Biomedical subjects

Eleftherios T Papoutsakis

Publications and source records attributed to Eleftherios T Papoutsakis.

16 recordsLinked to original sources

PubMed2006

Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes.

Megakaryocytic (Mk) cell maturation involves polyploidisation, and the number of platelets produced increases with Mk DNA content. Ploidy levels in cultured human MK cells are much lower than those observed in vivo. This study demonstrated that adding the water-soluble vitamin nicotinamide (NIC) to mobilised peripheral blood CD34+ cells cultured with thrombopoietin (Tpo) more than doubled the percentage of high-ploidy (> or = 8N) MK cells. This was observed regardless of donor-dependent differences in Mk differentiation. Furthermore, MK cells in cultures with NIC were larger, had more highly lobated nuclei, reached a maximum DNA content of 64N (vs. 16N with Tpo alone), and exhibited more frequent and more elaborate cytoplasmic extensions. NIC also increased the ploidy of cultured primary murine MK cells and a cell line model (CHRF-288) of Mk differentiation. However, NIC did not alter Mk commitment, apoptosis, or the time at which endomitosis was initiated. Despite the dramatic phenotypic differences observed with NIC addition, gene expression microarray analysis revealed similar overall transcriptional patterns in primary human Mk cultures with or without NIC, indicating that NIC did not disrupt the normal Mk transcriptional program. Elucidating the mechanisms by which NIC increases Mk maturation could lead to advances in the treatment of Mk and platelet disorders.

Animals↗

PubMed2005

A comparative genomic view of clostridial sporulation and physiology.

Clostridia are anaerobic, endospore-forming prokaryotes that include strains of importance to human and animal health and physiology, cellulose degradation, solvent production and bioremediation. Their differentiation and related developmental programmes are not well understood at the molecular level. Recent genome sequencing and transcriptional-profiling studies have offered a glimpse of their inner workings and indicate that a better understanding of the orchestration of the molecular events that underlie their unique physiology, capabilities and diversity will pay major dividends.

Bacillus subtilis↗

PubMed2005

Intracellular butyryl phosphate and acetyl phosphate concentrations in Clostridium acetobutylicum and their implications for solvent formation.

It has been suggested (L. H. Harris, R. P. Desai, N. E. Welker, and E. T. Papoutsakis, Biotechnol. Bioeng. 67:1-11, 2000) that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum. Here, we determined BuP and acetyl phosphate (AcP) levels in fermentations of C. acetobutylicum wild type (WT), degenerate strain M5, a butyrate kinase (buk) mutant, and a phosphotransacetylase (pta) mutant. A sensitive method was developed to measure BuP and AcP in the same sample. Compared to the WT, the buk mutant had higher levels of BuP and AcP; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production. Consistent with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains. For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry weight), and higher and sustained levels corresponded to higher butanol formation fluxes. The BuP levels never exceeded 40 to 50 pmol/g (dry weight) in strain M5, which produces no solvents. The BuP profiles were bimodal, and there was a second peak midway through solventogenesis that corresponded to carboxylic acid reutilization. AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutilization. As expected, in the pta mutant the AcP levels were very low, yet this strain exhibited strong butanol production. These data suggest that BuP is a regulatory molecule that may act as a phosphodonor of transcriptional factors. DNA array-based transcriptional analysis of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress genes.

Bacterial Proteins↗

PubMed2005

Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum.

