The stringent stress response controls proteases and ...May 23, 2020 · 1 1 The stringent stress...
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The stringent stress response controls proteases and global 1
regulators under optimal growth conditions in Pseudomonas 2
aeruginosa 3
Daniel Pletzer1,2,#, Travis M. Blimkie1, Heidi Wolfmeier1, Yicong Li1, Arjun Baghela1, Amy 4
H. Y. Lee1,3, Reza Falsafi1, and Robert E. W. Hancock1,# 5
Running title: Non-stressed stringent response regulon 6
Keywords: ppGpp, relA, spoT, aprA, global transcriptional regulator 7
1 Centre for Microbial Diseases and Immunity Research, Department of Microbiology and 8
Immunology, University of British Columbia, Vancouver, Canada 9
2 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand 10
3 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, 11
Canada 12
# Corresponding authors: Daniel Pletzer - email address: [email protected], Tel + 64 3 13
479 7478, Mailing address: University of Otago, Microbiology Building, PO Box 56, Dunedin 14
9054, New Zealand. 15
Robert E.W. Hancock – email address: [email protected], Tel +1 604 822 2682, Fax 604 16
827 5566, Mailing address: Room 232, 2259 Lower Mall Research Station, University of British 17
Columbia, Vancouver, British Columbia, Canada, V6T 1Z4. 18
19
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Abstract 20
The bacterial stringent stress response, mediated by the signaling molecule guanosine tetra-21
phosphate, ppGpp, has recently gained attention as being important during normal cellular growth 22
and as potential new therapeutic target, which warrants detailed mechanistic understanding. Here, 23
we used intracellular protein tracking in Pseudomonas aeruginosa PAO1, which indicated that 24
RelA was bound to the ribosome, while SpoT localized at the cell poles. RNA-Seq was used to 25
investigate the transcriptome of a ppGpp-deficient strain under non-stressful nutrient-rich broth 26
conditions where the mutant grew at the same rate as the parent strain. In exponential growth 27
phase, the lack of ppGpp led to >1,600 transcriptional changes (fold-change cut-off ±1.5), 28
providing further novel insights into the normal physiological role of ppGpp. The stringent 29
response was linked to gene expression of various proteases and secretion systems including aprA, 30
PA0277, impA, and clpP2. The previously observed reduction in cytotoxicity towards red blood 31
cells, in a stringent response mutant, appeared to be due to aprA. Investigation of an aprA mutant 32
in a murine skin infection model, showed increased survival rates of the aprA mutant consistent 33
with previous observations that stringent-response mutants have reduced virulence. In addition, 34
the overexpression of relA, but not induction of ppGpp with serine hydroxamate, dysregulated 35
global transcriptional regulators as well as >30% of the regulatory networks controlled by AlgR, 36
OxyR, LasR, and AmrZ. Together these data expand our knowledge about ppGpp and its 37
regulatory network and role in environmental adaptation. It also confirms its important role 38
throughout the normal growth cycle of bacteria. 39
Significance Statement 40
Microorganisms need to adapt rapidly to survive harsh environmental changes. Here, we showed 41
the broad influence of the highly studied bacterial stringent stress response under non-stressful 42
conditions that indicate its general physiological importance and might reflect the readiness of 43
bacteria to respond to and activate acute stress responses. Using RNA-Seq to investigate the 44
transcriptional network of Pseudomonas aeruginosa cells revealed that >30% of all genes changed 45
expression in a stringent-response mutant under optimal growth conditions. This included genes 46
regulated by global transcriptional regulators and novel downstream effectors. Our results help to 47
understand the importance of this stress regulator in bacterial lifestyle under relatively unstressed 48
conditions. As such it draws attention to the consequences of targeting this ubiquitous bacterial 49
signaling molecule. 50
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Introduction 51
To deal with stress and/or harmful environmental conditions, microbes can adopt versatile 52
adaptive lifestyles. To enable such lifestyle changes to occur rapidly, bacteria have evolved 53
complex hierarchical regulatory networks to trigger diverse molecular responses that alter gene 54
expression and protein activity. Global regulatory systems enable the coordination of downstream 55
effectors that help recognize and appropriately respond to new environments. In particular 56
microbial life depends on the ability to rapidly switch from favorable conditions such as rapid 57
growth in nutrient rich media, to recognize and counteract external threats, and switch into a 58
survival mode (1). Here we wondered whether such stress adaptations might also operate under 59
optimal, rapid-growth conditions that are not usually considered “stressful”. 60
As long as sufficient and appropriate nutrients are provided and toxic agents are absent, 61
bacteria continue to replicate, although in normal culture they eventually stop growing (e.g. in 62
Escherichia coli at around two billion bacteria per ml). On a cellular and molecular level, the 63
processes that they undergo during rapid growth are, however, likely quite stressful with rapid 64
replication, protein synthesis, cell division and reorganization of the cell (2). For example, there is 65
a disconnect between bacterial division every 20-40 minutes under optimal conditions and 66
replication and segregation of the chromosomal DNA, which is 1000 times the length of the cell 67
and thus highly condensed, that requires 60-90 minutes (2). Furthermore, as the density of bacteria 68
increases they start to experience depletion of one or more essential nutrients/growth requirements, 69
and/or the formation of inhibitory products such as organic acids, which eventually leads to the 70
stationary phase (3). It is known that maintenance of bacteria in stationary phase is guided by the 71
alternative stress/starvation sigma factor (σS) (4, 5), and the stringent stress response (3) which 72
indicates that cessation of growth in broth culture occurs under stressful circumstances. However, 73
it is worth asking about the mechanistic impacts of such factors during rapid, apparently 74
uninhibited growth. 75
One major mechanism for dealing with stress is through the stringent stress response 76
intermediated by the second messenger guanosine tetra-phosphate (ppGpp) (6, 7). The activation 77
of the stringent stress response during amino acid starvation is due to the accumulation of 78
uncharged, deacylated tRNA molecules in the cytosol that enter the ribosome A site and ultimately 79
cause ribosome stalling (8). In most Gram-negative bacteria, two enzymes, RelA and SpoT, 80
mediate ppGpp homeostasis. Recently, Winther et al. (9) showed that RelA binds to empty tRNA 81
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molecules in the cytosol and the tRNA-RelA complex further loads into the A-site of the ribosome. 82
RelA becomes activated and synthesizes ppGpp after interaction with the large Sarcin-Ricin loop 83
of the 23S rRNA (9). On the other hand, SpoT, a bifunctional enzyme with weak ppGpp synthetase 84
activity as well as a ppGpp degradative function, controls the balance of cellular ppGpp levels 85
through its hydrolase activity (10). SpoT is mainly regulated by other environmental stress and 86
starvation signals, including carbon, phosphate, iron or fatty acid starvation (7). The stringent 87
stress response leads to the dysregulation of a third or more of bacterial genes enabling stressed 88
cells to divert resources away from growth and division and towards stress coping mechanisms to 89
promote survival until nutrient conditions improve (7). 90
Mutants lacking RelA and SpoT are unable to produce ppGpp and consequently have multiple 91
defects in coping with stress and show reduced virulence in animal models (11-15). Interestingly, 92
during exponential growth, basal levels of ppGpp in Bacillus subtilis act as one of the major 93
regulators of GTP homeostasis (16), while in E. coli ppGpp influences negative supercoiling (17) 94
and modestly affects bacterial growth rates in Luria Broth but not M9 glucose minimal medium 95
(18). Recently, ppGpp has been shown to bind to two separate sites on the RNA polymerase: 1) 96
the β’-ω subunit and 2) the interface between the β’ subunit and the transcription factor DksA. 97
DksA works in concert with ppGpp as a co-regulator that amplifies the impact of ppGpp (19). 98
During starvation, cells accumulate ppGpp that interferes with σ70 binding to promoters, and 99
consequently allows for the binding of alternative factors such as σS and σ32 that can re-direct the 100
RNA polymerase to energy-saving processes (20, 21). Given that ppGpp has been shown to 101
interfere with binding of a variety of σ factors to the core RNA polymerase (21, 22), this raises the 102
question as to what happens at the transcriptional level when ppGpp is absent. We hypothesized 103
that the absence of a regulatory element impacting on RNA polymerase should have substantial 104
transcriptional consequences even under non-stressful conditions. This was further supported by 105
the recent work of Sanchez-Vazquez et al. (23) that demonstrated that overproduction of ppGpp 106
in E. coli, after only 5 minutes and in the absence of any obvious stress, led to the altered expression 107
of more than 750 transcripts dependent on ppGpp binding to the RNA polymerase. 108
Here, we investigated the promoter activity of the relA and spoT genes and examined their 109
intracellular localization in P. aeruginosa PAO1. We used a stringent response mutant and 110
compared its transcriptional landscape to wild-type expression during an unstressed (rich medium) 111
bacterial lifestyle where cells would be likely to mainly focus on growth and replication. Our 112
