The Pseudomonas aeruginosa phosphodiesterase gene nbdA is transcriptionally regulated by RpoS and AmrZ

Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5’-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa possesses a network of genes encoding proteins for the turnover of the second messenger c-di-GMP involved in regulating-among others-the lifestyle switch between planktonic, motile cells and sessile biofilms. Insight into the transcriptional regulation of these genes is important for the understanding of the protein function within the cell. Determination of the transcriptional start site of the phosphodiesterase gene nbdA revealed a new promoter region and consequently a shortened open reading frame for the corresponding protein. Binding sites for RpoS and AmrZ were identified in silico and confirmed experimentally. Previously reported regulation by nitric oxide was reevaluated and a strong influence of the moonlighting protein NirS identified.

14 formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-15 di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for 16 enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight 17 regulation of expression, subcellular localized function and protein interactions control the 18 activity of these enzymes. In this work we elucidated the transcriptional regulation of the 19 gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a 20 transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in 21 biofilm dispersal. NO is released from macrophages during infections but can also be 22 produced by P. aeruginosa itself during anaerobic denitrification. Recently however, 23 contradictory results about the role of NbdA within NO-induced biofilm dispersal were 24 published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain 25 insights into this discrepancy. Determination of the transcriptional start site of nbdA by 26 5'-RACE and subsequent identification of the promoter region revealed a shortened open 27 reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for 28 RpoS and AmrZ were discovered in the newly defined promoter region. Employing 29 chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-30 dependent activation and AmrZ repression of nbdA transcription. In order to investigate the 31 impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO 32 were applied. While neither exogenous nor endogenous NO had an influence on nbdA 33 promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA 34 transcription independently of its enzymatic activity during denitrification. The latter supports 35 a role of NirS in P. aeruginosa apart from its enzymatic function. 36

IMPORTANCE 37
The opportunistic pathogen Pseudomonas aeruginosa possesses a network of genes 38 encoding proteins for the turnover of the second messenger c-di-GMP involved in regulating-39 among others-the lifestyle switch between planktonic, motile cells and sessile biofilms. 40 Insight into the transcriptional regulation of these genes is important for the understanding of AmrZ were identified in silico and confirmed experimentally. Previously reported regulation 45 by nitric oxide was reevaluated and a strong influence of the moonlighting protein NirS 46 identified. 47

INTRODUCTION 49
The opportunistic human pathogen Pseudomonas aeruginosa is able to form acute and 50 chronic infections, the latter associated with biofilm formation (1). Within biofilms, bacteria 51 are embedded in a self-produced matrix and are highly protected against the host immune 52 system and antibiotic treatments (2, 3). Therefore, biofilm associated infections are difficult to 53 treat and the P. aeruginosa biofilm lifecycle has become a well-studied topic in the last 54 decades. Environmental cues like changes in nutrient availability or the diatomic gas nitric 55 oxide (NO) are able to induce biofilm dispersal by promoting a switch between the sessile 56 and planktonic lifestyle of the bacteria (4-6). In general, the biofilm lifecycle is dependent on 57 the second messenger bis-(3,5)-cyclic diguanosine-monophosphate (c-di-GMP). However, c-58 di-GMP does not only regulate the biofilm lifecycle, but rather is involved in various bacterial 59 processes e.g., motility, secretion systems, virulence and cell cycle progression (7). The 60 intracellular level of c-di-GMP is dependent on diguanylate cyclases (DGC) that build c-di-61 GMP from two molecules of GMP and c-di-GMP-specific phosphodiesterases (PDE) that 62 hydrolyze c-di-GMP to either pGpG or GMP (8-12 demonstrated that the deletion of neither nbdA, rbdA nor dipA led to a loss of biofilm 102 dispersal in response to NO (26). These contrary findings underline, that c-di-GMP 103 modulating proteins are tightly regulated in P. aeruginosa and changes in environmental 104 conditions might impact expression or activity of those enzymes. Therefore, we decided to 105 reevaluate the transcriptional regulation of nbdA to obtain insights into this discrepancy and 106 to gain a better understanding of NbdA's role within the c-di-GMP network of P. aeruginosa 107 PAO1. 108

