Changes in protein synthesis and identification of proteins specifically induced during solventogenesis in Clostridium acetobutylicum

Abstract

At the end of the exponential growth phase the Gram-positive bacterium Clostridium acetobutylicum performs a metabolic switch from classical sugar fermentation accompanied by the production of acetate and butyrate to reinternalization and oxidation of these acids to acetone and butanol. Protein synthesis in acidogenic and solventogenic C. acetobutylicum cells was compared by two-dimensional gel electrophoresis of radioactively labeled proteins. The results show that the switch from acid to solvent production is accompanied by dramatic changes in the protein pattern. During solventogenesis, the synthesis of 52 proteins out of 130 analyzed was increased more than twofold, the synthesis of 34 proteins decreased to less than half as compared with synthesis in acidogenic cells. The changes in protein synthesis were generally reflected by changes in the abundance of the respective proteins, as determined from quantitative analysis of silver-stained second-dimensional gels. Nine proteins induced during solventogenesis were identified by N-terminal microsequencing. One of these proteins, the acetoacetate decarboxylase, is directly involved in solventogenesis. Other proteins synthesized in higher amounts during solventogenesis were three general stress proteins (DnaK, GroEL, Hsp 18), two enzymes involved in serine biosynthesis (serine aminotransferase and 3-phosphoglycerate dehydrogenase) as well as a seryl-tRNA synthetase. mRNA analysis provided evidence that the latter three are encoded by genes organized in an operon and are transcriptionally induced at the onset of solventogenesis. The proteins acetoacetate decarboxylase and Hsp 18 occurred in two variants, indicating possible covalent modification of these proteins.

Transcriptional Program of Early Sporulation and Stationary-Phase Events in Clostridium acetobutylicum

“…Expression of spo0A (CAC2071), the master regulator of solventogenesis and sporulation in solvent-forming clostridia (29,59), is discussed later. Schaffer and colleagues have previously identified several proteins through two-dimensional gel electrophoresis whose expression was induced during solventogenesis in a transient continuous culture (69): solventogenic acetoacetate decarboxylase (gene adc); heat shock protein Hsp18 (CAC3714); isopropylmalate dehydrogenase LeuB (CAC3171); serine biosynthesis proteins SerA (CAC0015), SerC (CAC0014), and SerS (CAC0017); and pyridoxine biosynthesis protein PdxY (CAC0594). The expression of all these genes (Fig.…”

Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum

“…In C. acetobutylicum, hsp90 has a CIRCE promoter, and, thus, is apparently HrcAregulated unlike B. subtilis (Tomas et al, 2003b). hsp18 is an a-crystallin-type heat shock protein whose expression at the RNA and/or protein level has been shown to be induced by heat shock (Pich et al, 1990;Sauer and Dürre, 1993), the onset of solventogenesis Sauer and Dürre, 1993;Schaffer et al, 2002), and butanol stress (Alsaker et al, 2004;Tomas et al, 2004). It contains a highly conserved CtsR operator sequence in its promoter (Schumann et al, 2002;Tomas et al, 2004).…”

“…In addition, a locus of genes related to the uptake and conversion of sulfate to cysteine was upregulated 60-120 min after acetate stress. Cysteine biosynthesis requires serine, and the serine-biosynthesis operon (Schaffer et al, 2002), which includes serC (CAC0014) and serA (CAC0015), was differentially upregulated 10-60 min post-acetate stress. Finally, genes in a putative operon orthologous to microbial methionine transporters (CAC0984-CAC0986) were differentially upregulated after acetate stress.…”

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

“…There is little information available on the changes in the proteome during the metabolic shift in phosphate-limited continuous cultures (Schaffer et al 2002). Accordingly, because their deduction from transcriptional data might be misleading (Keene 2010;Straub 2011;Zhang et al 2014), here we assign constant, but phenotype-specific, intracellular enzyme concentrations to the subpopulations.…”