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findings strongly suggest that, in addition to its function in managing stress and growth rate, the 113
stringent response also plays an important role during normal growth. 114
Results and Discussion 115
RelA-YFP was observed in a helical arrangement in P. aeruginosa 116
Bacterial growth rate is regulated and controlled in part by the availability and quantity of 117
ribosomes and ribosomal RNA. In E. coli, ribosomal gene expression is controlled by ppGpp, 118
which helps to maintain the association of charged tRNAs with the ribosome (24). In E. coli 119
Natagawa et al. (25) and Sarubbi et al. (26) demonstrated that relA gene expression is under dual 120
promoter control, with one promoter constitutively expressed and one activated temporarily during 121
the transition from exponential to stationary growth phase. Here, we confirmed the dual-promoter 122
activity of relA in P. aeruginosa PAO1 and the requirement of both promoters for maximum 123
expression of RelA during amino acid starvation and in the stationary phase (SI Appendix, 124
Additional Data 1). At the translational level, the current working model indicates that RelA 125
becomes activated when uncharged tRNA molecules enter the A-site of the ribosome. Activation 126
of RelA, and thus synthesis of ppGpp, leads to the deactivation and release of RelA from the 127
ribosome until it is reactivated by another stalled ribosome; a mechanism known as ‘hopping’ 128
between stalled ribosomes (8). This paradigm for the association between the ribosome and RelA 129
has recently been challenged, and it has been suggested that the interaction between RelA and 130
uncharged tRNA does not necessarily involve the ribosome. Nevertheless, activation of RelA only 131
occurs when a RelA-tRNA complex enters the A-site of a stalled ribosome (9). This is supported 132
by cryo-EM structures of ribosome-bound RelA-tRNA complexes shown by Li et al. (27) and 133
others (28, 29) where they revealed that RelA synthesizes ppGpp only when bound to the 70S 134
subunit of the ribosome. 135
This prompted us to further investigate, in P. aeruginosa, the subcellular localization of RelA 136
when fused to yellow fluorescence protein (YFP). At single cell resolution we identified helical 137
spatial distribution of RelA-YFP (twisted with alternating bands around the DNA-rich nucleoid 138
area) (Figure 1A), similar to that observed for ribosomes in E. coli (30-32), indicating that, as for 139
E. coli, RelA was likely bound to the ribosomes in P. aeruginosa when overexpressed. Our data 140
was also in accordance with those of English et al. (33) where they used single-molecule 141
experiments to show that RelA was bound to the ribosome under non-starvation conditions. We 142
further investigated the subcellular localization of RelA-YFP upon stress induction using either 143
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serine-hydroxamate (SHX) to mimic amino acid starvation, or stationary phase-growth of P. 144
aeruginosa cultures. While a very similar distribution of RelA-YFP was observed after stress 145
induction, there was a significantly higher coefficient of variation (Figure 1C) consistent with 146
increased fluorescence distribution as spots within the cell (as opposed to equally distributed 147
signal). This was consistent with an interpretation that RelA was bound to the ribosome but 148
dissociated to some extent during stress. 149
SpoT-GFP localized at the cell pole in P. aeruginosa 150
We utilized a SpoT-GFP fusion protein to visualize its subcellular localization and found that 151
under normal growth conditions, the fusion protein localized at the cell pole and the septal ring of 152
elongated cells (Figure 1B). Bacterial cells are well organized factories where cellular asymmetry 153
and compartmentalization plays a vital role in many cellular processes (34). In recent years, the 154
importance of localization of certain proteins at the pole of rod-shaped bacteria has become 155
increasingly evident, with such proteins being crucial for fundamental cellular and regulatory 156
processes (including chromosome segregation, cell cycle, chemotaxis, adhesion, and motility), as 157
well as virulence (34, 35). Intriguingly the manifestation of pili or flagella at a specific pole allows 158
bacteria to quickly move through the environment and associate with e.g. mucosal surfaces, which 159
is interesting given the polar localization of SpoT and the role of ppGpp in such processes (36, 160
37). The localization at the division septum was consistent with results in Caulobacter, where 161
SpoT was found to be involved in blocking the initiation of DNA replication and regulation of 162
DnaA, a conserved replication initiator protein (38), although we did not investigate this potential 163
function here. 164
Intriguingly, upon stress induction by either carbon- or iron (dipyridyl treatment) limitation, 165
the median signal intensity of the SpoT-GFP complex decreased by 5.7-fold and 4-fold, 166
respectively, and the signal became more uniformly distributed. The decreased coefficient of 167
variation correlated with irregular fluorescence observed across the cells (Figure 1B, D). This 168
observation might relate to the ppGpp hydrolyzing activity of SpoT, which presumably maintains 169
ppGpp homeostasis; indeed, inside the cell, ppGpp molecules are broadly distributed and bind to 170
ribosomes that are found around the nucleoid and at the cell poles (30). 171
Transcriptional changes dependent on ppGpp occurred during normal growth 172
Since both RelA and SpoT were obviously present during logarithmic growth in nutrient-rich 173
conditions (SI Appendix, Fig S1) (17, 18) we investigated a role for ppGpp under such 174
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circumstances. Interestingly there were no obvious or significant differences, for the studied 175
strains, on growth during the logarithmic phase in three different media (Appendix I; Fig. S2), 176
which contrasted with results in E. coli (18) where they used a different growth methodology to 177
avoid spontaneous suppressor mutants. To understand underlying mechanisms, Gaca et al. (39) 178
used microarrays to show that a ppGpp-deficient strain, in the Gram-positive bacterium 179
Enterococcus faecalis, altered expression in the exponential phase of growth of 246 genes, 180
including genes influencing pyruvate production and GTP homeostasis. Here we utilized the more 181
comprehensive method of RNA-Seq to test the effect that the absence of ppGpp had on the 182
transcriptome under nutrient-rich, non-stressful conditions in P. aeruginosa. 183
RNA-Seq was performed on the P. aeruginosa PAO1 wild-type and a strain lacking the ability 184
to produce ppGpp (relAspoT double mutant) during exponential growth (OD600 of 0.5) under 185
nutrient-rich conditions (2xYT broth). This revealed 1,669 dysregulated genes with a fold-change 186
of ±1.5 and an adjusted (for false discovery) p-value of <0.05 (Dataset 1). Expression of a subset 187
of 15 genes (with a fold-change around the ±1.5 cut-off) was validated using qRT-PCR, 188
demonstrating similar expression trends and an overall R2 correlation coefficient of 0.66 (p-value 189
<0.01) (SI Appendix, Fig. S3). 190
Gene Ontology (GO) and pathway enrichment (KEGG) were used for functional enrichment 191
analysis. This demonstrated the upregulation of six pathways (including biosynthetic and 192
metabolic processes, as well as the type 3 secretion system and cell surface signaling pathways) 193
and downregulation of six pathways (including cell transport, type 2 and 6 secretion systems, and 194
acetyltransferase activity) in GO (Fig. 2A), while KEGG analysis indicated the downregulation of 195
six pathways (including chemotaxis, quorum sensing, and amino acid and fatty acid metabolism) 196
(Fig. 2B). Upregulation, in the stringent response mutant, of the Type 3 Secretion System was 197
interesting since this system requires bacterial cells to contact with the host to directly inject its 198
substrates that include cytotoxins (40). In contrast, several genes encoding Type 2 secretion 199
systems (PA3095-3103, Xcp genes that mediate exoprotease secretion and PA0677-PA0689, Hxc 200
genes, and effector LapA) and Type 6 secretion systems (T6SS) (Figure 2A) were downregulated 201
in the mutant. The T6SS, found on three different loci on the chromosome (H1-T6SS: PA0074-202
PA0091, H2-T6SS: PA1656-PA1671, and H3-T6SS:PA2359-PA2371), target other bacteria by 203
secreting effector proteins that can inhibit or kill them through a contact-delivery system (41). The 204
H2 and H3 systems are important P. aeruginosa pathogenesis mediators (42). Although GO 205
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analysis indicated that the T6SS were overall downregulated in the stringent response mutant 206
(Figure 2A), the H1 system was actually upregulated. This agrees with others who showed that 207
H1-T6SS is repressed by when quorum sensing is highly active, whereas such circumstances 208
activate H2 and H3 (42). Indeed, regulation of the T6SSs might be under quorum sensing control 209
since quorum sensing pathways were downregulated in our KEGG analysis (Fig. 2B). 210
Quorum sensing is a complex network that cells use for cell-to-cell communication. The 211
quorum sensing network follows a hierarchy with LasR at the top directly influencing 212
transcriptional regulators such as qscR, vqsR, and rhlR, which regulate another set of regulators 213
such two component response systems (e.g., gacAS, pprAB), which in turn further regulate quorum 214
sensing (43, 44). The stringent response is known to activate quorum sensing in Pseudomonas 215
through increased expression of lasR and rhlR when relA was overexpressed, indicating that 216
quorum sensing can be activated independently of cell density (45), which is in accordance with 217
our data. 218