DNA microarray analysis of Clostridium acetobutylicum was used to examine the genomic-scale gene expression changes during the shift from exponential-phase growth and acidogenesis to stationary phase and solventogenesis. Self-organizing maps were used to identify novel expression patterns of functional gene classes, including aromatic and branched-chain amino acid synthesis, ribosomal proteins, cobalt and iron transporters, cobalamin biosynthesis, and lipid biosynthesis. The majority of pSOL1 megaplasmid genes (in addition to the solventogenic genes aad-ctfA-ctfB and adc) had increased expression at the onset of solventogenesis, suggesting that other megaplasmid genes may play a role in stationary-phase phenomena. Analysis of sporulation genes and comparison with published Bacillus subtilis results indicated conserved expression patterns of early sporulation genes, including spo0A, the sigF operon, and putative canonical genes of the sigma(H) and sigma(F) regulons. However, sigE expression could not be detected within 7.5 h of initial spo0A expression, consistent with the observed extended time between the appearance of clostridial forms and endospore formation. The results were compared with microarray comparisons of the wild-type strain and the nonsolventogenic, asporogenous M5 strain, which lacks the pSOL1 megaplasmid. While some results were similar, the expression of primary metabolism genes and heat shock proteins was higher in M5, suggesting a difference in metabolic regulation or a butyrate stress response in M5. The results of this microarray platform and analysis were further validated by comparing gene expression patterns to previously published Northern analyses, reporter assays, and two-dimensional protein electrophoresis data of metabolic genes (including all major solventogenesis genes), sporulation genes, heat shock proteins, and other solventogenesis-induced gene expression.

Amino Acids↗

PubMed2004

Molecular understanding of oxygen-tension and patient-variability effects on ex vivo expanded T cells.

Immunotherapy with ex vivo cultured T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving the proliferation and efficacy of the expanded cells. Low oxygen tension (5% pO(2)) was previously reported to improve T-cell expansion and alter cellular phenotypic characteristics compared to T cells cultured at 20% pO(2). Here we report the use of DNA-array based transcriptional analysis coupled with protein-level analysis to provide molecular insights into pO(2) and patient-variability effects on expanded primary human T cells. Analysis of seven blood samples showed that reduced pO(2) results in higher expression of genes important in lymphocyte biology, immune function, and cell-cycle progression. 20% pO(2) resulted in higher expression of genes involved in stress response, cell death, and cellular repair. Expression of granzyme A (gzmA) was found to be significantly regulated by oxygen tension with cells at 5% pO(2) having greater gzmA expression than at 20% pO(2). Protein-level analysis of gzmA was consistent with transcriptional analysis. Granzyme K (gzmK) was coexpressed with gzmA, whereas Granzyme B (gzmB) expression was found to precede the expression of both gzmA and gzmK in 15-day cultures. Temporal gene expression patterns for seven blood samples demonstrate that most genes are expressed by all patient samples in similar temporal patterns. However, several patient-specific gene clusters were identified, and one cluster was found to correlate well with cell proliferation and may potentially be used to predict patient-specific T-cell expansion.

Cell Culture Techniques↗

PubMed2004

A model-based optimization framework for the inference on gene regulatory networks from DNA array data.

MOTIVATION: Identification of the regulatory structures in genetic networks and the formulation of mechanistic models in the form of wiring diagrams is one of the significant objectives of expression profiling using DNA microarray technologies and it requires the development and application of identification frameworks. RESULTS: We have developed a novel optimization framework for identifying regulation in a genetic network using the S-system modeling formalism. We show that balance equations on both mRNA and protein species led to a formulation suitable for analyzing DNA-microarray data whereby protein concentrations have been eliminated and only mRNA relative concentrations are retained. Using this formulation, we examined if it is possible to infer a set of possible genetic regulatory networks consistent with observed mRNA expression patterns. Two origins of changes in mRNA expression patterns were considered. One derives from changes in the biophysical properties of the system that alter the molecular-interaction kinetics and/or message stability. The second is due to gene knock-outs. We reduced the identification problem to an optimization problem (of the so-called mixed-integer non-linear programming class) and we developed an algorithmic procedure for solving this optimization problem. Using simulated data generated by our mathematical model, we show that our method can actually find the regulatory network from which the data were generated. We also show that the number of possible alternate genetic regulatory networks depends on the size of the dataset (i.e. number of experiments), but this dependence is different for each of the two types of problems considered, and that a unique solution requires fewer datasets than previously estimated in the literature. This is the first method that also allows the identification of every possible regulatory network that could explain the data, when the number of experiments does not allow identification of unique regulatory structure.

Algorithms↗

PubMed2004

Molecular insights into the pleiotropic effects of plasma on ex vivo-expanded T cells using DNA-microarray analysis.

