Stage 0 sporulation gene A as a molecular marker to study diversity of endospore‐forming Firmicutes

Abstract

In this study, we developed and validated a cultureindependent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endosporeforming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our cultureindependent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes.

A Combination of Extreme Environmental Conditions Favor the Prevalence of Endospore-Forming Firmicutes

“…Soil, sediment, and biofilm samples were subjected to an indirect DNA extraction procedure as previously described (Wunderlin et al, 2013). This modified protocol included the separation of the biomass from soil and sediment using a 1% (w/v) hexa-meta-phosphate solution followed by the collection of the biomass for DNA extraction.…”

“…There are at least two potential explanations to this. On the one hand, a methodological bias against molecular detection of Firmicutes could explain their under detection (Filippidou et al, 2015) and, accordingly, it has been shown that a tailored DNA extraction method allows for a better assessment of the abundance and diversity of EFF in environmental samples (Wunderlin et al, 2013). On the other hand, the poor detection in molecular ecology studies might accurately reflect the relative distribution of EFF in environmental samples.…”

Genome Diversity of Spore-Forming Firmicutes

“…(93), Macrococcus caseolyticus (94), and many others (44), see Table 5. Still, Spo0A can be used as a molecular marker for evaluating the abundance of spore-formers in natural environments (134). …”

Estimating the Abundance of Endospores of Sulfate-Reducing Bacteria in Environmental Samples by Inducing Germination and Exponential Growth

“…This is valuable when quantitative DNA extraction from the target population requires laborious processing, e.g. for endospores (Wunderlin et al 2013), or when the target population is difficult to access due to low relative abundance and a complex matrix such as sediment. A trait-based approach is also advantageous when probes or primers are not available to fully encompass the diversity of the target group, or when its diversity is simply unknown.…”

“…For example, it has recently been estimated that endospores comprise half of all cells in subsurface marine sediments Lomstein et al 2012). Despite their ecological potential and apparent numerical importance, little is known about the metabolic and physiological diversity or the abundance of dormant populations within microbial communities due to a paucity of methods to distinguish and study dormant cells Wunderlin et al 2013;Wunderlin et al 2014). One difficulty concerns unambiguously differentiating between active, dormant and dead cells; combinations of methods are required to target these various physiological states (Blagodatskaya and Kuzyakov 2013).…”