Staphylococcus aureus can survive in the absence of c-di-AMP upon inactivation of the main glutamine transporter

The nucleotide second messenger c-di-AMP negatively regulates potassium and osmolyte uptake in Staphylococcus aureus and many other bacteria. c-di-AMP is also important for growth and an S. aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously, we have shown that an S. aureus dacA mutant can grow after the acquisition of inactivating mutants in opuD, encoding the main glycine-betaine osmolyte transporter, or mutations in alsT, encoding a predicted amino acid transporter. Using the size of bacterial cells as a proxy for their osmotic balance, we show that inactivation of OpuD helps bacteria to re-establish their osmotic balance, while inactivation of AlsT does not and bacteria remain enlarged, a characteristic of S. aureus cells unable to produce c-di-AMP. We show that AlsT is the main glutamine transporter in S. aureus, thus revealing that S. aureus can survive without c-di-AMP when glutamine uptake is prevented. Using a bioinformatics approach combined with uptake assays, we identified GltS as the main glutamate transporter in S. aureus. Using WT and mutant strain, we show that glutamine is preferred over glutamate for bacterial growth and that its uptake represses c-di-AMP production. Glutamine and glutamate are important players in osmotic regulation, but their cellular levels also serve as a key indicator of nitrogen availability in bacterial cells. Therefore, we not only provide a further connection between the c-di-AMP signalling network and osmotic regulation in S. aureus but also to central nitrogen metabolism. Graphical abstract


Introduction
4 al., 2015, Whiteley et al., 2017. c-di-AMP binds to and negatively regulates a number of 75 different potassium and osmolyte importers (Rocha et al., 2019, Quintana et al., 2019 depending on their intracellular c-di-AMP levels (Zeden et al., 2018, Corrigan et al., 2011. Bacteria of the high c-di-AMP level S. aureus mutant strain LAC*gdpP show a decrease in 8 constructed by phage transducing the alsT::tn region from the Nebraska Transposon Mutant 187 Library (NMTL) strain NE142 (Fey et al., 2013) into the S. aureus LAC* strain background.
determining the levels of the individual amino acids in the culture supernatant at the start of the experiment (T = 0 h) as compared to 6, 10 and 12 h following their growth in TSB 192 medium. While no significant differences were observed for most amino acids (Fig. S1), a 193 slight increase in the utilization of aspartate and a slight decrease in the uptake of serine 194 was observed ( Fig. 2B and 2C), suggesting that AlsT could potentially be a serine 195 transporter. To test this, uptake assays were performed with radiolabelled serine using the 196 WT LAC* strain, the alsT mutant strain LAC*alsT::tn piTET as well as the complementation 197 strain LAC*alsT::tn piTET-alsT. However, no significant differences in the uptake rate of 198 serine were observed between the strains (Fig. 3A), indicating that AlsT is not the main 199 serine transporter in S. aureus. While slight differences in the amino acid uptake profile were 200 observed between the WT and alsT mutant strain, this analysis did not allow us to identify 201 the main substrate for AlsT. However, it is of note that using this method one cannot 202 distinguish between glutamine and glutamate or asparagine and aspartate utilization.

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Additionally, tryptophan was not measured due to the limitations of the method used.
knowledge, this has not yet been experimentally verified. Expression of alsT is controlled in al., 2005), whose production is induced in the presence of glutamine as nitrogen source by 219 the two-component system GlnKL. To test if S. aureus AlsT is a potential glutamine or 220 glutamate transporter, uptake assays were performed with radiolabelled glutamine and 221 glutamate using the WT S. aureus strain LAC*, the alsT mutant LAC*alsT::tn piTET and the 222 complementation strain LAC*alsT::tn piTET-alsT. Uptake of glutamine, but not of glutamate, 223 was severely reduced in the alsT mutant when compared to the WT (Fig. 3B). This defect 224 was restored upon expression of alsT in the complementation strain (Fig. 3C). To confirm 225 that alsT also functions as main glutamine transporter in the LAC*dacA/alsT suppressor 226 strain, uptake assays were also performed with strain LAC*dacA/alsT and compared to that

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The resulting LAC*glnQ::tn mutant strain displayed no difference in glutamine or glutamate   1999, Gunka & Commichau, 2012. In addition, differences in cellular c-di-AMP levels were 267 reported in B. subtilis depending on the presence of glutamine or glutamate as available 268 nitrogen source. More specifically, an increase in cellular c-di-AMP levels was observed in However, none of the transporters (AlsT, SAUSA300_0914 and GlnPQ) investigated so far major high-affinity Na + -coupled glutamate/aspartate symporter and can also mediate the uptake of glyphosate (Wicke et al., 2019). An additional two paralogs, DctP and GltP are 276 found in B. subtilis of which GltP has also been shown to be a glutamate transporter (Tolner 277 et al., 1995)

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Next, the uptake of radiolabelled glutamine and glutamate was assessed for the WT LAC* 286 strain and the LAC* gltT::tn and LAC* gltS::tn mutants. No difference in the uptake of 287 glutamine was observed between the strains (Fig. 5A) and in the case of LAC*gltT::tn, also 288 no difference in the uptake of glutamate was observed. However, a significant reduction in 289 glutamate uptake was observed for strain LAC*gltS::tn when compared to the WT (Fig. 5B).

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The glutamine uptake defect could be restored in a complementation strain harbouring  conditions. Taken together, these data indicate that glutamine is preferred over glutamate for 314 the growth of S. aureus in CDM lacking ammonium as nitrogen source.

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Glutamine uptake leads to a reduction in the cellular c-di-AMP levels in S. aureus

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In a previous study, it has been reported that the presence of glutamine or glutamate in the 318 growth medium can affect cellular c-di-AMP levels in B. subtilis and it was proposed that 319 glutamate uptake leads to an activation of c-di-AMP synthesis in this organism (Gundlach et 320 al., 2015b). To assess if the presence of glutamine or glutamate would also affect c-di-AMP aureus strain LAC* following growth in CDM+Gln or CDM+Glu medium. c-di-AMP levels phosphodiesterase GdpP (strain LAC*gdpP::kan) c-di-AMP levels were increased as 326 compared to a WT strain (Fig. 7A). Interestingly and similar as observed for the WT strain, c-   piTET-alsT (Fig. 7C). Taken together, these results highlight that glutamine uptake blocks c-345 di-AMP production in S. aureus and that eliminating glutamine from the medium or 346 preventing its uptake stimulates c-di-AMP production. Such an activation is likely achieved allowing bacteria to re-establish their osmotic balance. Bacteria unable to produce c-di-AMP 356 are larger than WT cells but bacteria that are unable to produce c-di-AMP and also lack 357 OpuD, the main transporter for the osmolyte glycine betaine in S. aureus, are similar in size 358 to WT bacteria. This indicates that in these cells the osmotic balance has been restored.

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Furthermore, we show that mutations in alsT, which we identify as part of this study to 360 encode for the main glutamine transporter in S. aureus, suppress the essentiality of c-di-

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AMP in a different way and potential mechanisms for this are discussed here.

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Over the last decade, considerable evidence has emerged that c-di-AMP plays a 363 major role in osmotic regulation, primarily by positively regulating potassium export or far are themselves not essential. Therefore, the essentiality of c-di-AMP is likely due to its 370 ability to regulate multiple target proteins simultaneously. Furthermore, in the absence of this 371 molecule, many transporters are activated rather than inactivated, likely leading to 372 accumulation of toxic levels of metabolites, such as potassium and osmolytes. Consistent 373 with this idea, inactivating mutations in potassium uptake systems, oligopeptide and 374 osmolyte transporters have been reported to rescue the growth defect of bacteria unable to 375 produce c-di-AMP (Whiteley et al., 2015, Whiteley et al., 2017, Gundlach et al., 2017b, (Zeden et al., 2018. Using bacterial cell size as a proxy for the osmotic balance of cells, we 380 show here that inactivation of OpuD likely helps an c-di-AMP null strain survive by allowing 381 bacteria to re-establish their osmotic balance, as dacA/opuD mutant bacteria, which cannot produce c-di-AMP but are also lacking the main glycine betaine transport, are similar in size conditions in S. aureus, indicating that glutamine also plays an important role in osmotic 407 regulation (Anderson & Witter, 1982). However, under the osmotic stress conditions tested 408 in this previous study, glutamine accumulation was proposed to be due to synthesis rather 409 than uptake (Anderson & Witter, 1982). In terms of other functions of the glutamine transporter AlsT in S. aureus; it is interesting to note that in a recent study investigating genetic determinants required for eDNA during biofilm formation, it was found that 412 inactivation of GdpP as well as AlsT results in a significant decrease in eDNA release 413 (DeFrancesco et al., 2017). Since we show here that in an alsT mutant, which is unable to 414 import glutamine, cellular c-di-AMP levels can be significantly higher as compared to a WT 415 strain (Fig. 7), similar to a gdpP mutant, this could mean that the underlying mechanistic 416 bases for the decrease in eDNA release observed for the gdpP and alsT mutant strains 417 might be related.