OBJECTIVE: Immunotherapy with ex vivo-expanded T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving cell expansion and efficacy. We used DNA-microarray and flow-cytometric analysis coupled with functional assays to investigate mechanistic aspects of plasma supplementation in ex vivo T-cell expansion. METHODS: The effect of plasma supplementation on 18 primary T-cell cultures over a 15-day expansion was investigated. Transcriptional analysis of 5 samples was done with time points every 2 to 3 days throughout the 15-day expansion. Quantitative RT-PCR analysis was used to confirm selected microarray data. The expression of granzyme A and vimentin were analyzed using intracellular flow cytometry. T-cell functionality was assessed using a mixed leukocyte reaction (MLR). RESULTS: We show that the increased expansion of plasma-supplemented cultures of primary human T cells is mostly due to increased cell survival. T cells from plasma-supplemented cultures show higher expression of immunoglobulin genes, integrins, and genes of cytotoxic granules, suggesting a possible enhanced immune function. This was confirmed using a mixed leukocyte reaction and intracellular granzyme-A measurements. A distinct gene expression pattern was correlated to viability differences between plasma-supplemented and serum-free cultures. Ontological analysis of genes in this pattern suggests that the decreased viability of serum-free cultures correlates with higher expression of actin-cytoskeleton and lipid-metabolism genes. Vimentin was found to be expressed higher in serum-free cultures. CONCLUSIONS: These results indicate that the observed decreased cytotoxicity of T cells cultured in serum-free media may be due to increased oxidative stress and cytoskeleton degradation.

Cell Culture Techniques↗

PubMed2004

Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell's response to butanol stress.

Spo0A is the regulator of stationary-phase events and is required for transcription of solvent formation genes in Clostridium acetobutylicum. In order to elucidate the role of spo0A in differentiation, we performed transcriptional analysis of 824(pMSPOA) (a spo0A-overexpressing C. acetobutylicum strain with enhanced sporulation) against a plasmid control strain. DNA microarray data were contrasted to data from a spo0A knockout strain (SKO1) that neither sporulates nor produces solvents. Transcripts of fatty acid metabolism genes, motility and chemotaxis genes, heat shock protein genes, and genes encoding the Fts family of cell division proteins were differentially expressed in the two strains, suggesting that these genes play roles in sporulation and the solvent stress response. 824(pMSPOA) alone showed significant downregulation of many glycolytic genes in stationary phase, which is consistent with metabolic flux analysis data. Surprisingly, spo0A overexpression resulted in only nominal transcriptional changes of regulatory genes (abrB and sigF) whose expression was significantly altered in SKO1. Overexpression of spo0A imparted increased tolerance and prolonged metabolism in response to butanol stress. While most of the differentially expressed genes appear to be part of a general stress response (similar to patterns in two plasmid control strains and a groESL-overexpressing strain), several genes were expressed at higher levels at early time points after butanol challenge only in 824(pMSPOA). Most of these genes were related to butyryl coenzyme A and butyrate formation and/or assimilation, but they also included the cell division gene ftsX, the gyrase subunit-encoding genes gyrB and gyrA, DNA synthesis and repair genes, and fatty acid synthesis genes, all of which might play a role in the immediate butanol stress response, and thus in enhanced butanol tolerance.

Bacterial Proteins↗

PubMed2004

Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum.

The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response. Strain 824(pGROE1) was generated by groESL overexpression. The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h. A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol). Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA. A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes. Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease. Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.

Bacterial Proteins↗

PubMed2003

Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains.

Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms.

Adaptation, Physiological↗

PubMed2003

Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program.

DNA array and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824. Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter. The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del). Overexpression of groESL resulted in increased final solvent titers 40% and 33% higher than those of the wild type and plasmid control strains, respectively. Active metabolism lasted two and one half times longer in 824(pGROE1) than in the wild type. Transcriptional analysis of 824(pGROE1) revealed increased expression of motility and chemotaxis genes and a decrease in the expression of the other major stress response genes. Decreased expression of the dnaKJ operon upon overexpression of groESL suggests that groESL functions as a modulator of the CIRCE regulon, which is shown here to include the hsp90 gene. Analysis of the plasmid control strain 824(pSOS95del) revealed complex host-plasmid interactions relative to the wild-type strain, resulting in prolonged biphasic growth and metabolism. Decreased expression of four DNA gyrases resulted in differential expression of many key primary metabolism genes. The ftsA and ftsZ genes were expressed at higher levels in 824(pSOS95del), revealing an altered cell division and sporulation pattern. Both transcriptional and Western analyses revealed elevated stress protein expression in the plasmid-carrying strain.

