Salt‐sensitivity of σ^H and Spo0A prevents sporulation of Bacillus subtilis at high osmolarity avoiding death during cellular differentiation

Abstract

Summary The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete.

Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing

“…However, the portion of differential expression within the σ E , σ F , σ G , and σ K regulons was relatively low and can be explained by very low expression levels, non-sporulation-related gene functions, and/or different degradation rate of dormant spore transcripts in stressed vs. non-stressed outgrowing spores (Dataset S1 ). All differentially expressed σ H -genes except the σ W -dependent spo0M were downregulated (Table 3 ), consistent with the role of σ H in sporulation initiation that is blocked at high salinity (Ruzal et al, 1998 ; Widderich et al, 2016 ).…”

“…In its natural habitats, B. subtilis is frequently exposed to increases in environmental salinity, which has profound influences on cellular physiology and triggers adaptive responses (Bremer, 2002 ; Hoffmann and Bremer, 2016 ). High salinity exerts detrimental effects on B. subtilis spore formation (Ruzal et al, 1998 ; Widderich et al, 2016 ), spore germination (Nagler et al, 2014 , 2015 , 2016 ; Nagler and Moeller, 2015 ), and, as shown here and previously, spore outgrowth (Tovar-Rojo et al, 2003 ; Nagler et al, 2014 , 2016 ). Although it seems counter-intuitive that high salinity inhibits the formation of desiccation resistant spores, blocking this costly cellular differentiation program most likely reflects the inability of starving cells to gather sufficient resources (e.g., for the massive production of osmoprotective proline) required for sporulation during simultaneous salt stress (Brill et al, 2011a ; Widderich et al, 2016 ).…”

“…High salinity exerts detrimental effects on B. subtilis spore formation (Ruzal et al, 1998 ; Widderich et al, 2016 ), spore germination (Nagler et al, 2014 , 2015 , 2016 ; Nagler and Moeller, 2015 ), and, as shown here and previously, spore outgrowth (Tovar-Rojo et al, 2003 ; Nagler et al, 2014 , 2016 ). Although it seems counter-intuitive that high salinity inhibits the formation of desiccation resistant spores, blocking this costly cellular differentiation program most likely reflects the inability of starving cells to gather sufficient resources (e.g., for the massive production of osmoprotective proline) required for sporulation during simultaneous salt stress (Brill et al, 2011a ; Widderich et al, 2016 ). In contrast, spore germination and outgrowth can be initiated under non-growth-permissive salt conditions, likely resulting in a survival disadvantage and indicating the lack of a counteracting sensory and regulatory response system (Boch et al, 1994 ; Nagler et al, 2014 , 2016 ).…”

Identification of the first glyphosate transporter by genomic adaptation

“…The Göttingen Genomics Laboratory (Göttingen, Germany) performed library preparation and sequencing on Illumina instruments. The reads were mapped on the B. subtilis and E. coli reference genomes NC_000964 and U00096, respectively, from GenBank (Blattner et al, 1997;Barbe et al, 2009) as previously described (Widderich et al, 2016) using the Geneious software package (Biomatters) (Kearse et al, 2012). All identified mutations were verified by performing PCRs and Sanger sequencing.…”

Impact of high salinity and the compatible solute glycine betaine on gene expression of Bacillus subtilis

“…Previous studies of salt‐adapted B. subtilis cells had revealed alterations in gene expression that affect multiple aspects of cellular physiology, namely uptake and synthesis of osmoprotectants (Kappes et al ., 1996; von Blohn et al ., 1997; Kappes et al ., 1999; Brill et al ., 2011; Hoffmann et al ., 2013), cell wall metabolism and cell division (Dartois et al ., 1998; Steil et al ., 2003; Fischer and Bremer, 2012), iron metabolism (Hoffmann et al ., 2002; Steil et al ., 2003), degradative enzyme synthesis (Kunst and Rapoport, 1995), endospore formation (Kunst and Rapoport, 1995; Ruzal et al ., 1998; Widderich et al ., 2016) and motility (Steil et al ., 2003). In one of these studies global gene expression profiling of high‐salinity adaptation of B. subtilis was performed using a first generation DNA array with PCR products spotted on a nylon membrane (Steil et al ., 2003).…”