Plasmid and strain construction 120
Oligonucleotides used for plasmid construction are listed in Table 2. Markerless deletion 121 mutants were produced as described previously with minor modifications (31). DNA 122 fragments were generated for each deletion via splicing-by-overlap extension (SOE) PCR 123 using the corresponding Up and Down primer pairs (Table 2) and integrated into the allelic 124 exchange vector pEXG2 (29). The vector was transferred from E. coli S17-I to P. aeruginosa 125 PAO1 via biparental mating. Pseudomonas isolation agar supplemented with gentamicin was 126 used to select cells which integrated the allelic exchange vector by homologous 127 recombination. Those cells were streaked twice on LB medium containing sucrose (15 % 128 w/v) to force the second crossover event. Truncation of target genes was verified via colony 129 PCR and sequencing. 130 Transcriptional nbdA-lacZ fusion was generated in the vector mini-CTX1-lacZ (30). A 171 bp 131 fragment of the nbdA promoter region and 279 bp of the coding sequence was amplified via 132 PCR and integrated in front of the promoterless lacZ gene encoded on the vector. The 133 transcriptional fusion and the empty vector control were transferred to P. aeruginosa strains 134 via biparental mating and chromosomally integrated into the attB site on the genome via 135 integrase-mediated chromosomal integration. 136

137
Primer β-galactosidase activity was indicated by a color change due to the formation of the yellow 149 colored product ortho-nitrophenol, the reaction was stopped by addition of 500 µl 1 M 150 Na 2 CO 3 . Cell debris was precipitated by centrifugation and product formation was measured 151 in the supernatant at OD 420 . The activity of β-galactosidase was calculated as follows: Miller 152 units (MU) = (OD 420 / (OD 600 * volume * incubation time)) * 1000. 153

RNA extraction and semi quantitative RT-PCR 154
Bacteria were grown to exponential and early stationary phase as described above.  Table 2. 163

Determination of transcriptional start sites by 5'-RACE 164
P. aeruginosa cells were inoculated 1:100 from an overnight culture in LB medium and 165 incubated 5 h at 37 °C. Total RNA isolation was performed as previously described (33). 166 Primers used for 5'-RACE are listed in Table 2. cDNA was synthesized at 42 °C for 60 min 167 with M-MLV-RT (Promega) using gene-specific primer SP1_nbdA. The cDNA was treated 168 with shrimp alkaline phosphatase (New England Biolabs) and purified with MinElute kit 169 (Qiagen). A deoxyadenosine tail was added to the 3' end of the cDNA using terminal  In order to investigate the role of the alternative sigma factor RpoS and the transcription 203 factor AmrZ on nbdA expression, the promoter region of nbdA was transcriptionally fused to 204 the reporter gene lacZ and integrated in the φCTX attachment site of PAO1 wt, ΔrpoS and 205 ΔamrZ. Activity of the β-galactosidase in the respective strains was determined in 206 exponential (4 h) and early stationary (7 h) growth phase. In the wt strain, a 4-fold increase in 207 nbdA transcription was observed when cells entered the early stationary phase, which 208 suggests transcriptional activation by RpoS. Deletion of rpoS resulted in a loss of nbdA 209 promoter activity in both, exponential and stationary growth phase (Fig. 2), confirming the 210 role of RpoS as transcriptional activator of nbdA. In the ΔamrZ strain a strong increase of 211 nbdA promoter activity was observed in both, exponential and early stationary growth phase 212 (Fig. 2). AmrZ is therefore likely acting as a transcriptional repressor for nbdA. 213

221
As there is a sharp oxygen gradient present in biofilm macrocolonies (37), O 2 might also 222 have an impact on the expression of genes active in biofilms. Although there is no hint for an 223 FNR-like, ANR, or DNR regulator binding site in the promoter region, we tested nbdA 224 promoter activity also under anaerobic conditions. Induction of the nbdA promoter was 225 observed when cultures reached stationary phase in all tested strains, similarly to the aerobic 226 growth conditions (Fig. 3). Overall, the values for promoter activity under oxygen limitation 227 were significantly lower than in aerobic conditions. The nbdA promoter activity in the amrZ 228 deletion strain was significantly increased compared to the wild-type background. Therefore, 229 AmrZ seems to repress nbdA transcription similarly in aerobic and anaerobic growth 230 conditions. 231

Impact of nitric oxide on the transcription of nbdA 243
We previously reported increased amounts of nbdA transcript in dispersed cells after  induced biofilm dispersal compared to untreated planktonic cells and suggested a NO-245 dependent transcriptional regulation of nbdA (23 The denitrification deficient strains showed normal growth under aerobic conditions in LB 258 medium complemented with KNO 3 (Fig. 4A). In contrast, under anaerobic denitrifying 259 conditions the growth of PAO1 ∆nirS and ∆nirF was reduced compared to the wt PAO1 (Fig.  260 4B). The ∆norCB strain was no longer able to grow. For the analysis of nbdA transcription, 261 the strains containing the nbdA promoter lacZ-fusion were grown under aerobic/microaerobic 262 conditions and ß-galactosidase assays were performed with samples of the exponential (4 h) 263 and early stationary growth phase (7 h) (Fig. 4C). Compared to the wt, the nirS deletion had 264 a severe activating effect on nbdA expression in both, exponential and early stationary 265 growth phase. Surprisingly, nbdA transcription in the ∆nirF strain, which produces an 266 enzymatically inactive NirS, was not as high as in the ∆nirS strain but comparable to the level 267 of transcription in the wt background. The transcription of nbdA in the ∆norCB strain is 268 slightly decreased compared to the wt background. Due to the impaired growth in anaerobic 269 conditions of ∆norCB strain, we could not test for the effect of accumulation of endogenous 270 NO on nbdA transcription. In order to confirm the findings for the ∆nirS strain, a semi 271 quantitative RT-PCR experiment was performed with cDNA of wt and deletion mutant in both 272 tested growth phases (Fig. 4D). While the control PCR with recA primers showed equally 273 strong bands for all samples, nbdA expression in the wt in exponential growth phase was 274 weaker than in early stationary growth phase. In the nirS deletion strain, there was more 275 transcript of nbdA detectable than in wt, which is consistent to the findings of the β-276 galactosidase assay.

289
In addition to the influence of intrinsic nitric oxide on nbdA transcription, the effect of 290 exogenous NO was investigated. Therefore, the PAO1 wt harboring the nbdA-lacZ fusion 291 was grown with increasing amounts of the NO donor sodium nitroprusside (SNP) and nbdA 292 promoter activity was determined by β-galactosidase activity (Fig. 5A). Low concentration of 293 added SNP to the growth medium had no effect on nbdA promoter activity, whereas the 294 addition of 500 µM SNP led to a decrease of nbdA transcription. This effect is comparable to 295 the observed decrease of ndbA transcription in the NO-accumulating strain ∆norCB. As 296 P. aeruginosa is able to detoxify nitric oxide via flavohemoglobin (42, 43) under aerobic 297 conditions, the influence of short-term NO stress on nbdA transcription was analyzed. 298 Therefore, PAO1 wt nbdA-lacZ was grown to stationary phase in LB and then stressed for 30 299 min by the addition of 500 µM SNP. Compared to the untreated control, no changes in the 300 nbdA promoter activity were observed (data not shown).

307
In order to figure out whether the strong increase of nbdA expression in the ∆nirS strain was 308 based on nitrite accumulation due to interrupted denitrification (4), β-galactosidase assays 309 were performed with different amounts of nitrite in the growth medium (Fig. 5B). None of the 310 tested nitrite concentrations had a comparable effect on the nbdA promoter as the deletion of 311 nirS. The addition of nitrite to the medium rather decreased expression of nbdA slightly, 312 probably due to bacteriostatic effect of nitrite. 313 314 315

DISCUSSION 316
In this study we analyzed the transcriptional regulation of nbdA coding for the 317 phosphodiesterase NbdA, involved in the c-di-GMP modulating network in P. aeruginosa. 318 Determination of the transcription initiation site of nbdA by 5'-RACE revealed an erroneous 319 annotation of the ORF in the databases. A new promoter region was identified, containing 320 putative binding sites for RpoS and AmrZ. Gene expression of nbdA was shown to be 321 activated in stationary growth phase by the alternative sigma factor RpoS (σ S ). A further level 322 of regulation is introduced through the repression by the ribbon-helix-helix transcription factor 323 AmrZ. Oxygen limitation, supplementation with nitrite, and endogenous or exogenous nitric 324 oxide did not affect the transcription of nbdA. Surprisingly, deletion of the nitrite reductase 325 NirS showed a strong activating effect on nbdA transcription, while a strain with an 326 enzymatically inactive NirS (ΔnirF) showed no transcriptional changes. 327 The sigma factor RpoS is known as the master regulator of gene expression during 328 stationary growth phase. Furthermore, it is responsible for the activation of genes in 329 response to different stresses, e.g. starvation, heat, oxygen or osmotic stress (44)(45)(46) GMP modulating enzymes, are either transcriptionally activated or repressed by RpoS (Table  339 3 and references therein). Thus, similar to E. coli, RpoS-dependent regulation significantly 340 affects the c-di-GMP network of P. aeruginosa. RpoS regulated genes are often subject to 341 further regulatory mechanisms. Activator or repressor proteins might be involved, as well as 342 post-transcriptional regulation. The psl operon coding for matrix polysaccharide biosynthesis 343 genes in P. aeruginosa is controlled transcriptionally by RpoS and post-transcriptionally by 344 RsmA (58). In P. putida KT2440 the exopolysaccharide cluster pea is activated by RpoS and 345 repressed by AmrZ (59). Actually, when we evaluated and compared the data of the PA14 346 RpoS regulon (35) and the PAO1 AmrZ regulon (34) we found 18 out of 40 genes encoding 347 for c-di-GMP modulating enzymes in P. aeruginosa presumably regulated by both proteins, 348 RpoS and AmrZ (Table 3, (34,35)). 349 The transcriptional regulator AmrZ controls a large regulon containing 398 gene regions in 350 PAO1. Transcription of amrZ itself is in a great extend dependent on the alternative sigma 351 factor AlgT (σ 22 ) which is known to regulate coversion to mucoidity and stress responses in 352 P. aeruginosa (60, 61). AmrZ was shown to regulate genes important for P. aeruginosa 353 virulence, including type IV pili, extracellular polysaccharides, and the flagellum (34). It 354 particularly influences genes required for alginate production and twitching-motility (34, 62-355 64). Within the c-di-GMP network of P. aeruginosa, AmrZ activates transcription of 14 genes 356 and represses 10 genes encoding GGDEF/EAL-domain proteins ((34), Table 3). With these 357 numbers, AmrZ appears to be one of the major regulators for genes coding for c-di-GMP 358 modulating enzymes in P. aeruginosa, possibly affecting the cellular c-di-GMP level. This 359 role for AmrZ was previously also observed in P. fluorescens F113, where the cellular c-di-360 GMP level was affected by AmrZ through the regulation of a complex network of genes 361 encoding DGCs and PDEs (65). From our work we conclude that nbdA transcription is 362 repressed by AmrZ during aerobic as well as anaerobic planktonic growth while a condition 363 in which the nbdA promoter is de-repressed remains uncertain. Repression through AmrZ is 364 described to be dependent on the C-terminus mediated tetramerization of the protein (66). In 365 some cases, e.g. pilA repression, the expression level of AmrZ plays an important role for its 366 function, as binding efficiency of AmrZ to different promoter regions differs (64). Additionally, 367 a competition of the activator RpoS and the repressor AmrZ upon binding to the nbdA 368 promoter might be possible. 369  influence of NO on nbdA transcription was unlikely. These findings in addition to the 390 contradictory results in the literature concerning the involvement of NbdA in NO-induced 391 biofilm dispersal of P. aeruginosa (23,26) led to the reevaluation of the transcriptional 392 regulation of nbdA by NO. In this study, no direct stimulation of nbdA promoter activity by 393 NO, neither by addition of exogenous NO nor by accumulation of intrinsic NO in 394 planktonically grown cells was observed. The previously observed induction of nbdA 395 expression in our qRT-PCR experiments (23) might be due to more complex regulatory 396 processes during biofilm formation and dispersal. From the present data, we conclude that 397 nbdA expression in planktonic cells is not directly induced by NO at the transcriptional level. 398

Effect of the nitrite reductase NirS on nbdA promoter activity 399
In this study, we observed a strong increase in the nbdA transcription level when the nitrite 400 reductase NirS was deleted. At first, we assumed that the upregulation of nbdA expression 401 might be due to accumulation of intrinsic nitrite from interrupted denitrification. However, 402 addition of nitrite to the growth medium did not change nbdA promoter activity. Further, the 403 ΔnirF strain producing an enzymatically inactive NirS protein (41) did not enhance nbdA 404 transcription. Therefore, we suggest that the presence of the periplasmic protein NirS affects 405 nbdA transcription independently of its enzymatic activity. The moonlighting role of NirS was 406 previously described for the type III secretion system in P. aeruginosa (70). Additionally, NirS 407 was shown to affect flagellum biogenesis by the formation of a complex with the flagellar 408 subunit FliC and the chaperone DnaK (4, 60, 71). Suggesting this complex role for NirS 409 besides denitrification in P. aeruginosa, the increase of nbdA promoter activity in the ΔnirS 410 strain is probably derived from a global regulatory change in the cell.

411
All in all, we were able to reannotate the nbdA gene and revealed consensus sequences for 412 the alternative sigma factor RpoS and the transcription factor AmrZ within the nbdA promoter 413 region. Our data confirmed RpoS as activator and AmrZ as repressor for nbdA transcription, 414 however, no transcriptional regulation by endogenous or exogenous NO or nitrite was 415 observed in planktonically grown cells. 416 417

AUTHORS STATEMENTS 418
Authors and contributors 419 KG, SZ, NFD conceived the study, KG and SZ performed experiments, KG and SZ analyzed 420 the data, KG wrote first draft of manuscript, all authors revised and approved the final version 421 of the manuscript. 422

Conflict of interests 423
The authors declare no conflict of interest. 424

Funding information 425
This work was funded by the SPP 1879 "Nucleotide Second Messenger Signaling in 426 Bacteria" of the DFG. 427