Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Abstract

Low-G؉C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competencemediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

“…The strains used in this study are listed in Table 2. Kanamycin marker deletions were made by transforming T. saccharolyticum cells with deletion vectors, using a protocol described previously (41). In brief, the deletion vectors consist of a kanamycin marker flanked by upstream and downstream homology regions of the target gene.…”

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum

“…C. thermocellum strains LL1160 and LL1161 were constructed by transforming respective integration plasmids pSH016 and pSH019 into strain LL1111 (Table 1; see Table S1); transformation and colony selection were carried out as previously described (24). T. saccharolyticum strains LL1193 and LL1194 were constructed by transforming the respective vectors pCP14 and pCP14* into wild-type T. saccharolyticum by using a natural-competence-based system (25) (Table 1; see Table S1), and transformants were selected by resistance to the antibiotic kanamycin.…”

Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

“…5-Fluoro-2=-deoxyuridine (FUDR) was used in the subsequent negative selection step to remove the htk marker. Transformation of T. saccharolyticum was performed as described previously (27). The primers and plasmids used for genes deletions are listed in Tables 2 and 3, respectively.…”