We also observed that differential expression often involved co-expressed neighbouring genes. 219
This could be visualized by projecting differentially expressed genes onto the circular chromosome 220
(Fig. 2C), which revealed 34 clusters of genes (total 289 genes, 17% of differentially expressed 221
genes) with multiple genes with a similar direction of expression that had a broad array of 222
functions. Indeed within these clusters, we observed differential expression of genes involved in 223
various secretion systems, transporters, quorum sensing, pyocin synthesis, vitamin B12 synthesis, 224
heme export and cytochrome C synthesis, ethanol oxidation, sulfur and carbon metabolism, 225
biofilm formation, fatty acid and LPS biosynthesis, polyamine transport, adherence, ribosomes, 226
and other biosynthetic gene synthesis systems (Dataset 2). Intriguingly only genes in 8 out the 34 227
clusters were organized in operons. 228
The stringent response is required for environmental adaptations 229
Since the ΔrelAspoT stringent response double mutant grew normally during the logarithmic 230
phase, we postulated that during normal growth the stringent response might control processes that 231
prepare cells for more stressful situations including environmental adaptations. Therefore, we 232
examined the RNA-Seq data with this in mind. 233
Once bacteria have invaded host tissues, they must immediately deal with stresses imposed by 234
host responses. One element assisting the initiation of colonization of P. aeruginosa and 235
counteracting host responses is direct cytotoxicity towards host cells that processes epithelial 236
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surfaces, enables adhesion, and counteracts the action of phagocytic cells (46). Stringent response 237
mutants demonstrate decreased cytotoxicity towards human bronchial epithelial cells as well as 238
minimal hemolytic activity, cf. wild type (12). This was consistent with the finding here that many 239
prominent cytolytic proteases were under the control of the stringent response under rapid growth 240
conditions, as judged by their downregulation in the double mutant, including LasA elastase (-7.3 241
fold), LasB elastase (-7.8 fold), Protease IV (piv -3.2 fold), alkaline protease AprA (-9.6 fold) and 242
PA3535 (-2.1 fold); similarly the heat stable hemolysin/rhamnolipid (synthesized by rhlABC = -243
4.5, -8.3 and -8.7 fold respectively) was substantially down regulated (Dataset 1). This is also 244
generally consistent with the role of the stringent response in adjusting to amino acid deprivation 245
since extracellular proteases could digest proteins in the environment creating an additional source 246
of needed amino acids. Nevertheless protease upregulation by ppGpp under normal growth 247
conditions could be considered preparatory to more stressful circumstances. 248
Conversely, as mentioned above there was upregulation by about two-fold of the Type 3 249
Secretion System (T3SS) machinery and regulatory genes in several adjacent operons (PA1699-250
PA1725) (Fig. 2) as well as the effector/toxin exoT. Both of these observations could be related 251
in part to stringent regulation of quorum sensing genes under rapid growth conditions since we 252
observed down regulation in the ΔrelAspoT stringent response double mutant of rhlI (-3.0 fold), 253
rhlR (-2.6 fold), lasR (-1.6 fold), rsaL (-3.0 fold), pqsH (-2.0 fold) and pqsL (-3.6 fold; cf. pqsA-E 254
that were 3.9-4.6 fold upregulated). Exo-proteases and hemolysin are upregulated during quorum 255
sensing which is in turn upregulated by ppGpp, whereas T3SS genes are negatively controlled by 256
RhlI (47). 257
The ability to respond rapidly is necessary to accommodate sudden environmental changes 258
and the failure to adapt can have negative or even lethal consequences to the organism. Consistent 259
with a role in preparing for environmental adaptation, the stringent response was shown to be 260
required for swarming motility, rhamnolipid production, adherence, and pyoverdine and 261
pyocyanin production in strain PAO1 (SI Appendix Fig. S4), in accordance with previous studies 262
(12, 37, 48). P. aeruginosa cells that encounter semi-viscous conditions with a poor nitrogen 263
source are driven to swarm rapidly across surfaces (49). Intriguingly, while the stringent response 264
double mutant was unable to swarm, we found that, in rich medium liquid broth, where even the 265
wild type fails to swarm, genes required for swarming, such as LasB (50), were strongly 266
dysregulated in the ΔrelAspoT mutant. Indeed, 29% (60 out of 207) of the genes that are known 267
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(PAO1 orthologues from Yeung et al. (49)) to influence swarming motility (49) were either up- 268
(30 genes) or downregulated (30 genes) in the ΔrelAspoT stringent response mutant. Included in 269
these were regulators required for normal swarming motility, with five (rhlIR, lasR, rsaL, mvaT 270
and oruR) that were down regulated in the mutant (i.e. positively regulated by the stringent 271
response), and three that were upregulated (gbuR, phoQ and nosR). Intriguingly, the two-272
component response regulator gacA actually suppresses swarming and mutants in this gene 273
demonstrate hyperswarming phenotypes (49); correspondingly it was 1.8 fold upregulated in the 274
mutant (i.e. suppressed in the WT by ppGpp). We hypothesize that this large proportion of gene 275
expression changes, especially in regulatory genes, prepares for conditions for rapid adaptation to 276
swarming motility. 277
Swarming and the expression of flagella is also necessary to prepare cells for cell-to-cell or 278
cell-to-surface adherence (49). The Pseudomonas permeability regulator (PprB, PA4296) controls 279
the tad (tight adherence) locus, positively regulates fimbriae assembly (51) and flp pilus assembly, 280
and is therefore a key regulator required for adhesion (52). The tad locus (PA4302-PA4306, 281
Cluster 33), the type IVb pilin flp, and the usher- and chaperone-encoding cupE1 were >2-fold, 282
7.1 fold, and 2.8 fold downregulated in the stringent response mutant (Dataset 1-2, Fig. 2C). This 283
is in accordance with results for their corresponding regulator PprB, which was 4.7 fold 284
downregulated. Induction of the stringent response led to the expression of pprB and tadG (Table 285
1) suggesting that the stringent response regulates pilus assembly via PprB. Adaptation and 286
attachment are the first steps before bacterial biofilm formation which is also regulated by the 287
stringent response (74). Consistent with this, we found that 31% (228 out of 734) of genes that are 288
known to be required for biofilm formation in Pseudomonas (53) were either up- (92 genes) or 289
downregulated (136 genes) in the ΔrelAspoT mutant. Intriguingly, 11 biofilm regulators were 290
also identified with 4 upregulated (including efflux pump repressor mexR, T3SS assembly 291
regulator pcrH and repressor ptrB, and anti-sigma factor vreR) and 7 downregulated (including the 292
two-component sensory protein pprA, the chloramphenicol resistance activator cmrA, and 293
mycobactin siderophore uptake regulator femR). Overall these data support the proposal that the 294
stringent response regulates rapid adaptation to environmental changes. 295
The metalloprotease AprA, a novel downstream effector of the stringent stress response, was 296
required for full virulence 297
Since the stringent response has been shown to influence P. aeruginosa infection in multiple 298
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models (12, 14, 37, 48, 54, 55) and strongly regulated cytolytic proteases under normal growth 299
conditions, we further investigated the importance of one of these, AprA, in a high-density murine 300
skin infection model. The alkaline protease aprA (PA1249) was 4.1 fold upregulated upon SHX 301
induction and 9.6 fold downregulated in the mutant. The deletion of the aprA gene did not affect 302
lesion sizes (Figure 3B) or bacterial counts (Figure 3C) in the abscess. However, after three days 303
there was a significant enhancement of survival (77%) of the aprA mutant cf. the wild-type PAO1 304
(~33% survival of mice) (Figure 3D). Therefore, AprA acts as a novel downstream effector of the 305
stringent response and is required for full virulence under high-density infections; previous data 306
has implicated this protein in the regulation of virulence and destruction of host defence systems 307
(56). 308
Based on this result, we further examined protease-encoding genes using the combined lists 309
of peptidases/proteases from MEROPS (57) and the Pseudomonas Genome Database (58). It was 310
found that 37.6% (73 of 194 genes; p-value <0.001) of genes were significantly dysregulated in 311
the stringent response mutant (Figure 3A). We then focused on genes that were dysregulated in 312
the double mutant, according to RNA-Seq, and showed an inverse correlation, using qRT-PCR, 313
when ppGpp production was induced by SHX or relA overexpressed (Table 1). 314
The zinc-dependent protease PA0277 (5.2 fold downregulated by SHX induction, 9.9-fold 315
upregulated in the mutant) is directly controlled by the post-transcriptional regulator RsmA (59), 316
and the expression of rsmA is directly activated by AlgR (60) and indirectly via GacA through 317
RsmY and RsmZ (61). All of these were further controlled by the stringent stress response under 318
normal conditions. The immunomodulating metalloprotease impA (PA0572) was 2.8 fold 319
upregulated by SHX and 9.1 fold downregulated in the mutant. ImpA is important during infection 320
and protects P. aeruginosa from neutrophil attack, by cleaving the P-selectin glycoprotein ligand-321
1 on neutrophils as well as targetting CD43 and CD44 involved in leukocyte homing (62). ImpA 322
contains a LasR-regulated Xcp-dependent signal sequence (63), but SHX induction did not 323
influence the expression of lasR. Therefore, impA might be under direct control of the stringent 324
response. The putative caseinolytic peptidase clpP2 (PA3326) was 2.4 fold upregulated by SHX, 325
and 10.9 fold downregulated in the stringent response mutant; it is known to be involved in 326
motility, biofilm formation, pigmentation and iron scavenging (64). We thus investigated impact 327
of these proteases in the high-density murine skin infection model. There was no effect when 328
mutants in PA0277, ImpA, or ClpP2 were tested (data not shown). 329
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Stringent regulation of global transcriptional regulators 330
The large number of differentially expressed genes was apparently controlled in a hierarchical 331
process. Thus no fewer than 132 regulators were differentially expressed in the ΔrelAspoT double 332
mutant, with 79 being downregulated and 53 upregulated. More highly dysregulated and 333
prominent regulators included alternative sigma factor and regulator of the general stress response 334
and quorum sensing rpoS (-3.8-fold), quorum sensing regulators rhlIR (-3.0 fold, -2.6 fold) and 335
rsaL (-3.0 fold), the global regulator of virulence and quorum sensing vqsR (-2.5 fold), virulence 336
sigma factor vreI (3.0 fold), cold-shock protein cspD (-3.0 fold), upregulated T3SS repressor ptrB 337
(8.1 fold) and regulator pcrG (2.3 fold), DNA-damage-inducible regulator lexA (2.3 fold), the 338
alternative sigma factor involved in alginate biosynthesis algU (1.9-fold), the aerobic ethanol 339
oxidation system regulator erbR (6.8 fold) and two-component system eraSR (6.2 fold, 4.1 fold), 340
the nitrous oxide reduction regulator nosR (5.4 fold), the efflux pump activator brlR (2.4 fold), 341
dowregulated two-component regulators of CupE fimbrial assembly activation pprBA (-1.8 fold, 342
-4.7 fold), and PA4074 (-3.2 fold). 343
The potential that regulators such as these were mediating the very substantial effects of RelA 344
and SpoT during logarithmic growth phase, prompted us to further investigate global regulation in 345
the ppGpp-deficient strain. The 1,669 DE genes were used to search for transcriptional regulators 346
(Figure 4A) that influenced their expression by filtering to retain the first individual differentially 347
expressed in an operon; this reduced the list to 1,201 unique genes. Based on the location of the 348
start of the gene, we then extracted the region 250-bp upstream of the transcriptional initiation 349
sites for these genes (65) and searched for potential transcription factor binding site motifs. Motif-350
based sequence analysis (MEME) identified four significant motifs (Figure 4B) that were further 351
processed using the motif comparison tool Tomtom, which revealed several known global 352
regulators from CollecTF, namely: VqsM, AlgR, AmrZ, PvdS, LasR, and OxyR (Figure 4B). In 353
the next step, the regulon of each regulator extracted from CollecTF (66) was further tested for 354
statistical enrichment in the original list of differentially expressed genes, which revealed four 355
significantly dysregulated regulons: AlgR (p < 0.02), OxyR (p < 0.02), LasR (p < 0.01) and AmrZ 356
(p < 0.01); of these only LasR was itself modestly 1.57 fold downregulated. The relevance of this 357
analysis is indicated by the fact that global regulators like these control many downstream targets 358
affected by the stringent response, as typified by LasR, a mediator of the 3-oxo-C12-359
acylhomoserinelactone mediated quorum sensing response (45). 360
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Global transcriptional regulators responded to relA overexpression but not SHX induction 361
To further explore regulation by the stringent-response mediator, we overexpressed the relA 362
gene in WT as well as induced the production of ppGpp with serine hydroxamate (SHX) (Table 363
1). Intriguingly, the expression of several transcriptional regulators including argR, lasR vqsM, 364
amrZ, algR, and to some extent oxyR were only increased when relA was overexpressed but not 365
when the stringent response was induced with SHX (Table 1). SHX treatment triggers stress since 366
it is basically equivalent to amino acid starvation, while relA overexpression should in principle 367
only effect ppGpp levels. So these situations are not precisely equivalent and we would argue that 368
relA overexpression more precisely mimics effects during normal growth while SHX reflects an 369
alarm response. Alterations in ppGpp levels due to relA overexpression could potentially lead to 370
the shrinkage and depletion of the GTP and c-di-GMP pools that might interfere with 371
transcriptional and translational downstream processes (67), although the added impact of SHX-372
induced starvation would be a major consideration. 373
In P. aeruginosa, low levels of c-di-GMP can trigger the activation of the global regulators 374
such as AmrZ or AlgR. AmrZ, involved in biofilm production, iron homeostasis, and motility, has 375
been shown to regulate diguanylate cyclases and phosphodiesterases to modulate c-di-GMP (68, 376
69). Under conditions where ppGpp was not synthesized in the double mutant, we found that ~44% 377
(59 out of 144) of genes of the AmrZ regulon were dysregulated (Figure 4F), including upregulated 378
genes in polysaccharide production, psl (cluster 16) and pel (cluster 21), and downregulated genes 379
in the Type VI secretion hsi (cluster 10) (Figure 2). A comparison to the Chip-Seq analysis by 380
Jones et al. (68) further revealed that ~29% of the regulon (115 out of 398) was dysregulated in 381
the stringent response mutant (with 74 genes in common between the two regulons). However, 382
AmrZ-mediated regulation is itself quite complex since AmrZ belongs to the regulon of the 383
quorum sensing regulator LasR (70). 384
In the stringent response mutant, ~61% (51 of 83) genes in the LasR network were 385
dysregulated (Figure 4E) including rhamnolipid rhl (cluster 25), biofilm-matrix polysaccharide psl 386
(cluster 16), type VI secretion hsi (cluster 10), quinolone quorum sensing pqs (cluster 4), anti-387
metabolit amb (cluster 18), alkaline protease apr (cluster 7) (Figure 2C, Figure 4E), and various 388
other exoenzymes mentioned above. Furthermore we observed altered expression in the 389
ΔrelAspoT double mutant of 7 of the 10 genes with direct LasR binding sites in their promoters 390
(70), namely aprX-A, pqsH, rhlR, rsaL, pslA-M, ambB-E, and clpP2 (Dataset 1). A comparison to 391
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the Chip-Seq analysis by Gilbert et al. (70), showed similar dysregulation of ~64% (47 out of 74) 392
of genes in the LasR regulon (with an overlap of 72 genes in both regulons). 393
The stringent response has been suggested to affect OxyR and might modify the cellular redox 394
state (71). OxyR uses a redox-sensing mechanism to sense oxidative damage such as caused by 395
H2O2 in the cytoplasm in E. coli (72). When oxidized, it induces the expression of a catalase that 396
further reduces H2O2. The stringent response is required for optimal catalase activity (71), which 397
is in accordance with our finding that katA (PA4236) was 2.4-fold downregulated in the stringent 398
response mutant. The OxyR regulon also controls quorum sensing, protein synthesis, oxidative 399
phosphorylation and iron homeostasis (73) and ~34% (30 of 89) of regulon genes were 400
dysregulated in the stringent response mutant (Figure 4D). Similarly the double mutant 401
demonstrated altered expression of ~34% (19 out of 56) of genes of the OxyR regulon identified 402
by Chip-Seq by Wei et al (73), including ahpC, katA, dpS, PA2050, pntAA, aspS and PA1541 403
(with a total overlap of 44 genes in the two published versions of the OxyR regulon). 404
AlgR is important for type IV pili and alginate production, and synthesis of c-di-GMP via the 405
diguanylate cyclase MucR (74). Of the small known AlgR regulon in CollectTF, there were 65% 406
(13 of 20) genes dysregulated in the double mutant (Figure 4C), including genes encoding pilus, 407
hydrogen cyanide production (hcn), and cytochrome c oxidase (cco). In addition, a Chip-Seq 408
analysis by Kong et al. (74) revealed a much larger regulon and we found ~31% (48 of 155) of 409
those genes were dysregulated in the stringent response mutant only one of which overlapped with 410
the CollectTF regulon. 411
The stress σ factor RpoS is interrelated with the stringent response and is known to operate 412
during normal growth (75). Intriguingly, the regulatory networks of RpoS, as well as RpoN, RpoD, 413
and AlgU were significantly dysregulated in the stringent stress response mutant (Appendix I; Fig. 414
S5) and alternative σ factors PA0149, PA0762 (algU), PA2050, PA2896 (sbrI), PA3410 (hasI), 415
and PA3899 (fecI) were all upregulated in this mutant (Table 1, Dataset 1). However, 416
overexpression of relA or induction of the stringent response did not influence the expression of 417
any of these σ factors, suggesting that their dysregulation in the mutant might be due to binding 418
and stability difficulties to the RNA polymerase due to missing ppGpp. 419
Conclusion 420
In conclusion, we find that the P. aeruginosa stringent stress response not only influences 421
global transcriptional regulators under normal growth conditions, but also the expression of 422
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downstream effectors that potentially enable rapid adaptations. Our findings expand our 423
knowledge about the stringent response and suggest it has an important role throughout the 424
bacterial life. The stringent response, originally discovered as a stress response during amino acid 425
starvation, influences the expression of hundreds of genes (15). It is now clear that the stringent 426
response is more than just a response to starvation and that the production of the signaling molecule 427
ppGpp strongly influences global gene expression of bacterial cells. Here, we further defined the 428
stringent response as being required under nutrient-rich rapid-growth conditions. This reinforces 429
proposals that the stringent response is an excellent, novel target for the development of new 430
antimicrobials (12, 76, 77). Further mechanistic understanding of downstream processes after 431
stringent response signal blockage will help in the development of novel compounds for clinical 432
use, and reveal new targets amongst the downstream effectors such as, for example, the alkaline 433
protease AprA. 434
Materials and Methods 435
Bacterial strains and growth conditions 436
Bacterial strains and plasmids are listed in SI Appendix; Table S1 and S2. All organisms were 437
cultured at 37°C in double Yeast Tryptone (2YT) or Basal Medium 2 (BM2) (78). Liquid cultures 438
were placed with shaking at 250 rpm. Cultures harboring individual vectors were supplemented 439
with 15 µg/ml gentamicin (Gm), 100 µg/ml ampicillin (Ap) for E. coli, 50 µg/ml Gm, and 250 440
µg/ml carbenicillin (Cb) for P. aeruginosa. Bacterial growth was monitored at an optical density 441
at 600 nm (OD600) using either a spectrophotometer or a 96-well microtiter plate reader (Synergy 442
H1; BioTek). 443
Molecular methods 444
PCRs were carried out using the Phusion DNA polymerase (Thermo Scientific) in accordance 445
with the manufacturer’s instructions and optimized annealing temperatures for each primer set. 446
PCR reactions were supplemented with 5% dimethyl sulfoxide. Restriction digestions were 447
performed using Thermo Scientific FastDigest restriction enzymes according to the 448
manufacturer’s instructions. All ligation reactions were carried out at room temperature using 449
Thermo Scientific T4 DNA ligase. DNA purifications were either performed using the GeneJET 450
PCR purification kit (Thermo Scientific) or the GeneJET Gel extraction kit (Thermo Scientific) 451
following the manufacturer’s instructions. 452
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Construction of the PAO1 arginine argB auxotroph and alkaline protease aprA mutant 453
The construction of both knockout vectors was based on the protocol by Zumaquero et al. (79) 454
and carried out as previously described (80). Briefly, primers argB_up_fwd(Bam)/argB_up_rev 455
and argB_down_fwd/argB_down_rev(Hind) were used to amplify the 500 bp knockout alleles for 456
the argB gene from P. aeruginosa PAO1 genomic DNA. Primers 457
aprA_up_fwd(Bam)/aprA_up_rev and aprA_down_fwd/aprA_down_rev(Hind) were used to 458
amplify the 500 bp knockout alleles for the aprA gene from P. aeruginosa PAO1 genomic DNA, 459
respectively (SI Appendix; Table S3). The obtained fragments were used in an overlapping PCR 460
reaction with up_fwd and down_rev primers. Next, each fusion fragment was cloned into the 461
suicide vector pEX18Gm via BamHI/HindIII restriction sites and verified by sequencing. The 462
generation of both mutants was based on the site-specific insertional mutagenesis strategy of 463
Schweizer and Hoang (81) and carried out as described previously (82). To confirm the deletions, 464
locus-specific primers that bind up- and downstream of the amplified knockout alleles were used 465
(argB_outA/argB_outB or aprA_outA/aprA_outB) and the resulting knockout fragments verified 466
by sequencing. 467
Cloning of the PAO1 relA, cysM, and rpoZ transcriptional promoter reporter fusions 468
Transcriptional fusions between the relA promoter region and the mCherry gene were created 469
using the primers relA-Pro_fwd(Xba)/relA-Pro_rev-mCherry_fwd to amplify the 84 bp relA 470
promoter from PAO1 genomic DNA and mCherry_fwd-relA-Pro_rev/mCherry_rev_t0(Kpn-Apa) 471
to amplify mCherry from plasmid pUCP23.mCh. The resulting fragments were gel-purified and 472
fused in another PCR reaction with relA-Pro_fwd/mCherry_rev primers. The fusion product was 473
cloned onto pUCP22 in opposite direction of the lac promoter via XbaI/KpnI restriction sites. 474
Transcriptional fusions between the promoter regions of cysM (270 bp) and the mCherry gene 475
were created on plasmid pUCP22. Therefore, the mCherry gene was flipped onto pUCP22 via 476
EcoRI/BamHI yielding mCherry in opposite direction of Plac. Next, the cysM promoter was 477
amplified from PAO1 genomic DNA via cysM-Pro_fwd(Xba)/cysM-Pro_rev(Bam), gel-purified 478
and cloned onto pUCP22.mCherry via XbaI/BamHI (yielding pUCP22.cysM-Pro.mCherry). 479
Transcriptional fusions of the cysM promoter, the relA promoter, and the mCherry gene were 480
created via amplification of the cysM promoter (270 bp) from genomic DNA using cysM-481
Pro_fwd(Xba)/cysM-Pro_rev(Spe). The obtained fragment was ligated via XbaI/SpeI in front of 482
the relA promoter on plasmid pUCP22.relA-Pro.mCherry to yield pUCP22.cysM-relA-483
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Pro.mCherry. All created constructs were confirmed by sequencing and transformed into PAO1 484
wild type as well as the auxotroph arginine mutant ΔargB as described earlier (82). 485
Transcriptional fusions between the promoter regions of rpoZ and the eGFP gene were created 486
on plasmid pUCP23. The rpoZ promoter region (88 bp) was amplified via rpoZ-487
Pro_fwd(SacI)/rpoZ-Pro_rev-egfp_fwd from PAO1 genomic DNA and the eGFP gene from 488
pBBR.TIR.egfp.t0 via egfp_fwd-rpoZ-Pro_rev/egfp_rev(Kpn). Both fragments were fused with a 489
subsequent PCR using rpoZ-Pro_fwd/egfp_rev and further transferred onto pUCP23 via SacI/KpnI 490
in opposite direction of the lac promoter. All created constructs were confirmed by sequencing and 491
transformed into PAO1 as described earlier (82). 492
Promoter fusion constructs - fluorescence and growth curve experiments 493
Overnight grown bacteria (16-18 h) in 2YT medium at 37°C with shaking (250 rpm) were 494
washed (8000 rpm, 3 min), resuspended in BM2, and further diluted to an OD600 of 0.1. 200 µl 495
were transferred to a flat bottom 96-well polystyrene microtiter plate (Corning). Plates were 496
incubated at 37°C with continuous fast linear shaking at 567 cycles per minute (cpm) in a 497
microplate reader (Synergy H1; BioTek). OD600 and fluorescence (mCherry and eGFP were 498
detected at 580 to 610 m and 488 to 509 m, respectively) were taken every hour over a 16-hour 499
period. Experiments were performed three times with at least three technical replicates. Amino 500
acid starvation growth experiments were performed with the PAO1 wild type and the auxotroph 501
arginine mutant ΔargB, both carrying promoter fusion constructs, in minimal medium BM2 502
without NH4Cl, and supplementation of 6.25 µM or 12.5 µM arginine. Statistical analysis was 503
performed using a paired one-sided t-test where each time point was compared to the starting 504
timepoint to identify a significant increase in promoter activity. 505
Cloning of the inducible relA and spoT constructs 506
The relA gene was PCR-amplified from PAO1 genomic DNA with primers 507
relA_fwd(Xba)/relA_rev(Hind). The amplified product was gel-purified and cloned into 508
pHERD20T via XbaI/HindIII to allow expression from the pBAD promoter, and subsequently 509
sequenced. The spoT gene was PCR-amplified from PAO1 genomic DNA with primers 510
spoT_fwd/spoT_rev(Hind). The amplified product was gel-purified, digested with HindIII and 511
cloned into pHERD20T via SmaI/HindIII to allow expression from pBAD and was subsequently 512
sequenced. Each plasmid was transformed into P. aeruginosa PAO1 wild-type as previously 513
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described (82). 514
Cloning of the RelA-YFP and SpoT-GFP translational reporter fusions 515
The relA gene was PCR-amplified from PAO1 genomic DNA with primers 516
relA_fwd/relA_rev. Both primers had an EcoRI restriction site incorporated. The reverse primer 517
lacked the RelA stop codon. The amplified product was cloned onto pBAD24.yfp via EcoRI and 518
orientation verified via restriction digest and sequencing. The resulting pBAD24.relA-linker-yfp 519
plasmid was digested with BamHI, treated with S1 nuclease, and further digested with HindIII. 520
The resulting fragment was transferred onto pHERD20T via HindIII/SmaI. 521
The spoT gene was PCR-amplified from PAO1 genomic DNA with primers 522
spoT_fwd/spoT_rev-link-gfp_fwd. The reverse primer lacked the SpoT stop codon and had an in-523
frame linker sequence (atggtgtctatcactaaagatcaaatc) fused to the forward sequence of egfp. The 524
egfp gene was amplified from pBBR1.TIR.egfp.t0 with primers gfp-fwd-link-spoT-rev/egfp-rev 525
whereby the gfp-fwd primer was the reverse complement primer of the spoT-rev primer to allow 526
the fusion of both fragments in a second PCR with spoT_fwd and gfp_rev primers. The amplified 527
product was gel-purified, digested with HindIII and cloned into pHERD20T via SmaI/HindIII 528
before sequencing. Reporter fusions were verified as described below. 529
Functional verification of the RelA-YFP and SpoT-GFP reporter fusions using swarming 530
Since the fluorescence fusion were in-frame at the C-terminal end of the gene of interest, a 531
fluorescent signal would indicate a correctly folded fusion protein. However, overexpression of 532
fusion proteins can lead to misfolding and subsequent sequestering into insoluble inclusion bodies 533
(83). To verify that both fusion proteins were correctly folded and functional, we complemented 534
the ΔrelAΔspoT double mutant with either RelA-YFP or SpoT-GFP and tested the ability of the 535
strain to swarm on semi-solid agar plates (BM2 plates containing 0.4% agar). The PAO1 stringent 536
response double mutant is unable to swarm (SI Appendix, Fig S4 and (37)). The PAO1 wild-type 537
strain as well as the ΔrelA/spoT mutant were transformed with an empty pHERD20T vector control 538
and RelA-YFP and SpoT-GFP fusions. All strains were scraped from overnight grown plates and 539
suspended in sterile demineralized water to an OD600 of 0.025. Ten µl of a bacterial cell suspension 540
was applied onto a swarming agar plate and incubated at 37°C for 18 h. Experiments were repeated 541
at least three times. The motility complementation further verified an intact fusion protein (SI 542
Appendix, Fig S6). 543
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Growth and confocal microscopy of protein-fusions 544
Cultures harboring the RelA-YFP construct were grown in BM2 (at 250 rpm) to an OD600 of 545
0.3 prior to induction with 5% L-arabinose. After 30 minutes of induction, cultures were split and 546
one received with 1 mM SHX for another 30 minutes. Stationary phase cultures at an OD of 547
2.5±0.3 were induced with 5% L-arabinose for 30 minutes. Cultures harboring SpoT-GFP were 548
grown to an OD600 of 0.3 prior induction with 5% L-arabinose for 30 min. Cultures were washed 549
one time with PBS (8000 rpm, 5 min) before resuspending in BM2 with or without carbon source 550
for 30 min. Iron starvation was induced with 1 mM dipyridyl for 30 minutes. 551
To visualize the fluorescent protein localization in cells, the fluorescent proteins were cross-552
linked in order to achieve minimal cell movement and preserve the physiological state. Cells were 553
cross-linked with 3.7% formaldehyde at room temperature for 1 hour. Cells were washed twice 554
with PBS by centrifugation (8000 rpm, 5 min) and the subsequent pellet resuspended in 1 ml of 555
PBS. Cells were visualized on a Zeiss microscope. 556
Growth, induction, and RNA isolation for quantitative real-time (qRT)-PCR and RNA-Seq 557
P. aeruginosa PAO1 wild-type, ΔrelAspoT double mutant, as well as relA and spoT 558
overexpressing strains were grown to an OD600 of 0.5 in 2YT broth. Induction experiments were 559
carried out with either 1 mM serine hydroxamate (SHX) to chemically induce stringent conditions 560
in the wild-type or via overexpression of relA or spoT from pHERD20T through the addition of 561
1% L-arabinose into the culture medium. Induced cultures were left for 30 minutes at 37°C with 562
shaking (250 rpm). 563
Bacteria were harvested in RNAprotect Bacteria Reagent (QIAGEN) by centrifugation 564
(13,000 rpm, 2 min). Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) following 565
the manufacturer’s instructions. The obtained RNA was DNAse-treated (Ambion/Life 566
Technologies) and subsequently quantified using a Nanodrop ND-2000 spectrophotometer 567
(Thermo Fischer Scientific) and RNA integrity determined by agarose gel electrophoresis. 568
For qRT-PCR experiments, high-quality RNA was reverse transcribed and amplified with a 569
Roche LightCycler 96 instrument, in combination with the qScriptTm One-Step SYBR® Green 570
qRT-PCR Kit (QuantaBio) according to the manufacturer’s protocol. Template RNA (5 571
ng/sample) was used in a standard 25 μl qRT-PCR reaction with specific primers. Each sample 572
was analyzed for gene expression in at least triplicate. Quantification of mRNA transcripts was 573
performed by the comparative Ct method (84) using the 16S gene as normalizer. 574
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For RNA-Seq experiments, ribosomal RNA was further depleted using the RiboZero Bacteria 575
Kit (Illumina). Library preparation was done with the KAPA Stranded Total RNA Kit (Kapa 576
Biosystems), and sequencing performed on the Illumina HiSeq2500 instrument at the University 577
of British Columbia’s Sequencing and Bioinformatics Consortium (generating single end reads 578
1×100 bp). The read quality and alignment of sequencing samples was carried out as previously 579
described (85). Briefly, FastQC v0.11.6 and MultiQC v1.6 were used for quality, STAR v2.6 for 580
the alignment of transcriptomic reads to the PAO1 reference genome (obtained from the 581
Pseudomonas Genome Database (58)) and read counts generated using HTSeq v0.11.2. Library 582
sizes had a minimum of 1.2 million, median of 3.1 million, and maximum of 7.6 million uniquely 583
mapped reads. Differentially expressed (DE) genes between the double mutant and wild type were 584
determined using DESeq2 v1.24.0 with thresholds of adjusted p-value ≤ 0.05 and absolute fold 585
change ≥ 1.5. 586
Visualization of RNA-Seq data 587
Relative expression of DE genes were plotted using the circular visualization software package 588
CIRCOS (86). Since we found stretches of genes that were dysregulated in the same direction, we 589
used an arbitrary cut-off of five consecutive genes to extract gene information for these regions. 590
The rationale for using at least five genes in a row was that four genes almost revealed 100 clusters 591
and six in a row also showed 34 clusters. 592
Regulatory elements and regulator enrichment 593
Operon information for P. aeruginosa PAO1 was downloaded from DOOR2 (87) and gene 594
annotations obtained from the Pseudomonas Genome Database (58). The following methodology 595
was then applied to all 1669 DE genes identified in the RNA-Seq experiment: The transcriptional 596
start site (TSS) of each gene was obtained from Gill et al. (65). For any TSS type denoted as 597
antisense, the strand for that gene was switched. In case that there was no TSS available, the start 598
codon as listed in the genome annotations was used. Then the list of 1669 genes filtered to include 599
the first gene from a given operon (or single genes for those not in an operon), yielding 1201 genes. 600
Once the starting location had been determined, the R package BSGenome v1.48.0 (88) was used 601
to extract the 250-bp upstream region for each of the 1201 genes. These sequences were then 602
submitted to MEME (89) for identification of novel motifs with a significance threshold of E-603
value ≤ 0.05. The program was set to find up to five motifs while all other settings were left at 604
their default. These five de novo motifs found by MEME were then submitted to Tomtom (90) to 605
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identify potential matches to characterized motifs and their corresponding regulators within the 606
CollecTF database (66). Matches identified by Tomtom were considered significant with q-value 607
≤ 0.5. 608
The list of all PAO1 regulators and their controlled genes (regulon) was downloaded from 609
CollecTF (66) or recent Chip-Seq manuscripts as indicated in the text. This list of regulons was 610
then tested for enrichment in the list of 1669 DE genes using Fisher’s Exact Test, implemented via 611
a custom script in R. Multiple test correction was performed using the Benjamin-Hochberg (BH) 612
method. Significance of enrichment for each regulon was determined using a threshold of BH-613
corrected p-value ≤ 0.05. 614
Functional enrichment of DE genes 615
Enrichment of GO terms were performed using GofuncR (Steffi Grote, 2018), testing the DE 616
genes against a custom set of GO annotations downloaded from the Pseudomonas Genome 617
Database (58). The full list of 1,669 DE genes was split into up and down regulated, with GO 618
enrichment being performed independently on each of these sets. Results were filtered using a 619
significance threshold of family-wise error rate (FWER) ≤ 0.1. Enrichment of KEGG Pathways 620
was done using Gage v2.3.0 (91) on the full list of 1,669 DE genes. Results were filtered for 621
significance based on q-value ≤ 0.2. 622
Enrichment of cellular functions, based on manually curated lists from various sources 623
(Dataset 3) was performed on the full list of 1,669 DE genes using Fisher’s Exact Test, 624
implemented via a custom script in R. Multiple test correction was performed using the BH method 625
and filtered on a significance of ≤ 0.05. 626
Growth curves experiments, pyoverdine production, and pyocyanin measurements 627
PAO1 strains were grown overnight (16-18 h) in dYT medium at 37°C with shaking (250 628
rpm). The overnight culture was resuspended in dYT broth to an OD600 of 0.1, then 200 µl 629
transferred to a flat bottom 96-well polystyrene microtiter plate (Corning) and incubated at 37°C 630
with continuous fast linear shaking at 567 cycles per minute (cpm) in a microplate reader (Synergy 631
H1; BioTek). OD600 and fluorescence readings were taken every hour over a 24-hour period as 632
previously described (85). Experiments were performed three times with at least three technical 633
replicates. 634
Pyocyanin production of PAO1 and PAO1.ΔrelAspoT was measured as previously described 635
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22
(85). Briefly, cells were grown in LB overnight and subsequently washed in SCFM broth, adjusted 636
to OD600 of 0.1, and further cultivated at 37°C with aeration (250 rpm) for 20 h and pyocyanin 637
extracted from filter-sterilized supernatants. Experiments were performed three times with at least 638
two technical replicates. 639
Swarming motility assays 640
Swarming motility was examined on KB plates containing 0.4% agar. Strains were adjusted 641
in KB medium to an OD600 of 0.1 and incubated for 24 h at 37°C as previously described (92). 642
All experiments were performed at least three times. 643
Adherence experiments 644
Adherence experiments were performed as previously described (85). Briefly, strains were 645
streaked onto dYT agar plates and grown overnight at 37C. Bacteria were scraped from the plates 646
and resuspended in dYT medium to an OD600 of 0.5 and 100d l added into polystyrene microtiter 647
plates (Falcon) and incubated at room temperature for 1 h. Then each well was washed and adhered 648
cells stained with crystal violet. Afterwards, plates were washed and crystal violet dissolved in 649
70% ethanol at room temperature and absorbance measured at 595 nm with a microplate reader 650
(Synergy H1; BioTek). Data analysis was performed to calculate the mean and standard deviation, 651
after removal of outliers that were more than one standard deviation from the mean. Data was 652
further normalized to the wild type. Experiments were performed at least three times with up to 653
six technical replicates. 654
Ethics statement 655
Animal experiments were performed in accordance with The Canadian Council on Animal 656
Care (CCAC) guidelines and were approved by the University of British Columbia Animal Care 657
