1

Basal Level Effects of (p)ppGpp in the Absence of Branched Chain Amino Acids in 1

Actinobacillus pleuropneumoniae 2

3

Gang Lia, Qian Zhao,

a Tian Luan,

a Yangbo Hu,

b Yueling Zhang,

a Ting Li,

a Chunlai 4

Wang,a Fang Xie,

a Wanjiang Zhang

a, Paul R. Langford,

c Siguo Liu

a* 5

6

aState Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, 7

Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 8

Harbin, China 9

bKey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, 10

Chinese Academy of Sciences, Wuhan, China 11

cSection of Paediatric Infectious Diseases, Department of Infectious Disease, Imperial 12

College London, St. Mary’s Campus, London, United Kingdom 13

14

Running Head: Effects of (p)ppGpp in Actinobacillus pleuropneumoniae 15

16

#Address correspondence to Siguo Liu, liusiguo@caas.cn 17

18

Gang Li, Qian Zhao and Tian Luan contributed equally to this work. Author order was 19

determined in order of joining experiment. 20

21

22

23

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JB Accepted Manuscript Posted Online 3 February 2020J. Bacteriol. doi:10.1128/JB.00640-19Copyright © 2020 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 26

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory 27

mechanism in bacterial pathogens, enabling adaptation to adverse environments and 28

linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs 29

of pigs, it's only known natural host. Pig lungs are known to have a low concentration of 30

free branched chain amino acids (BCAAs) compared to plasma. We had investigated the 31

role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae. Now, we 32

sought to determine whether (p)ppGpp was a trigger signal for the SR in A. 33

pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic 34

analyses of wild type (WT) and relAspoT double mutant (which does not produce 35

(p)ppGpp), we found that (p)ppGpp could repress de novo purine biosynthesis and 36

activate antioxidant pathways. There was a positive correlation between GTP and 37

endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology 38

and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the 39

biosynthesis of BCAAs were constitutively up-regulated regardless of the existence of 40

BCAAs without accumulation of (p)ppGpp beyond basal level. Collectively, our study 41

shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. 42

pleuropneumoniae. (p)ppGpp-mediated regulation in A. pleuropneumoniae is different to 43

that described for the model organism Escherichia coli. Further work will establish 44

whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved 45

among other veterinary pathogens, especially those in the Pasteurellaceae family. 46

47

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IMPORTANCE 48

(p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional 49

challenges. Here, we present a transcriptional and phenotypic differences of A. 50

pleuropneumoniae grown in different chemically defined media in the absence of 51

(p)ppGpp. We show that the deprivation of branch-chain amino acids (BCAAs) does not 52

elicit a change in the basal level (p)ppGpp, but this level is sufficient to regulate the 53

expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is 54

different to that of the model organism Escherichia coli, but similar to that found in some 55

Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp, but 56

also further validates the complexity and multiplicity of (p)ppGpp regulation in 57

microorganisms that occupy different biological niches. 58

59

KEYWORDS Actinobacillus pleuropneumoniae, Stringent response, BCAAs, (p)ppGpp, 60

GTP 61

62

INTRODUCTION 63

The alarmone (p)ppGpp was first identified 50 years ago as accumulating in response 64

to amino acid starvation in Escherichia coli (1). Mounting evidence has indicated that 65

(p)ppGpp is a key factor in bacterial physiology including rapid response to diverse 66

stresses, which profoundly affects cellular processes including transcription, replication 67

and translation, and is important for virulence (2, 3), differentiation and persistence (4-7). 68

The (p)ppGpp-induced starvation response is called the stringent response (SR), and this 69

signaling pathway is utilized by the vast majority of bacterial species to mediate the stress 70

response to amino acid starvation (8-10). The RelA/SpoT Homologue (RSH) proteins, 71

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which synthesize or degrade (p)ppGpp, have been found throughout the tree of life (11, 72

