Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system.

The terrestrial serpentinite-hosted ecosystem known as "The Cedars" is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars ('Paeni-Cedars'), Alishewanella sp. BS5-314 ('Ali-BS5-314'), and Anaerobacillus sp. CMMVII ('Anaero-CMMVII'). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7-10 (minimum pH tested was 7), temperatures 20-40°C, and 0-3% NaCl concentration. The addition of 10-20 mM CaCl2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10-12 and temperatures 10-40°C, with limited growth observed 1-5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9-12, with the addition of 0.01-1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.