The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus Delta H

This study is a continuation by the Environmental Biotechnology Group of the University of Tubingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus Delta H are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus Delta H. For this, we expanded our system for genetic modification of M. thermautotrophicus Delta H using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus Delta H compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus Delta H strains compared to the wild-type strain.IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus Delta H with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus Delta H. Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools.