Cyclic di-AMP regulation of osmotic homeostasis is essential in Group B Streptococcus

Abstract

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.

Identification of the main glutamine and glutamate transporters inStaphylococcus aureusand their impact on c-di-AMP production

“…Over the last decade, considerable evidence has emerged that c-di-AMP plays a major role in osmotic regulation, primarily by positively regulating potassium export or negatively regulating potassium and osmolyte uptake (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c, Gundlach et al ., 2019). However, individual c-di-AMP target proteins identified thus far are themselves not essential.…”

“…Furthermore, in the absence of this molecule, many transporters are activated rather than inactivated, likely leading to accumulation of toxic levels of metabolites, such as potassium and osmolytes. Consistent with this idea, inactivating mutations in potassium uptake systems, oligopeptide and osmolyte transporters have been reported to rescue the growth defect of bacteria unable to produce c-di-AMP (Whiteley et al ., 2015, Whiteley et al ., 2017, Gundlach et al ., 2017b, Gundlach et al ., 2017c, Pham et al ., 2018, Devaux et al ., 2018, Zeden et al ., 2018). We have previously shown that in S. aureus inactivation of the main glycine betaine transporter OpuD bypasses the requirement of c-di-AMP for the growth of S. aureus in rich medium (Zeden et al ., 2018).…”

“…There is now considerable evidence that one such messenger, cyclic di-adenosine monophosphate (c-di-AMP) plays a significant role in osmoregulation in bacteria (Pham et al ., 2018, Pham & Turner, 2019, Quintana et al ., 2019, Zarrella et al ., 2018, Teh et al ., 2019, Fahmi et al ., 2019, Devaux et al ., 2018, Bai et al ., 2014, Zeden et al ., 2018, Corrigan et al ., 2011, Rocha et al ., 2019, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Witte et al ., 2013, Whiteley et al ., 2015, Whiteley et al ., 2017). c-di-AMP binds to and negatively regulates a number of different potassium and osmolyte importers (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c). c-di-AMP is essential for bacterial growth under standard growth conditions but it is also toxic at high levels in many Firmicutes, hence its cellular levels must be tightly regulated (Gundlach et al ., 2015b, Mehne et al ., 2013, Corrigan et al ., 2011, Corrigan et al ., 2015, Woodward et al ., 2010, Witte et al ., 2013).…”

“…c-di-AMP is essential for bacterial growth under standard growth conditions but it is also toxic at high levels in many Firmicutes, hence its cellular levels must be tightly regulated (Gundlach et al ., 2015b, Mehne et al ., 2013, Corrigan et al ., 2011, Corrigan et al ., 2015, Woodward et al ., 2010, Witte et al ., 2013). In S. aureus and Listeria monocytogenes, deletion of the diadenylate cyclase gene dacA , the enzyme responsible for the synthesis of c-di-AMP, was only possible in chemically defined medium (Whiteley et al ., 2015, Zeden et al ., 2018, Devaux et al ., 2018), whereas in Bacillus subtilis all three c-di-AMP cyclases could only be inactivated in minimal medium also containing low amounts of potassium (Gundlach et al ., 2017b).…”

“…Previously, we found that inactivation of the main glycine betaine transporter OpuD as well as the predicted amino acid transporter AlsT (SAUSA300_1252) allows an S. aureus dacA mutant to grow in rich medium in the absence of c-di-AMP (Zeden et al ., 2018). In several other Firmicutes, including B. subtilis, Lactococcus lactis, Streptococcus pneumoniae, Streptococcus pyogenes and L. monocytogenes , inactivating mutations have also been identified in osmolyte and potassium transport systems that allow these bacteria to grow in the absence of c-di-AMP (Pham et al ., 2018, Pham & Turner, 2019, Quintana et al ., 2019, Zarrella et al ., 2018, Teh et al ., 2019, Fahmi et al ., 2019, Devaux et al ., 2018, Bai et al ., 2014, Zeden et al ., 2018, Corrigan et al ., 2011, Rocha et al ., 2019, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Witte et al ., 2013, Whiteley et al ., 2015, Whiteley et al ., 2017). This is consistent with the idea that in the absence of c-di-AMP, potassium and osmolyte transporters are more active, resulting in the accumulation of toxic levels of potassium and osmolytes in the cell.…”