Abstract
Background:Chlamydomonas reinhardtii, a green micro-alga, is normally cultured in laboratories in Tris-Acetate Phosphate (TAP), a medium which contains acetate as the sole carbon source. Acetate in TAP can lead to occasional bacterial and fungal contamination. We isolated a yellow-pigmented bacterium from a Chlamydomonas TAP plate. It was named Clip185 based on the Chlamydomonas strain plate it was isolated from. In this article we present our work on the isolation, taxonomic identification and physiological and biochemical characterizations of Clip185. Methods: We measured sensitivities of Clip185 to five antibiotics and performed standard microbiological tests to characterize it. We partially sequenced the 16S rRNA gene of Clip185. We identified the yellow pigment of Clip185 by spectrophotometric analyses. We tested tolerance of Clip185 to six heavy metals by monitoring its growth on Lysogeny Broth (LB) media plates containing 0.5 mM -10 mM concentrations of six different heavy metals. Results: Clip185 is an aerobic, gram-positive rod, oxidase-negative, mesophilic, alpha-hemolytic bacterium. It can ferment glucose, sucrose and mannitol. It is starch hydrolysis-positive. It is very sensitive to vancomycin but resistant to penicillin and other bacterial cell membrane- and protein synthesis-disrupting antibiotics. Clip185 produces a C50 carotenoid, decaprenoxanthin, which is a powerful anti-oxidant with a commercial demand. Decaprenoxanthin production is induced in Clip185 under light. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of Clip185 revealed a 99% sequence identity to that of Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals. Conclusions: Our results show that Clip185 belongs to the genus Microbacterium. In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new Microbacterium species or a novel strain of Microbacterium binotii, and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.
Highlights
Chlamydomonas reinhardtii is a green unicellular alga in the phylum Chlorophyta
Our results show that Clip[185] belongs to the genus Microbacterium
Tris-Acetate Phosphate (TAP) medium recipes are described in Mitra et al, 2020.9 4A+ liquid TAP cultures were grown on a shaker under low light (70-80 μmol m-2s-1) for 3 days for aeration as described in Mitra et al, 2020.8,9 Clip[185] stock was maintained in the lab under dim light (15-30 μmol m-2s-1) at 22°C on Lysogeny Broth (LB) agar medium (Cold Spring Harbor Protocols)
Summary
Chlamydomonas reinhardtii is a green unicellular alga in the phylum Chlorophyta. It is an excellent experimental system to plant biologists, medical and bioenergy researchers.[1,2,3] Chlamydomonas is normally cultured in the lab in Tris-AcetatePhosphate (TAP), a medium which contains acetate as the sole carbon source.[4]. The primary goal of the high school student project was to identify a specific antibiotic and its right concentration that will eliminate Clip[185] contamination, without affecting Chlamydomonas growth on TAP media plates. This high school microbiology research project was further extended by two undergraduates from the University of West Georgia, to characterize Clip[185] on biochemical and physiological levels and determine the genus identity of Clip[185]. Whole genome sequencing data analyses will help us to identify genes in Clip[185] that are responsible for antibiotic-resistance, metal tolerance/detoxification and C50 carotenoid production, but will help to clarify whether Clip[185] is a new Microbacterium sp. We present our research on the isolation of Clip[185] and its physiological and biochemical characterizations
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