12). There are three main groups of enzymes in the RSH superfamily, which are long-73

RSH enzymes, small alarmone synthetases (SAS) and small alarmone hydrolases (SAH). 74

In most β- and γ-proteobacteria , (p)ppGpp is synthesized by the monofunctional 75

synthetase RelA and degraded by the bifunctional protein SpoT (13). While there is good 76

data on the role of (p)ppGpp in growth regulation with the model microorganisms 77

Escherichia coli and Bacillus subtilis (14-21), there is comparatively less with bacterial 78

pathogens of veterinary origin. 79

Actinobacillus pleuropneumoniae is a γ-proteobacterium which is the causative agent 80

of porcine pleuropneumonia, a highly contagious respiratory disease that causes 81

significant economic losses throughout the worldwide porcine industry (22). Typically it 82

is isolated from the swine respiratory tract (23, 24) with acute and chronic disease caused 83

by A. pleuropneumoniae being associated with lung tissue damage. Field isolates are 84

classified into two biotypes dependent on their requirement for NAD, and further 85

subdivided into 18 serovars based on capsular antigens (25-27). Some major virulence 86

factors required for the development of clinical disease have been identified, e.g, the 87

RTX toxins, iron-acquisition proteins, and components of pathways required for survival 88

in anaerobic environments (reviewed in (23)). In addition, an A. pleuropneumoniae 89

knockout mutant of ilvI, which encodes the enzyme acetohydroxyacid synthase (AHAS) 90

required for branched-chain amino acid (BCAA) biosynthesis, was attenuated for 91

virulence in pigs (28, 29), reflecting that the BCAA content in porcine pulmonary 92

epithelial lining fluid is only 10 to 17% of the concentration in plasma (28, 29). Whether 93

the low availability of BCAAs in the porcine lung is a signal that triggers the SR in A. 94

pleuropneumoniae is unknown, and there is relatively little data from other pathogens 95

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(30-32). The aim of this study was to determine whether there was a link between BCAA 96

availability and the SR in A. pleuropneumoniae, and the effect on phenotypic 97

characteristics. 98

In our previous work, we showed that deletion of A. pleuropneumoniae relA alone 99

had phenotypic effects, e.g. on the ability to form biofilms (33). In this study, we have 100

additionally constructed a relAspoT double mutant ((p)ppGpp0) mutant that does not 101

produce (p)ppGpp, and compared the global transcriptional profile with wild-type (WT) 102

in chemically defined medium (CDM), and determined phenotypic characteristics. The 103

transcriptome results strongly indicate that the basal level of (p)ppGpp blocks de novo 104

synthesis of purine nucleotides, and there were significant changes in gene expression in 105

antioxidant metabolic pathways, and the balance of GTP homeostasis and H2O2 content 106

was altered. Thus, in A. pleuropneumoniae, the basal level of (p)ppGpp appears to 107

constitutively regulate the transcription of genes involved in biosynthesis of BCAAs and 108

purine. Interestingly, the direction of regulation of these two pathways is opposite to that 109

described in the model organism E. coli (34). Thus researchers are advised to be cautious 110

in extrapolating data obtained with model microorganisms to their specific pathogen of 111

interest. 112

113

RESULTS 114

The growth characteristics of WT and (p)ppGpp0 are the same in rich medium 115

but (p)ppGpp0

growth is impaired upon sudden starvation. Firstly, we tested the 116

ability of WT and mutant strains to synthesize (p)ppGpp. Quantification of extracted 117

nucleotides separated by thin-layer chromatography (TLC) (Fig. 1A) indicated that no 118

detectable (p)ppGpp was produced in the (p)ppGpp0

mutant. No significant difference in 119

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the growth rates and viability was observed between WT and (p)ppGpp0 (Fig. 1B, 1C). 120

However, when the (p)ppGpp0 was subjected to sudden amino acid starvation its viability 121

decreased sharply, unlike the WT. Within 120 min of amino acid starvation, only 8% of 122