1-Butanol↗

PubMed2003

Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations.

Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB.

Alcohol Dehydrogenase↗

PubMed2003

DNA array-based transcriptional analysis of asporogenous, nonsolventogenic Clostridium acetobutylicum strains SKO1 and M5.

The large-scale transcriptional program of two Clostridium acetobutylicum strains (SKO1 and M5) relative to that of the parent strain (wild type [WT]) was examined by using DNA microarrays. Glass DNA arrays containing a selected set of 1,019 genes (including all 178 pSOL1 genes) covering more than 25% of the whole genome were designed, constructed, and validated for data reliability. Strain SKO1, with an inactivated spo0A gene, displays an asporogenous, filamentous, and largely deficient solventogenic phenotype. SKO1 displays downregulation of all solvent formation genes, sigF, and carbohydrate metabolism genes (similar to genes expressed as part of the stationary-phase response in Bacillus subtilis) but also several electron transport genes. A major cluster of genes upregulated in SKO1 includes abrB, the genes from the major chemotaxis and motility operons, and glycosylation genes. Strain M5 displays an asporogenous and nonsolventogenic phenotype due to loss of the megaplasmid pSOL1, which contains all genes necessary for solvent formation. Therefore, M5 displays downregulation of all pSOL1 genes expressed in the WT. Notable among other genes expressed more highly in WT than in M5 were sigF, several two-component histidine kinases, spo0A, cheA, cheC, many stress response genes, fts family genes, DNA topoisomerase genes, and central-carbon metabolism genes. Genes expressed more highly in M5 include electron transport genes (but different from those downregulated in SKO1) and several motility and chemotaxis genes. Most of these expression patterns were consistent with phenotypic characteristics. Several of these expression patterns are new or different from what is known in B. subtilis and can be used to test a number of functional-genomic hypotheses.

Bacterial Proteins↗

PubMed2003

Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum.

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

Acetone↗

PubMed2002

Differential expression and phosphorylation of distinct STAT3 proteins during granulocytic differentiation.

External stimuli act in concert with intracellular signals to regulate a cell's genetic program, activating genes important in granulocytic lineage commitment, proliferation, and maturation. Signal transducer and activator of transcription 3 (STAT3), a transcription factor, has been implicated in mediating granulocytic differentiation. We have examined the role of STAT3 as a physiologic mediator of granulocytic kinetics. Distinct isoforms--the long form STAT3 alpha, the truncated forms STAT3 beta and STAT3 gamma, and a putative novel form STAT3 delta--were expressed and activated in a maturation stage-specific manner. With the progression of differentiation, the ratio of isoforms shifted from predominantly STAT3 alpha to STAT3 beta. The kinetics of STAT3 gamma, generated through proteolytic cleavage of STAT3 alpha, coincided with but were inverse to those of STAT3 alpha. STAT3 delta was expressed at low levels and decreased with differentiation but was preferentially phosphorylated during an intermediate stage of maturation. Under different culture conditions (pH, O(2) tension [pO(2)], IL-3), we found that the expression and phosphorylation status of the different STAT3 isoforms displayed unique kinetic patterns that correlated with the effects on granulocyte differentiation. The evidence suggests that signals triggered by pH, pO(2), and IL-3 each converge on STAT3 through independent mechanisms, exploiting the flexibility granted by the diversity in expression and phosphorylation of the different STAT3 isoforms, to regulate distinct granulocytic cell responses. The selective expression of STAT3 isoforms and their activation is a major determinant of granulocytic cell development and provides a molecular basis for evaluating the effects of various environmental factors on the STAT3-mediated signaling pathway.

Antigens, CD34↗

PubMed2002

Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824.

The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.

Bacterial Proteins↗