Genes and Genetic System

The field of genetics has been restricted for many years to Escherichia coli and few other genera of aerobic or facul tative anaerobic bacteria such as Pseudomonas, Bacillus, and Salmonella. Anaerobic bacteria were known and studied since before 1900, but work on the genetics and molecular biology of anaerobic bacteria began to emerge only in the 1970s. The volume by Sebald is the most recent comprehensive review on genetics of anaerobes (46). Improvements in basic techniques for culturing anaerobes and recent advances in molecular biology techniques have helped in making rapid progress in understanding genes and genetic systems in anaerobic bacteria. Thermoanaerobic bacteria produce many thermostable enzymes but the yields are low. However, their enzymes can be overproduced by cloning the genes into mesophilic, industrially important aerobic bacteria. The genes can be overexpressed in both gram-positive and gram-negative hosts such as Bacillus subtilis and E. coli (47). One major advantage of cloning genes for thermostable enzymes in mesophiles is in the recovery of enzymes in greater than 95% purity by a singlestep high-temperature treatment that inactivates host proteins, including protease enzyme (47). Aerobic bacteria produce about 10-fold higher biomass yields than the anaerobic bacteria. Therefore, there is no advantage, except for biotransformation, in producing the enzymes in an anaerobic system.

Several species of the genus Clostridium are ofbiotech-nological interest because of the end products of their fermentative metabolism, the stereospecific reductions that they undertake, and the potentially important enzymes that they produce. Other species, such as C. botulinum, produce some of the most powerful toxins known today, which have been commercialized for medical treatments such as the correction of crossed eyes.

Various elements of gene-transfer technology have been developed in several species. Two gene-transfer procedures should prove widely applicable throughout the genus— electroporation and conjugal plasmid mobilization. In clos-tridia, mutants defective in a variety of functions, including purine, pyrimidine, vitamin, and amino acid biosynthesis, pathways of fermentative metabolism, and sporulation, have been isolated with ethyl methane sulfonate, A-methyl-A'-nitro-A-nitrosoguanidine, and ultraviolet light, and have been characterized (48-52). These mutant strains provide valuable information about gene regulation and function. Conjugal transfer of plasmids and transposons as well as phase transfection/plasmid transformation has now been documented in several species. In addition, recombinant DNA technology has also been employed to gain insights into the gene-transfer systems in Clostridium. Protoplasts of C. acetobutylicum can be trans-fected with phage DNA (53). The whole cells of C. acetobutylicum can be transformed with plasmid DNA using an electroporation procedure. Permealized cells of Thermoan-aerobacter thermohydrosulfuricus have been transformed with plasmid pUB110 (encoding resistance to kanamycin, KmR) and a derivative, pGS13, carrying a chloramphenicol resistance (CmR) marker (54).

A variety of cloning vectors have been developed for use in C. acetobutylicum. To date only pMTL500E has been used to introduce cloned heterologous genes into C. aceto-butylicum. These genes included the Clostridium pasteu-rianum leuB gene (55) and the C. thermocellum celA gene (see Ref. 56). Acquisition of the celA gene was demonstrated by an in situ plate assay using Congo red. In most cases, screening of clostridial gene banks has relied on expression of the heterologous genes in E. coli. Appropriate E. coli mutants have been employed for the isolation of C. acetobutylicum genes involved in the acetone-butanol fermentation. The butyraldeyde dehydrogenase gene of C. acetobutylicum has been isolated by complementation of an E. coli aldehyde dehydrogenase-deficient mutant. Endo-S-glucanases and xylanse of C. thermocellum and other cellulolytic clostridia are easily detectable on plates containing the appropriate substrate by the Congo red assay. Upon staining with Congo red, positive clones are surrounded by a yellow hydrolysis zone on a red background.

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