(p)ppGpp0

cells had survived, compared to 160% of WT cells (Fig. 1D). The result 123

indicates that (p)ppGpp is required for WT to survive amino acid starvation i.e. nutrient 124

stress. 125

(p)ppGpp is required for the maintenance of proper growth and viability in the 126

absence of BCAAs. That the alarmone (p)ppGpp has a major role in regulating growth of 127

bacteria had been reported elsewhere (4, 5, 16). In order to determine the precise amino 128

acid requirement of A. pleuropneumoniae, we examined its growth in 20 media i.e. 129

normal CDM and CDM lacking each amino acid. The results indicate that (p)ppGpp0 had 130

a strong requirement for isoleucine and valine, and a weaker requirement for leucine for 131

growth under the conditions tested (Fig. S1). Growth curve and viability data from both 132

WT and the (p)ppGpp0 mutant in CDM-BCAA medium, suggests that the WT grows 133

better in some amino acid deleted medium compared to complete CDM (Fig. 2A, 2C). 134

However, loss of (p)ppGpp resulted in decreased growth and viability regardless of the 135

existence of BCAA (Fig. 2B, 2D). To determine whether BCAA acted as a starvation 136

signal sufficient enough to be sensed by A. pleuropneumoniae, we determined the 137

formation of (p)ppGpp of WT grown in media. Our results show that deleting BCAA 138

from CDM did not increase the production (p)ppGpp, the basal level in A. 139

pleuropneumoniae WT remained unchanged (Fig. 2E). 140

(p)ppGpp production is associated with bacterial morphological changes. We 141

determined whether (p)ppGpp was associated with changes in A. pleuropneumoniae 142

morphology as determined by transmission electron microscopy. In nutrient-rich TSB 143

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medium, the morphology of WT and (p)ppGpp0 were similar (Fig. 3). However, in CDM 144

and CDM-BCAA deleted medium, there were differences in bacterial morphology. The 145

WT structure was similar in rich medium to that observed in CDM BCAA-deleted. In 146

contrast, in CDM BCAA-depleted media, (p)ppGpp0 had fewer septa when compared to 147

WT. Additionally, the smoothness of the surface appeared decreased, the cells were 148

longer, and some bacteria were severely deformed. Such changes were most apparent 149

when (p)ppGpp0 was cultured in CDM-val. These results suggest an association between 150

phenotype and the (p)ppGpp-mediated SR. 151

Availability of (p)ppGpp affects A. pleuropneumoniae virulence. In order to 152

assess the contribution of (p)ppGpp to virulence, we determined the LD50 of the WT and 153

(p)ppGpp0 strain for larvae of Galleria mellonella (35). The result suggested that the 154

absence of (p)ppGpp weakens the virulence of the (p)ppGpp0 mutant by two fold (Fig. 155

S2). 156

Overview of transcriptome differences between WT and (p)ppGpp0 grown in 157

CDM or CDM-BCAA. To gain a more thorough understanding of the genetic basis, 158

transcriptome analysis was done to delineate the mechanisms underlying (p)ppGpp-159

mediated function under BCAA-deleted conditions. Firstly, we compared the 160

transcriptome of WT which was cultured with or without BCAA in CDM medium (Fig. 161

4A upper table). Unexpectedly there was little difference in transcriptome. Similar results 162

were obtained with the (p)ppGpp0 mutant (Fig. 4A upper Table). This result supports the 163

observation that the WT did not induce accumulation of (p)ppGpp in the absence of 164

single BCAAs. Although the absence of BCAAs had little effect on the transcriptome of 165

WT and (p)ppGpp0, the response to (p)ppGpp was quite different when WT and the 166

(p)ppGpp0 mutant were grown in the same medium. When comparing (p)ppGpp

0 to WT 167

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in normal (see Data set S1 in the supplemental material), there were 114 up-regulated and 168

