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

Abstract

Environmental conditions unsuitable for microbial growth are the rule rather than the exception in most habitats. In response to this, microorganisms have developed various strategies to withstand environmental conditions that limit active growth. Endospore-forming Firmicutes (EFF) deploy a myriad of survival strategies in order to resist adverse conditions. Like many bacterial groups, they can form biofilms and detect nutrient scarcity through chemotaxis. Moreover, within this paraphyletic group of Firmicutes, ecophysiological optima are diverse. Nonetheless, a response to adversity that delimits this group is the formation of wet-heat resistant spores. These strategies are energetically demanding and therefore might affect the biological success of EFF. Therefore, we hypothesize that abundance and diversity of EFF should be maximized in those environments in which the benefits of these survival strategies offsets the energetic cost. In order to address this hypothesis, geothermal and mineral springs and drillings were selected because in these environments of steep physicochemical gradients, diversified survival strategies may become a successful strategy.We collected 71 samples from geothermal and mineral environments characterized by none (null), single or multiple limiting environmental factors (temperature, pH, UV radiation, and specific mineral composition). To measure success, we quantified EFF gene copy numbers (GCN; spo0A gene) in relation to total bacterial GCN (16S rRNA gene), as well as the contribution of EFF to community composition. The quantification showed that relative GCN for EFF reached up to 20% at sites characterized by multiple limiting environmental factors, whereas it corresponded to less than 1% at sites with one or no limiting environmental factor. Pyrosequencing of the 16S rRNA gene supports a higher contribution of EFF at sites with multiple limiting factors. Community composition suggested a combination of phylotypes for which active growth could be expected, and phylotypes that are most likely in the state of endospores, in all the sites. In summary, our results suggest that diversified survival strategies, including sporulation and metabolic adaptations, explain the biological success of EFF in geothermal and natural springs, and that multiple extreme environmental factors favor the prevalence of EFF.

Keywords: 16S rRNA gene; Clostridium; Firmicutes; endospores; geothermal springs; mineral springs; qPCR; spo0A.

FIGURE 1

Correlation of relative presence of EFF and selected environmental factors. Generalized additive models depicting the relative presence of Endospore-forming Firmicutes –EFF- (spo0A/16S rRNA gene ratio; y-axis) to the in situ measurements of temperature (A), pH (B). No significant correlation between relative presence of EFF and these two factors is observed.

FIGURE 2

Effect of multiple, single, and no limiting factors on relative presence of EFF. The boxplots represent the qPCR ratios of spo0A gene/16S rRNA gene, grouped by decision node.

FIGURE 3

Diversity analysis of eight samples: 4NAP1 (Nea Apollonia, Greece, multiple extreme environmental factors, biofilm, and water from thermal spring boring); Col, 49THE1 and 51THE3 (Los Volcanos, Colombia, biofilm and water from thermal spring; Thermia, sediment from hot spring, Thermia, sediment from mineral spring, respectively, single extreme environmental factor); 44AGP2, 25KAM5, NeFer and NeSul (Agia Paraskevi spring, sediment from the thermal spring; Kanava beach, Milos, Greece, water, and sand from the beach shore; Ponts-de-Martel water, biofilm and precipitates from the Iron and Sulfur springs, Switzerland, respectively, no extremity factor present). (A) Total community composition based on 16S rRNA gene sequencing data to phylum level. (B) Total community composition represented by the most abundant OTUs (more than 10%). (C) The fraction of the low frequency OTUs of the total community. (D) Community composition of Firmicutes, based on 16S rRNA gene sequencing data. (E) Community composition based on spo0A gene sequencing data. OTUs detected were classified to known endospore-forming genera.