Committee (certificate number A14-0363). 658
Cutaneous mouse infection model 659
Mice used in this study were female outbred CD-1. All animals were purchased from Charles 660
River Laboratories (Wilmington, MA), were 7 weeks of age, and weighed about 25 ± 3 g at the 661
time of the experiments. 1 to 3% isoflurane was used to anesthetize the mice. Mice were euthanized 662
with carbon dioxide. The cutaneous mouse abscess infection model was performed as described 663
earlier (93). Briefly, P. aeruginosa PAO1 and its alkaline protease AprA deficient mutant were 664
grown to an OD600 of 1.0 in 2YT broth, subsequently washed twice with sterile PBS, and further 665
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23
adjusted to 5 108 CFU/ml. A 50 l bacterial suspension was injected into the right side of the 666
dorsum. The progression of the infection was monitored daily and mice euthanized that reached 667
humane endpoint. Abscess lesion sizes (visible dermonecrosis) was measured on day three. Skin 668
abscess tissues were excised (including all accumulated pus), homogenized in 1 ml sterile PBS 669
using a Mini-Beadbeater-96 (Biospec products) for 5 min and bacterial counts determined by serial 670
dilution. Experiments were performed at least 2 times independently with 3 to 5 animals per group. 671
Data Availability 672
All fastq and count files are available under Gene Expression Omnibus (GEO) accession 673
number GSE147132. The full list of differentially expressed genes is included in the Dataset 4. 674
Competing interests 675
The authors declare no competing interests. 676
Acknowledgements 677
DP is supported by an Alexander von Humboldt – Feodor Lynen Postdoctoral Fellowship, a 678
Cystic Fibrosis Canada Postdoctoral fellowship, and a Research Trainee Award from the Michael 679
Smith Foundation for Health Research. REWH is supported by Canadian Institutes from Health 680
Research grant FDN-154287 and holds a Canada Research Chair and UBC Killam Professorship. 681
.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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24
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30
Tables 929
Table 1: Fold-changes of P. aeruginosa PAO1 mRNA expression in the ΔrelAΔspoT double 930
mutant compared to wild-type (RNA-Seq data), compared to the influence of overexpression 931
in the WT of relA and induction with 1 mM serine hydroxamate (SHX) (qRT-PCR). Changes 932
greater than two-fold are presented in bold typeface. 933
Locus Gene Description or Product
Fold change relative to WT
PAO1
ΔrelA
ΔspoT
WT +
SHX
WT +
relA++
Differential effect of loss (ΔrelAΔspoT) and overexpression of ppGpp (+SHX/relA++)
PA0277 Zn-dependent protease with chaperone function 9.9 -5.2 -1.1
PA0572 impA Hypothetical protein -9.1 2.8 1.3
PA1245 aprX Type I secretion system -2.8 4.3 2.3
PA1249 aprA Alkaline metalloprotease -9.6 4.1 2.0
PA1384 galE UDP-glucose 4-epimerase -1.9 4.4 1.2
PA1430 lasR Transcriptional regulator -1.6 -1.1 3.0
PA1477 ccmC heme exporter protein 1.6 -2.3 1.2
PA1656 hsiA2 Type VI secretion protein -1.8 1.2 1.8
PA1874 bapA Adhesion protein -4.3 3.0 2.0
PA2016 liuR Regulator of liu genes -1.6 2.0 -1.4
PA2050 RNA polymerase ECF-subfamily σ70 factor 2.5 -1.5 -1.1
PA2227 vqsM Transcriptional regulator -1.7 1.4 2.5
PA2259 ptxS Transcriptional regulator -1.9 2.4 -1.2
PA2302 ambE Non-ribosomal peptide synthetase -4.1 3.0 1.4
PA2911 TonB-dependent receptor 8.0 -3.0 1.3
PA3095 xcpZ General secretion pathway protein -2.4 1.9 1.1
PA3326 clpP2 ATP-dependent Clp protease proteolytic subunit -10.9 2.4 1.8
PA3327 Non-ribosomal peptide synthetase -21.0 8.0 2.0
PA3462 Sensor/response regulator -1.5 2.2 1.1
PA3607 potA Polyamine transport protein 2.9 -9.9 -1.1
PA4210 phzA1 Phenazine biosynthesis protein -9.8 4.2 2.5
PA4211 phzB1 Phenazine biosynthesis protein -7.9 12.7 1.9
PA4296 pprB Two-component response regulator -4.7 3.8 1.7
PA4297 tadG Pilus assembly -2.0 3.1 1.4
Smaller effect of either loss of ppGpp or overexpression (+SHX and/or relA++)
PA0149 RNA polymerase ECF-subfamily σ70 factor 1.8 1.2 1.0
PA0625 Pyocin production 6.8 1.2 1.2
PA0652 vfr Transcriptional regulator -1.7 1.1 1.2
PA0762 algU RNA polymerase σ factor AlgU 1.9 -1.2 1.0
PA0893 argR Transcriptional regulator 1.1 -1.0 2.1
PA1271 TonB-dependent receptor 3.4 -1.0 1.3
PA1272 cobO cob(I)alamin adenosyltransferase 2.4 -1.2 1.2
PA1541 Drug efflux transporter 10.9 2.4 1.0
PA1698 popN Type III secretion protein 3.3 -1.3 1.4
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PA2069 Carbamoyl transferase -8.1 1.4 1.4
PA2193 hcnA Hydrogen cyanide synthase -3.2 -1.5 -1.1
PA2231 pslA Exopolysaccharide biosynthesis 2.3 1.2 1.6
PA2360 hsiA3 Type VI secretion protein -1.9 1.0 1.2
PA2426 pvdS RNA polymerase ECF-subfamily σ70 factor -1.1 -18.1 -2.7
PA3385 amrZ Alginate and motility regulator -1.0 1.0 2.1
PA3410 hasI RNA polymerase ECF-subfamily σ70 factor 3.0 -1.4 1.0
PA3661 Hypothetical protein 17.2 5.1 5.4
PA3899 fecI RNA polymerase σ factor 1.9 -1.1 -1.1
PA5261 algR Alginate biosynthesis regulatory protein -1.4 -1.2 2.1
PA5344 oxyR Transcriptional regulator -1.1 1.2 1.9
934
935
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Figures 936
937
938 Figure 1: Sub-cellular localization of RelA-YFP and SpoT-GFP fusions in P. aeruginosa 939
PAO1. Cells were grown in BM2 to mid-exponential phase and fluorescent constructs induced 940
with 5% L-arabinose for 30 min prior to individual stress treatments. Cells were washed in PBS 941
and subsequently the DNA counter-stained with Hoechst 3342 DNA stain (blue). A) RelA-YFP 942
cultures were treated with or without (control) 1 mM SHX at an OD of 0.5 for 30 min. Stationary 943
phase cultures were grown to an OD of 2.5±0.3 before induction with 5% L-arabinose. B) SpoT-944
GFP cultures were either transferred into BM2 (control), BM2 without carbon source, or treated 945
with 1 mM of the iron chelator dipyridyl at an OD of 0.5 for 30 min. Stationary phase cultures 946
were grown to an OD of 2.5±0.3 before induction with 5% L-arabinose. A, B) The pictures to the 947
very right show one single cell under uninduced (control) conditions. Rectangle indicates the 948
identification of single cells that have been used to measure fluorescence intensity. C) RelA-YFP 949
fluorescence intensity (left) and coefficient of variation (right) with or without (control) 1 mM 950
SHX and in stationary phase. D) SpoT-GFP fluorescence intensity (left) and coefficient of 951
variation (right) without stress (control) and upon removal of the carbon source, addition of 1 mM 952
iron chelator dipyridyl, and stationary phase. C, D) Statistics were performed using One-way 953
ANOVA with Dunn’s multiple comparisons test; p-value < 0.01 (**). The percentage of 954
Coefficient of variation = standard deviation (intensity) / mean (intensity) * 100. The higher the 955
Coefficient percentage the higher the likelihood of individual spots in the cells, while a lower 956
percentage indicates a rather equally distributed signal. Rel-YFP (n = 520 – 1700 cells) and SpoT-957
GFP (n = 750 – 1500 cells). 958
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33
959 Figure 2: GO enrichment, KEGG analysis, and circular visualization of the differential 960
expressed genes between the P. aeruginosa PAO1 stringent stress response mutant 961
ΔrelAspoT and wild-type strain under planktonic conditions. A) Results of GO term 962
enrichment performed by GOfuncR on the list of differentially expressed genes, downregulated 963
(left) and upregulated (right). GO terms were considered significant with q-value ≤ 0.2. B) Results 964
of KEGG enrichment performed by GAGE analysis with a threshold of q-value ≤ 0.2. A, B) Dot 965
size indicates total number of genes annotated to a particular term / pathway. Gene ratio represents 966
the proportion of total genes assigned to a term that are differentially expressed. C) Neighbouring 967
genes within the DE genes across the chromosome (left). The inner track of the Circos plot shows 968
log2-fold changes (grey). All significant values with adjusted p-value < 0.05 are highlighted in red 969
(log2fc > 0.585) and green (log2fc < -0.585). The numbers around the expression pattern show 970
identified regions where a minimum of five consecutive genes were dysregulated in the same 971
direction. The outer track shows the location of the genes (PA0001 – PA5420) on the PAO1 972
chromosome without the ‘PA’ prefix. Heatmap of the 34 regions color-coded based on up- (red) 973
or downregulation (green) of the set of the five consecutive dysregulated genes (right). See Dataset 974
2 for the full list of genes and annotation. 975
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976 Figure 3: Differentially expressed protease genes of the stringent response mutant compared 977
to the wild type and alkaline protease mutant aprA in a high-bacterial density skin infection 978
model. A) Circos plot of proteases with downregulated (green) and upregulated (red) genes around 979
the chromosome. Proteases that were not significant are delineated as grey. B-D) PAO1 wildtype 980
(n=15) and PAO1 aprA-deficient strain (n=13) in a cutaneous mouse infection model. B) Abscess 981
sizes. C) Bacterial load recovered from abscess tissue three days post infection. D) Survival 982
percentage over the course of a three days experiment. p-value = 0.03 based on Gehan-Breslow-983
Wilcoxon test. 984
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985 Figure 4: Motif search and regulons of identified transcription factor binding sites from 986
CollecTF. A) Overview of the identification pipeline to find dysregulated regulators in RNA-Seq 987
data. B) Identified motifs of dysregulated genes by MEME and regulators by TomTom. C) AlgR 988
regulon (20 genes; 12 down-regulated, 1 up-regulated). D) OxyR regulon (89 genes; 20 down-989
regulated, 10 up-regulated). E) LasR regulon (83 genes; 36 down-regulated, 15 up-regulated. F) 990