111 down-regulated genes (Fig. 4A bottom Table). There were 209 up-regulated and 89 169

down-regulated genes compared to WT in the (p)ppGpp0 mutant when they were cultured 170

in CDM-leucine medium (Fig. 4A bottom Table). There were 118 up-regulated and 102 171

down-regulated genes compared to WT in the (p)ppGpp0 mutant when they were cultured 172

in CDM-isoleucine medium (Fig. 4A bottom Table). There were 104 up-regulated and 68 173

down-regulated genes compared to WT in the (p)ppGpp0 mutant when they were cultured 174

in CDM-valine medium (Fig. 4A bottom Table). 175

Among these differentially expressed genes, there were 56 up-regulated and 36 176

down-regulated in common, compared to the WT, under CDM and CDM-BCAAs (Fig. 177

4B). Differentially expressed genes were annotated according to the COG database and 178

are summarized in Fig. 4C. The complete list of these genes is shown in Data set S1 in 179

the supplemental material. 180

The gene expression profiles identified some common and some different processes 181

controlled by (p)ppGpp. We found abundant genes associated with the intracellular 182

ribonucleoprotein complex, ribosome biogenesis, membrane proteins, and the purine 183

biosynthesis pathway were all up-regulated in the (p)ppGpp0

mutant. In contrast, some 184

genes involved in metal transport, and antioxidant pathways were down-regulated. The 185

expression of all these genes was in the opposite direction to that found in WT. This 186

suggests that the lack of (p)ppGpp leads to a transcriptionally relaxed state in the 187

(p)ppGpp0

mutant. Finally, the transcriptional mis-regulation results in the slow growth 188

and decreased viability of the (p)ppGpp0

mutant. 189

We also found that the genes encoding the acetohydroxy acid synthase (AHAS) 190

isozyme (ilvIHM, B838_RS0101105, B838_RS0101110, B838_RS0108605), catalyzing 191

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the first step in the biosynthesis of the BCAAs, were all inhibited in (p)ppGpp0 regardless 192

of the presence or absence of BCAA. 193

Additionally, the zapB (B838_RS0106965) and ftsA (B838_RS0102230) genes, 194

which are required for cell division and proper Z-ring formation, were up-regulated in 195

(p)ppGpp0

except in CDM-valine medium. This may explain for filamentous phenotype 196

of (p)ppGpp0 in the absence of valine in CDM. 197

A response regulator (B838_RS0102415) of a two-component system involved in the 198

regulation of nitrate metabolism was also down-regulated. Two genes (B838_RS11255, 199

B838_RS0100090) that are associated with lysine biosynthesis were inhibited more than 200

4-fold. Another three hypothetical proteins (B838_RS0105755, B838_RS0105760, 201

B838_RS0100880) were also significantly down-regulated. All these different genes may 202

also be putative targets of (p)ppGpp. 203

The purine nucleotide de novo biosynthesis pathways are activated in (p)ppGpp0 204

including in

CDM medium. Enrichment analysis among the 56 commonly up-regulated 205

genes in the (p)ppGpp0 mutant indicated ten genes (purF, purD, purN, purL, purM, purK, 206

purC, purH, purE, guaB) involved in the de novo purine biosynthesis and salvage 207

pathways, to be significantly up-regulated (Fig. 5). Their action results in synthesis of 208

inosine 5’-monophosphate (IMP) from phosphoribosyl pyrophosphate (PRPP), which is a 209

key intermediate in the synthesis of purine nucleoside triphosphates GTP and ATP. 210

However, there were no detectable differences in gene expression of guaA, gmk, and 211

hprT. GuaA and Gmk are involved in downstream steps pathway from IMP to GTP. 212

HprT (hypoxanthine phosphoribosyltransferase) was a key enzyme of salvage pathway, 213

which converts purines with similar chemical structure to GMP and IMP respectively. 214

