Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

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.

Draft genome sequence of Microbacterium fakhimisp. nov., a novel bacterium associated with the algaChlamydomonas reinhardtii

“…4b). The typical previously described three peaks for decapornoxanthin at 413 nm, 437 nm, and 467 nm were observed (Mitra et al, 2021).…”

“…nov. was grown under continuous illumination (Photosynthetic Photon Flux Density (PPFD) of 60-90 µmol photon⋅m -2 ⋅s -1 ), 24ºC and 140 rpm. Pigments extraction was performed according to (Mitra et al, 2021). For this, 1 mL of cells were treated with 5mL of 100% methanol during 1 h in the dark at 20ºC for carotenoid extraction.…”

“…Microbacterium spp. are widely distributed and has been isolated from a broad range of habitats such as soils (Takeuchi and Hatano, 1998;Lu et al, 2019), deserts (Mandakovic et al, 2015;Yang et al, 2018;Mandakovic et al, 2020;Zhu et al, 2021), arctic zones (Reis-Mansur et al, 2019), waters (Yi et al, 2021), heavy metal and other contaminated ecosystems (Qu et al, 2013;Learman et al, 2019;Lu et al, 2019;Corretto et al, 2020;Martínez-Rodríguez et al, 2020;Mishra et al, 2021), dairy products (Vithanage et al, 2016;Bellassi et al, 2020;Bellassi et al, 2021), phyllosphere (Behrendt et al, 2001;Marquez-Santacruz et al, 2010a;Bano et al, 2022), algae and cyanobacteria cultures (Jones et al, 2016;Mitra et al, 2021;Zhou et al, 2021), and animal host (Gneiding et al, 2008;Amano et al, 2019;Heo et al, 2020). Interestingly, Microbacterium also appears to be a common laboratory contaminant (Salter et al, 2014).…”

“…Many Microbacterium spp. have been study due to their biotechnological interest in facilitating bioremediation (Qu et al, 2013;Corretto et al, 2015;Avramov et al, 2016;Learman et al, 2019;Lu et al, 2019;Ortet et al, 2019;Corretto et al, 2020;Heidari et al, 2020;Martínez-Rodríguez et al, 2020;Mishra et al, 2021;Mitra et al, 2021) or promoting plant growth (Vílchez et al, 2018;Singh and Singh, 2019;García-Fontana et al, 2020;Yang et al, 2021). Moreover, many Microbacterium spp.…”

Chlamydomonas reinhardtii and Microbacterium forte sp. nov.,a mutualistic association that favor sustainable hydrogen production

“…are among the most common inhabitants of the rhizosphere (Rosenblueth and Martínez-Romero, 2006; Marquez-Santacruz et al, 2010; Abadi et al, 2020). They are also recurrent bacteria frequently found within algal cultures (Burmølle et al, 2006; Li et al, 2013; Jones et al, 2016; Ji et al, 2019; Martelli et al, 2021; Mitra et al, 2021), which is probably reflecting frequent coexistence of these organisms in natural ecosystems.…”