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There are several (not mutually exclusive) possibilities how preventing glutamine 419 uptake could rescue the growth of a c-di-AMP null strain (Fig. 8). The cellular 420 glutamine/glutamate ratio serves as a key indicator of nitrogen availability in bacterial cells 421 with a high glutamine/glutamate ratio indicating nitrogen availability (Forchhammer, 2007).

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The cellular glutamate concentration is usually higher than the glutamine concentration and 423 excess glutamine can be readily converted to glutamate via the GOGAT pathway by the 424 glutamine oxoglutarate aminotransferase composed in S. aureus of the GltB and GltD 425 proteins subunits (Gunka & Commichau, 2012). Hence, glutamine uptake and its availability 426 in the cell will provide a flux towards glutamate synthesis and glutamate is the counterion of 427 potassium in the cell. Therefore, glutamine uptake and its conversion to glutamate could 428 indirectly facilitate further potassium uptake, which becomes toxic in a c-di-AMP null strain, 429 in which potassium influx is already increased. As a result, reduction in glutamine uptake, as

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monocytogenes, the absence of c-di-AMP could also boost the metabolism and potentially 446 TCA cycle activity in S. aureus. As part of this study, we provide evidence that in S. aureus 447 glutamine is preferred over glutamate for growth in CDM lacking ammonium as nitrogen 448 source. The growth of an S. aureus strain in this glucose-containing but ammonium free 449 medium was improved by the addition of glutamine but not glutamate and the uptake of 450 glutamine mediated by AlsT was required for this growth improvement (Fig. 6). Hence, the 451 lack of c-di-AMP combined with glutamine uptake could fuel the bacterial metabolism and 452 the resulting metabolic imbalance might become toxic to the cell, similar as observed for L.

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monocytogenes (Sureka et al., 2014, Whiteley et al., 2017. This futile cycle might be 454 blocked by preventing glutamine uptake and reducing the metabolic activity of cells.

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The actual stimuli and underlying molecular mechanisms that regulate c-di-AMP 456 production in bacterial cells are at the moment poorly understood. As part of this study, we 457 show that glutamine uptake negatively impacts c-di-AMP production in S. aureus and 458 bacteria grown in medium lacking glutamine or inactivated for the main glutamine transporter

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AlsT have significantly increased cellular c-di-AMP levels ( Fig. 7B and 7C). The increase in an increase in cellular c-di-AMP levels was also detected in a gdpP mutant strain when enzyme GlmM (Tosi et al., 2019, Zhu et al., 2016, Gundlach et al., 2015b 467 2016) (see Model Fig. 8). YbbR and GlmM are encoded in the same operon with DacA.

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Glutamine is a key precursor for the production of the GlmM substrate glucosamine-6-P 472 since it and fructose-6-P are converted by GlmS to glutamate and glucosamine-6-P.

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Therefore, the cellular glutamine levels will impact GlmS activity and hence also the   All strains used in this study are listed in Table 1 and primers used in this study are listed in 513

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The cell diameters of 50 cells were measured and the average cell diameter determined.

721
Amino acid uptake assays. Amino acid uptake assays were performed and the data plotted