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Our results demonstrated that there was gene repression upstream in the pathways 215

necessary for IMP biosynthesis. Combining previous observations, (p)ppGpp may affect 216

multiple layers of regulation at both the transcriptional level directly or possibly via 217

posttranscriptional control. Our data suggest that in the absence of the relA and spoT 218

genes in the (p)ppGpp0 mutant the metabolic flux progresses towards the accumulation of 219

GTP, and this results in the disruption of GTP homeostasis directly. Finally, we identified 220

the promoter motif associated with regulation of genes involved in the A. 221

pleuropneumoniae purine biosynthesis pathway (Fig. 6, Data set S2). The results indicate 222

that these promoter motifs contain more A and T nucleotides compared to (p)ppGpp 223

repressed promoters reported in E. coli, is similar to (p)ppGpp activated promoters (34). 224

This suggest the sequence preference recognized by the transcription factor of A. 225

pleuropneumoniae is different to that in E. coli. 226

The Reactive Oxygen Species (ROS) metabolic pathways are repressed in 227

(p)ppGpp0. Among the genes that were commonly down-regulated in (p)ppGpp

0 228

compared to WT after growth in CDM and CDM-BCAAs, most were classified into 229

amino acid, nucleotide, and carbohydrate metabolism, and environmental information 230

processing (Fig. 4C). Four important genes that are related to antioxidants were 231

substantially down-regulated, i.e., superoxide dismutase (Fe-Mn family) 232

(B838_RS0109035, log2=3.12), thioredoxin-disulfide reductase (B838_RS0108535, 233

log2=3.12), 2-Cys peroxiredoxin (B838_RS0110750, log2=3.12), and oxidoreductase 234

(B838_RS0107845, log2=3.12). The down-regulation of antioxidant genes would lead to 235

an increase in ROS levels in vivo. ROS are formed as a natural byproduct of the normal 236

metabolism of oxygen and have important roles in cell signaling and homeostasis. A. 237

pleuropneumoniae is a facultative anaerobe, but superoxide radicals and hydrogen 238

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peroxide (H2O2) can be generated during aerobic amino acid metabolism (36). ROS are 239

considered a primary source of intracellular oxidative stress, and can cause significant 240

damage to cells. Our transcriptome results suggest that transcriptional inhibition of 241

superoxide dismutase in the (p)ppGpp0

mutant will most likely result in an increase of 242

exposure to superoxide radicals, which in turn will affect cell growth. 243

The levels of H2O2 supports the altered transcriptome. The above observations let 244

us to reason that down-regulation of expression of antioxidant genes would result in H2O2 245

accumulation. To investigate whether (p)ppGpp could affect the production of H2O2, we 246

compared the intracellular concentrations of H2O2 of WT and (p)ppGpp0 in CDM and 247

CDM-BCAA media. The results show that there is more than 100-fold increase in 248

cytoplasmic H2O2 production in (p)ppGpp0

compared to WT (Fig. 7). H2O2 content is 249

inversely related to CFU. We also found that WT produced less intracellular H2O2 when 250

BCAA is absent from the normal CDM medium. The increased H2O2 production in the 251

(p)ppGpp0 mutant suggests that the ROS balance was significantly negatively affected, 252

and may be one of the main reasons for the decrease of viability in stationary phase. This 253

result also suggested that loss of (p)ppGpp impairs protection against superoxide-254

mediated toxicity during stationary phase. 255

The relationship between GTP/H2O2 level and viability. To test the causal 256

relationship between GTP levels and viability, we treated cells with the GMP synthase 257

(GuaA) inhibitor decoyinine to inhibit GTP biosynthesis, and found that the growth and 258

viability of both WT (Fig. 8A, 8C) and (p)ppGpp0 (Fig. 8B, 8D) were inhibited. 259

However, decoyinine seemed to delay the rapid decline in the number of viable cells of 260

(p)ppGpp0

in the stationary phase (Fig. 8D). In contrast, guanosine (5 mM) addition to 261

CDM medium, which increases GTP levels by the purine salvage pathway, significantly 262

