ABSTRACT
The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy of solR, restoring acetone and butanol production to wild-type levels. This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels. The promoter region upstream of alcohol dehydrogense E (adhE) was examined in efforts to identify sites that play major roles in the control of expression. A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was analyzed using a chloramphenicol acetyl-transferase reporter system. Our results show that a region beyond the 0A box is needed for full induction of the promoter. Additionally, we show that the presence of sequences around a possible processing site designated S2 may have a negative role in the regulation of adhE expression.
Subject(s)
Acetone/metabolism , Bacterial Proteins/genetics , Butanols/metabolism , Clostridium/genetics , Clostridium/metabolism , DNA-Binding Proteins/genetics , Repressor Proteins/genetics , Solvents/metabolism , Alcohol Dehydrogenase/genetics , Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Industrial Microbiology , Mutation , Plasmids , Promoter Regions, Genetic , Transcription Initiation SiteABSTRACT
The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.
Subject(s)
Clostridium/enzymology , Clostridium/genetics , Genes, Bacterial , Methyltransferases/genetics , Methyltransferases/metabolism , 1-Propanol/metabolism , Bacterial Proteins/metabolism , Butanols/metabolism , Butyric Acid/metabolism , Cloning, Molecular , Cyclopropanes/chemistry , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Ethanol/metabolism , Fatty Acids/biosynthesis , Fatty Acids/chemistry , Gene Expression , Genetic Complementation Test , Kinetics , Phosphorylation , Phosphotransferases (Carboxyl Group Acceptor)/genetics , Phosphotransferases (Carboxyl Group Acceptor)/metabolism , Plasmids/genetics , Repressor Proteins/genetics , Solvents/metabolism , Transcription Factors/metabolismABSTRACT
Growth-attained curves have shown only limited effectiveness in studies of long bone growth in archaeological populations. As an alternative, the length of long bone diaphyses in children aged 0 to 12 years from the Libben skeletal collection were first normalized by average adult long bone length, and derivatives of fitted curves were then used to determine age-specific growth velocity. The sample was compared to healthy Euroamerican children from Denver, Colorado (Maresh: American Journal of Diseases of Children 89:725-742, 1955). Results indicate almost identical patterns of growth in the two populations with one exception: the Libben sample shows significantly diminished velocity during the first three years of life. This period of depressed growth coincides with high levels of infectious disease.