AmrZ regulon (144 genes; 22 down-regulated, 37 up-regulated). C-F) red; up-regulated, green; 991
down-regulated, blue; regulator, black; no change. Adjusted p-value ≤ 0.05, fold-change ± 1.5.992
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36
SI Appendix; Table S1: Bacterial strains used in this study 993
Strains Relevant characteristics or genotypea Reference
or source
Escherichia coli
XL1-Blue
recA1 endA1 gyrA96 thi-1 hsdR17(rK- mK+)
supE44 relA1 lac, [F′ proAB lacIq
ZΔM15Tn10(Tetracycline resistance)]
Stratagene
Pseudomonas aeruginosa PAO1 strains
WT Laboratory wild type strain (94)
relAspoT ΔrelAspoT double deletion mutant (12)
relAspoT/relA+
ΔrelAspoT deletion mutant chromosomally
complemented with the relA gene including its
promoter region
(12)
PAO1.relAspoT/spoT+
ΔrelAspoT deletion mutant chromosomally
complemented with the spoT gene including the
rpoZ-spoT promoter region
(12)
PAO1/pUCP22.relA-Pro.mCherry PAO1 carrying relA promoter fusion to
mCherry
This study
PAO1/pUCP22.cysM-Pro.mCherry PAO1 carrying cysM promoter fusion to
mCherry
This study
PAO1/pUCP22.cysM-relA-
Pro.mCherry
PAO1 carrying cysM-relA promoter fusion to
mCherry
This study
PAO1/pUCP23.rpoZ-Pro.egfp PAO1 carrying rpoZ promoter fusion to egfp This study
PAO1/pHERD20T.relA PAO1 carrying relA overexpression plasmid This study
PAO1/pHERD20T.spoT PAO1 carrying spoT overexpression plasmid This study
PAO1/pHERD20T.relA-YFP PAO1 carrying relA-YFP overexpression
plasmid
This study
PAO1/pHERD20T.spoT-GFP PAO1 carrying spoT-GFP overexpression
plasmid
This study
PAO1.ΔargB ΔargB deletion mutant This study
PAO1.ΔargB/pUCP22.relA-
Pro.mCherry
PAO1.ΔargB carrying relA promoter fusion to
mCherry
This study
PAO1.ΔargB/pUCP22.cysM-
Pro.mCherry
PAO1.ΔargB carrying cysM promoter fusion to
mCherry
This study
PAO1.ΔargB/pUCP22.cysM-relA-
Pro.mCherry
PAO1 carrying cysM-relA promoter fusion to
mCherry
This study
994
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SI Appendix; Table S2: Plasmids used in this study 995
Plasmids Phenotype Reference
pEX18Gm Gmr, pEX18Gm sucrose suicide vector carrying
the cysM-mCherry promoter fusion
(81)
pEX18Gm.argB-ko Gmr, pEX18Gm sucrose suicide vector carrying
the argB knockout alleles
This study
pHERD20T Apr, Escherichia-Pseudomonas shuttle vector
carrying araC-PBAD cassette
(95)
pUCP22 AprGmr, Broad-host-range vector (96)
pUCP23 AprGmr, Broad-host-range vector (96)
pUCP23.mCherry AprGmr, pUCP23 carrying the mCherry red
fluorescent protein gene
(97)
pUCP22.relA-Pro.mCherry AprGmr, pUCP22 carrying the relA-mCherry
promoter fusion
This study
pUCP22.cysM-Pro.mCherry AprGmr, pUCP22 carrying the cysM-mCherry
promoter fusion
This study
pUCP22.cysM-relA-Pro.mCherry AprGmr, pUCP22 carrying the cysM-relA-
mCherry promoter fusion
This study
pHERD20T.relA Apr, pHERD20T carrying the relA gene from
PAO1 und PBAD
This study
pHERD20T.spoT Apr, pHERD20T carrying the spoT gene from
PAO1 und PBAD
This study
pBAD24.YFP Apr, pBAD24 carrying the yfp gene and N-
terminal linker sequence
(98)
pBBR1.egfp.TIR Cmr, pBBR1MCS carrying egfp-T0 cassette in
pBBR1MCS in opposite orientation of Plac
(99)
pHERD20T.relA-YFP Apr, pHERD20T carrying the relA gene without
stop codon fused to linker sequence and yfp
This study
pHERD20T.spoT-GFP Apr, pHERD20T carrying the spoT gene without
stop codon fused to linker sequence and egfp
This study
a Antibiotic resistance: Gmr, gentamicin; Apr, ampicillin / carbenicillin; Cmr, chloramphenicol
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SI Appendix; Table S3: PCR primers used in this study
Primer Sequence
PCR primers
argB_up_fwd AGAGGATCCGTGGAAGACCGGCCACTC
argB_up_rev GTGGCGGGCGATCAGTGAGGGGAGCTCCGACATGTC
argB_down_fwd GACATGTCGGAGCTCCCCTCACTGATCGCCCGCCAC
argB_down_rev TGCAAGCTTCCAGCCACTGGCTGGACA
argB_outA ATGCTGTTCGCCAAGGACGT
argB_outB ATCGAACTCATCCATGCCGC
aprA-up_fwd AAGTACGACTACCTCACGGC
aprA-up_rev TGTCAGCGCGTCAGACGACGATGTCCAATGTCCACAGGTTTC
aprA-down_fwd GAAACCTGTGGACATTGGACATCGTCGTCTGACGCGCTGACA
aprA-down_rev ATGATCGGTCGCTTCGTGGT
aprA_outA TTACGACCTGGACGACAAGG
aprA_outB GACCCATGAGATCGAAGGCA
relA-Pro_rev/mCherrry_fwd ATCCTCCTCGCCCTTGCTCACCATCTTGCCTACCCTTTACCACG
relA-pro_fwd(Xba) CATTCTAGAGATGCCTGCGTAATCCGA
relA-Pro_rev/mCherrry_fwd CGTGGTAAAGGGTAGGCAAGATGGTGAGCAAGGGCGAGGAGGAT
mCherry_rev_t0(KpnI-ApaI) CATGGTACCGGGCCCTGGACTCACAAAGAAAAAACGC
cysM-Pro_fwd(Xba) AGTTCTAGAACCACCTCGTTCTGATCGCC
cysM-pro_rev(BamHI) TCAGGATCCGAGGCACTCGTTCGGCACTGACG
cysM-pro_rev(SpeI) CATACTAGTGAGGCACTCGTTCGGCACTGACG
rpoZ-Pro_fwd CCAGGGCCCGAGCTCGTCTGCTCGCCTAAATCGG
rpoZ-Pro_rev/egfp_fwd CACTTTTCGTTACGAGGAACACCATGCTGAGCAAGGGCGAGGA
egfp_fwd/rpoZ-Pro_rev TCCTCGCCCTTGCTCAGCATGGTGTTCCTCGTAACGAAAAGTG
egfp_rev AAGCTGGGTACCAACGGTGG
relA_oe_fwd TCTAGAATGGTACAGGTGAGAGCGCA
relA_oe_rev AAGCTTGGGTATCTCGGGTCTTCAG
spoT_fwd TTGCCGGGCATAGACGCCTT
spoT_rev GCCAAGCTTGGAAAAAGCGGGTCAGCTACG
996
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Supplementary Figures
997
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SI Appendix, Fig S1: Genomic orientation of the ppGpp synthase relA (PA0934) in P. 998
aeruginosa PAO1 and promoter activity in a PAO1 WT and an arginine auxotroph mutant. 999
A) The relA gene shown in an operon with the cysteine synthase (cysM, PA0932) and RNA 1000
methyltransferase (ygcA, PA0933) (thin arrow below genes). σ factor binding sites for RpoH (σ32; 1001
heat shock) and RpoS (σ38; stationary phase, starvation) indicated by the dotted lines upstream of 1002
relA and cysM, respectively. B, C) The cysM and relA promoter, respectively, was fused to the 1003
mCherry reporter gene and grown over a period of 16 h in BM2 broth in a 96-well plate with 1004
continuous shaking (567 cpm) with 6.5 and 12.5 μM arginine in PAO1 wild-type (left panel) and 1005
PAO1.ΔargB (right panel). Measurements were taken every hour at B) OD600 for absorbance and 1006
C) red fluorescence (mCherry expression) at excitation 580 nm and emission 610 nm. B, C) The 1007
first significant timepoint of the signal over time point 1 is indicated for the relA-mCherry (*) and 1008
cysM-mCherry (+). The occurrence of the first significant difference between the two constructs 1009
indicated with #. D, E) The cysM-relA promoters were fused to the mCherry reporter gene and 1010
grown over a period of 16 hours in BM2 broth in a 96-well plate with continuous shaking (567 1011
cpm) with 6.5 and 12.5 μM arginine in PAO1 (left panel) and PAO1.ΔargB (right panel). 1012
Measurements were taken every hour at D) OD600 for absorbance and E) red fluorescence 1013
(mCherry expression) at excitation 580 nm and emission 610 nm. B-E) The values are the means 1014
of three biological and 6 technical replicates; error bars represent the standard error of the mean. 1015
Comparisons were analyzed with a two-tailed Student’s t-test and significant differences indicated 1016
at p < 0.05. 1017
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1018 SI Appendix, Fig S2: Deletion of relA and spoT had no obvious impact on growth in three 1019
different media. A) King’s B (KB). B) Double yeast tryptone (2xYT). C) Synthetic Cystic 1020
Fibrosis Medium (SCFM). A minor effect was observed in the rate of death during stationary phase 1021
only in SCFM minimal medium whereby PAO1 relAspoT died at a slower rate which could be 1022
complemented by introduction of the relA gene. A-C) Bacterial growth in a 96-well plate with 1023
continuous shaking (567 cpm) for 16 h at 37°C. OD600 absorbance was measured every hour. 1024
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1025 SI Appendix, Fig S3: Scatter plot of the correlation between RNA-Seq and qRT-PCR 1026
expression for genes around the threshold cut-off (FC ±1.5). Verification of RNA-Seq data was 1027
performed on the same RNA for the following 15 genes: PA0652, PA0675, PA0762, PA0893, 1028
PA1270, PA1282, PA1430, PA2237, PA2426, PA3385, PA3410, PA3462, PA3899, PA5261, and 1029
PA5344. 1030
y = 0.8318x + 0.1713
R² = 0.6594
-2.00
-1.00
0.00
1.00
2.00
3.00
4.00
-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00
Rel
ativ
e ex
pre
ssio
n i
n q
RT
-PC
IR
Relative expression in RNA-Seq
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1031 SI Appendix, Fig S4: Phenotypic confirmations of the P. aeruginosa PAO1 wild-type, the 1032
stringent response mutant and complements. A) Swarming on KB agar plates at 37°C for 24 h. 1033
B) Rhamnolipid production (yellow arrows indicate ring formation) at 37°C for 48 h. C) 1034
Adherence to 96-well polypropylene plates in dYT, KB, and SCFM medium at room temperature 1035
for 1 h. Data normalized to wild-type adherence. D) Pyoverdine production normalized to wild-1036
type production. E) Pyocyanin production. A-E) * indicates p-value < 0.05 compared to wild-type. 1037
Experiments were performed at least three times. Error bars indicate ± standard error. 1038
1039
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1040 SI Appendix, Fig S5: Manually curated list analysis of the differential expressed genes 1041
between the P. aeruginosa PAO1 stringent stress response mutant ΔrelAspoT and wild-type 1042
strain. Gene ratio and set size for a number of curated gene lists from various sources (see Dataset 1043
3) were calculated using Fisher’s Exact Test with multiple test correction (Benjamin-Hochberg 1044
method). Significant results were determined using an adjusted p-value threshold of 0.05. Gene 1045
ratio represents the proportion of total genes assigned to a term that are differentially expressed. 1046
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1047 SI Appendix, Fig S6: Verification of functional RelA-YFP and SpoT-GFP in the PAO1 1048
stringent stress response mutant ΔrelAspoT. Swarming on BM2 agar plates at 37°C for 24 h. 1049
Representative images of the swarming-deficient stringent stress response mutant (left), and the 1050
complementation with RelA-YFP (middle) or SpoT-GFP (right) are shown. Experiments were 1051
repeated three times. 1052
1053
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1054
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