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enhanced growth of WT in stationary phase (Fig. 8C). The survival of (p)ppGpp0 263

decreased compared to WT strain (Fig. 8D). There appears to be a positive correlation 264

between the GTP level and growth for WT. However, the viability of (p)ppGpp0 in 265

stationary phase showed a negative correlation with GTP level. At the same time, high 266

GTP levels reduced the accumulation of H2O2 in WT (Fig. 8E). This relationship seems 267

to be broken due to the lack of (p)ppGpp in the (p)ppGpp0 mutant. 268

269

DISCUSSION 270

In our previous study, we evaluated the role of the relA gene in regulating biofilm 271

formation and viability (33). In this study, in order to exclude the synthase activity of 272

SpoT which can produce (p)ppGpp in response to diverse stress signals (37), we 273

constructed a A. pleuropneumoniae relAspoT double mutant. We found that there was no 274

significant difference in the growth rate and viability of WT and the (p)ppGpp0

mutant in 275

nutrient-rich medium. Subsequently, we cultured WT and (p)ppGpp0

strains in media 276

with or without BCAA. Under such growth conditions, the growth rate, morphology and 277

viability of the (p)ppGpp0 mutant were affected severely. 278

To explain the observed phenomena, we carried out transcriptional analyses, and 279

found that presence or absence of BCAAs had little effect on the transcriptomes of WT or 280

the (p)ppGpp0 mutant when cultured in different media, supporting the growth 281

phenotypes observed in this study. 282

However, significant changes were seen when the transcriptomes of the WT and 283

(p)ppGpp0

mutant grown in the same medium were compared. Ten genes involved in de 284

novo and salvage pathway of purine nucleotide synthesis were repressed in the WT. In 285

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other bacteria, the gmk and hprT genes have been proved that its regulation occurs 286

posttranscriptionally (38-41), (p)ppGpp could inhibit Gmk and HprT directly, this may 287

explain why there are no change in transcript level for these two genes. Recently, some 288

putative (p)ppGpp targets have been identified in E. coli (42, 43). These include PurF, 289

which catalyzes the first step of GTP biosynthesis pathway, and the proteins encoded by 290

the purC and guaB genes, both differentially regulated in this study. In general, except 291

for the guaA, gmk and hprT genes which were not differentially expressed, the 292

transcription of genes of the GTP metabolic pathway were all highly down-regulated in 293

the WT, most likely through direct interaction between RNA polymerase (RNAP) with 294

(p)ppGpp (34). Our results indicate that a change in GTP level can indirectly affect the 295

growth, viability and virulence of A. pleuropneumoniae. Whether physiological 296

characteristics could be affected by the GTP level were not identified in E.coli, but GTP 297

is essential for survival of B. subtilis (38). In addition, we found the promoter sequence 298

features of the equivalent operon involved in this pathway in A. pleuropneumoniae was 299

different from that in E.coli (34). More importantly, inhibition of the operon can be 300

achieved at a basal level of (p)ppGpp. Future work will focus on cellular changes 301

associated with above basal levels of (p)ppGpp. 302

Like B. subtilis, changes in GTP level were associated with growth affects in the 303

(p)ppGpp0 mutant. However, the purine regulatory mechanism appears to be different to 304

that in B. subtilis, where the necessary 10 genes are in one operons (44). In A. 305

pleuropneumoniae the 10 genes are instead spread throughout the genome. In E. coli, 306

(p)ppGpp typically works in conjunction with DksA, a small transcription factor, to alter 307

the transcription of many genes by binding to RNAP. Comparison of the amino acid 308

sequences of DksA from A. pleuropneumoniae and E. coli identified that they share more 309

