Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants

Abstract

The proteomic profiles of several Clostridium acetobutylicum strains were compared by two-dimensional gel electrophoresis and mass spectroscopy. The proteomic profile of C. acetobutylicum wild type strain ATCC 824 with and without a commonly used control plasmid and with a spo0A overexpression plasmid pMSPOA was compared. A total of 2,081 protein spots were analyzed; 23 proteins were chosen to be identified of which 18 were unique and 5 were proteins located in more than one location. The proteins identified were classified into heat shock stress response, acid and solvent formation, and transcription and translation proteins. Spo0A was identified and its protein expression was confirmed to be absent in the spo0A knockout SKO1 strain as expected, as was the protein Adc, which is known to be regulated by Spo0A. The expression of six proteins was not detected in strain SKO1 indicating these proteins require Spo0A. Spo0A overexpression affected the abundance of proteins involved in glycolysis, translation, heat shock stress response, and energy production. Two features were identified: five of the 23 proteins identified were located in more than one position and clusters of protein spots resembled fingers of a straightened hand. Normally a protein localizes to only one spot on the gel; localization of a protein to more than one spot is indicative of post-translational modifications, suggesting that such modification of proteins may be a more prevalent mechanism in C. acetobutylicum than previously thought. The clusters of protein spots resembling fingers of a straightened hand were in the acidic high molecular weight areas. Two such protein spots were identified as variants of the same protein, GroEL.

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

“…Phosphate acetyltransferase (CAC1742) involved in the catalysis of acetyl CoA to acetyl phosphate leading to the production of acetate [10] and acetoacetate decarboxylase (CAP0165) [genes involved in the solvent formation resides on the plasmid and hence the prefix CAP] [19], which is essential for the solvent production that catalyzes the decarboxylation of acetoacetate to acetone [23], were found to be significantly lower in expression from the stationary phase in comparison to the exponential growth. In contrast, proteomic analysis of C. acetobutylicum using glucose reported that acetoacetate decarboxylase is upregulated from exponential to stationary phase [31]. These findings were consistent with the protein-abundance analysis, which showed that (abrB) stationary/sporulation gene expression regulator protein (CAC0310) as the most abundant in stationary phase of xylose utilized ABE fermentation and Spo0A protein as the most abundant using glucose [31].…”

“…Very few proteomic studies have been conducted in C. acetobutylicum using one-and/or two-dimensional gel electrophoresis-mass spectrometry (1D/2D-GE-MS) technique. Besides, most of them were from C. acetobutylicum grown on glucose substrate and focused specifically on proteins involved in acidogenic and solventogenic pathways [28,31]. A total of 564 proteins identified from the exponential growth of glucose utilized C. acetobutylicum DSM1731 strain were used to publish the proteome reference map [17].…”

“…Stationary/sporulation gene expression regulator (abrB) B. subtilis ortholog protein (CAC0310), which is one of the five most abundant proteins identified from the stationary phase of xylose utilized ABE fermentation was found to be less abundant in the exponential growth. Contrarily, proteomic analysis of C. acetobutylicum using glucose substrate showed that Spo0A protein is constitutively abundant from exponential to stationary phase [31].…”

“…The efficiency of C. acetobutylicum in xylose utilization and solvent yields were significantly lower when compared to glucose [20], suggesting the need for an in-depth investigation on this organism. The genome sequence of C. acetobutylicum ATCC 824 strain was published [19] followed by the proteomic studies [13,28,31]. Moreover, the proteome reference map of C. acetobutylicum DSM 1731 strain using glucose substrate was reported [17].…”

Activity of abrB310 promoter in wild type and spo0A-deficient strains of Clostridium acetobutylicum

“…DNA microarrays have shown signiWcant diVerences in the expression of at least 123 genes between wild type and strain 824(pMspo0A), as well as signiWcant transcriptional diVerences between the acidogenic and solventogenic phases in wild type C. acetobutylicum and the asolventogenic mutant strain, M5 [4,5]. 2D gel analysis of the proteome of wild type, SKO1 and 824(pMspo0A) revealed signiWcantly diVerent levels of heat shock proteins, metabolic regulators and translational proteins between strains [6]. Such surveys are useful in allowing us to gage the complexity of the lifecycle of C. acetobutylicum.…”