Characterization of iron reduction in Clostridium acetobutylicum mutants obtained by targeted and transposon mutagenesis

Iron reduction in Gram-positive bacteria is not well understood yet, even if it has been investigated in some extent for Gram-positive bacteria. The mechanisms involved in the delivery of electrons to a solid terminal electron acceptor like iron oxides have not been defined. Clostridium acetobutylicum is an appropriate Gram-positive bacterium to study those mechanisms as genetic tools have been developed due to its industrial interest, allowing easy targeted and random mutagenesis. In this Master’s project, phenotype of two mutants, whose dihydroorotate dehydrogenase 1B (pyrD) or ferredoxin hydrogenase (hydA) gene have been knocked out through ClosTron mutagenesis, have been characterized and no phenotype diverging from the wild strain has been detected. However, evidences of flavin presence in the spent growth medium have been observed during experiment. An attempt to measure their redox state, either by direct measurement or through the addition of AQDS, has been done but results are non-conclusive so far. A screening of mutant issued from untargeted mutagenesis has been tried, but the protocol could not furnish stable results during the test phase using the wild type strain. The protocol has been improved, but there still are problems regarding growth and iron reduction.