A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

Abstract

The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.

Keywords: DNA methylation; conjugation; heliobacteria; restriction.