Identification and Characterization of Two Functionally Unknown Genes Involved in Butanol Tolerance of Clostridium acetobutylicum

Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance

Group II Intron-Anchored Gene Deletion in Clostridium

Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established

Close-up views of the protein spots with differential expression.

<p>I, DSM 1731; II, DDC14; III, 1731(pIMP1); IV, 1731(p1518-1519). A) The differentially expressed PFOR in DSM1731 and DDC14. B) The differentially expressed PFOR in 731(pIMP1) and 1731(p1518-1519). C) The differentially expressed Hag in DSM1731 and DDC14. D) The differentially expressed Hag in 731(pIMP1) and 1731(p1518-1519).</p

Diagram of growth inhibition.

<p>A), DSM 1731 and its deletion or disruption mutants DDC14, DC93 and DC94. B) 1731(pIMP1) and 1731(p1518-1519). The growth inhibition level was determined by using the following formula: [(<i>A</i>600)+BuOH/<i>A</i>600]×100, <i>A</i>600+BuOH is referred to cell density in the presence of butanol.</p

Overexpression of SMB_G1518-1519 influenced the motilities of <i>C. acetobutylicum</i>.

<p>Overexpression of SMB_G1518-1519 influenced the motilities of <i>C. acetobutylicum</i>.</p

Growth profiles for 1731(pIMP1), 1731(p1518-1519) and DDC14(p1518-1519)

<p>. A) Growth profiles under normal condition. B) Growth profiles under 1% butanol stress.</p

Growth profiles for DSM 1731 and its deletion or disruption mutants.

<p>A) Growth profiles under normal condition. B) Growth profiles under 1% butanol stress.</p

Bacterial strains, plasmids and primers.

<p><b>Abbreviations</b>: Amp^r, ampicillin resistance; Cm^r, chloramphenicol resistance; Tet^r, tetracycline resistance; <i>Φ3t1</i>, Φ3t1 methyltransferase gene of <i>Bacillus subtilis</i> phage Φ3t1. DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.</p

The analysis of main fermentation products after 50% of growth inhibition degree was achieved.

<p>A) The analysis of main fermentation products after DSM 1731 and its deletion mutant DDC14 were treated by butanol for 6 h. B) The analysis of main fermentation products after the plasmid control strain 1731(pIMP1) and overexpression strain 1731(p1518-1519) were treated by butanol for 18 h.</p

Functional classification of different proteins.

<p>Note: The protein levels in DSM 1731 and 1731(pIMP1) under normal condition were used as the basis for comparison, respectively. The upregulation of fatty acid synthesis enzymes (acyl-ACP (acyl-carrier-protein) thioesterase and 3-oxoacyl-ACP synthase I) is more likely to be an indicator reflecting cell damage from butanol stress. The upregulation of HSPs is regarded as a common response to butanol stress, but not related with the function of SMB_G1518-1519.</p