas sporulation efficiency was the same as the wild type with the B. subtilis pdhC gene. Results from analysis of the expression of various spo-lacZ fusions revealed that the pdhB and pdhC deletion strains were probably blocked between late stage II and stage III. Using membrane stains (FM 4-64, MTG) to observe septal biogenesis confirmed that the block in sporulation occurred between stages II and III. A suppressor of the pdhC deletion strain which sporulated approximately ten times higher than the deletion strain was isolated. Two suppressor genes were identified as rpoE and pksR by constructing a transposon insertion library in the pdhC deletion strain, screening for higher sporulation efficiency strains and then subcloning and sequencing the transposon insertion region. RpoE, the δ subunit of RNA polymerise, may reduce the affinity of RNA polymerase for DNA and loss of its function could result in altered transcription of certain genes. pksR, involved in synthesis of polyketide antibiotics, may be one of the target genes that the E1β and E2 subunits regulate. Suppression by rpoE implies that E2 regulation occurs at the level of transcription. The absence of E2 may prevent transcription which is restored by an RNA polymerase lacking the δ subunit. Alternatively, the lack of E2 may result in the transcription of genes which are normally repressed but cannot be transcribed without RpoE. The E1β and E2 subunits seem to work together during regulation.

Degree

Ph.D.

Advisors

Aronson, Purdue University.

Subject Area

Microbiology|Biochemistry

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