A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

Baker, Patricia L.; Orf, Gregory S.; Khan, Zahid; Espinoza, Levi; Leung, Sabrina; Kevershan, Kimberly; Redding, Kevin E.

doi:10.1128/AEM.01287-19

Title: A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

Journal Article · Tue Sep 17 00:00:00 EDT 2019 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.01287-19· OSTI ID:1631806

Baker, Patricia L. ^[1];

^[1]; Khan, Zahid ^[2]; Espinoza, Levi ^[1]; Leung, Sabrina ^[1]; Kevershan, Kimberly ^[3];

^[1]

Arizona State Univ., Tempe, AZ (United States)
Arizona State Univ., Tempe, AZ (United States); Univ. of Peshawar (Pakistan)
Arizona State Univ., Tempe, AZ (United States); Marana High School, Tucson, Arizona (United States)

The heliobacteria are members of the bacterial orderClostridialesand form the only group of phototrophs in the phylumFirmicutes. 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. Although the speciesHeliobacterium modesticaldumis an excellent candidate as a model system for the familyHeliobacteriaceae, 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 withH. modesticaldum. Five heliobacterial DNA methyltransferase genes were expressed in anEscherichia colistrain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation intoH. modesticaldumis 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. Moreover, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants. Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyllgas 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.

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Research Organization:: Arizona State Univ., Tempe, AZ (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division

Grant/Contract Number:: SC0010575

OSTI ID:: 1631806

Journal Information:: Applied and Environmental Microbiology, Vol. 85, Issue 19; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 8 works

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Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum Orf, Gregory S.; Redding, Kevin E. Photosynthesis Research, Vol. 142, Issue 3 https://doi.org/10.1007/s11120-019-00672-3	journal	September 2019

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Title: A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum

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