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than 64% identity. Additionally, the proposed two ppGpp binding sites of RNAP of E. 310

coli (45) are also conserved in RNAP from A. pleuropneumoniae. Although another 311

binding site of Region 2 β’ subunit （R362 ） had been replaced by H363 in A. 312

pleuropneumoniae (data not shown), it seems likely that (p)ppGpp is binding 313

RNAP/DksA to regulate transcription directly. In other bacteria, (p)ppGpp does not 314

directly affect RNAP (4, 5). One possibility considered was that whether A. 315

pleuropneumoniae possesses a homologue of the global regulatory factor CodY found in 316

Gram-positive bacteria (46-48). BLAST searches did not identify a CodY homolog or 317

ortholog in the A. pleuropneumoniae genome. Despite nearly five decades of study, 318

precisely how (p)ppGpp regulates cell growth remains poorly understood because the 319

complete set of target proteins is unknown. Although, some proteins have been reported 320

to bind or be inhibited by ppGpp in vitro (42), the physiological relevance of these targets 321

remains largely untested (with the exception of RNA polymerase (RNAP)(49) and PurF 322

(42) from E. coli, HprT (40) and GMK (41) from B. subtilis ). 323

Similar to what is seen in B. subtilis, disruption of GTP homeostasis in an equivalent 324

B. subtilis (p)ppGpp0 mutant results in metabolic changes and decreased cell viability, 325

even in the absence of starvation (38). Although, we demonstrated that (p)ppGpp is 326

associated with inhibition of transcription of enzymes involved in GTP biosynthesis in A. 327

pleuropneumoniae, we were uncertain if this would lead to dysregulation of GTP 328

homeostasis and changes in viability. So, to decrease or increase GTP levels transiently, 329

the GMP synthetase (GuaA) inhibitor decoyinine or guanosine, which can be converted 330

to GTP via the purine salvage pathway, were added to CDM medium. When the 331

intracellular GTP level was reduced by adding decoyinine, the growth of both WT and 332

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(p)ppGpp0 were decreased. The growth of (p)ppGpp

0 in stationary phage was elevated 333

sharply by adding guanosine, suggesting that GTP content was important for growth and 334

viability of A. pleuropneumoniae. Although, the underlying mechanism of death by 335

reducing GTP content is unclear, we demonstrated a negative correlation between GTP 336

and endogenous hydrogen peroxide content appears to exist, and this relationship seemed 337

to depend on the basal level of (p)ppGpp. 338

Genome-wide effects on transcription from ppGpp in E. coli have shown that the 339

basal level of ppGpp can down-regulate the transcript of genes involved in BCAA 340

biosynthesis, and up-regulate the transcription of genes involved in the purine 341

biosynthesis pathway, while after accumulation of ppGpp beyond the basal level, the 342

genes involved in these two pathways were regulated in opposite directions (34). This is 343

consistent with the observation in E. coli that expression of the BCAA operons can be 344

achieved by elevation of the basal ppGpp levels (32). In contrast, such regulation is 345

possible with only a basal level of (p)ppGpp in A. pleuropneumoniae, with the 346

transcription of genes involved in BCAA and purine biosynthesis being up-regulated and 347

down-regulated, respectively. This may result from the long-term adaptation of A. 348

pleuropneumoniae to the BCAA restricted environment of the lung, analagous to 349

adaptation of Helicobacter pylori in humans which has lost a large number of genes 350

associated with de novo synthesis of purine nucleotide in the stomach niche (44). 351

Collectively, although we have a relatively good understanding of the underlying 352

mechanisms by which (p)ppGpp modulates gene expression in the model organisms B. 353

subtilis and E. coli, much less is known about the process in other species that ordinarily 354

reside in different environmental niches. Our study indicates that A. pleuropneumoniae 355

(p)ppGpp-mediated regulation show differences to that of the model organism E. coli, 356

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and important for virulence, and further work will establish whether the results can be 357

applied to other veterinary pathogens, especially those in the Pasteurellaceae family. 358

MATERIALS AND METHODS 359

Bacterial strains, media, and growth conditions. The bacterial strains, primers and 360

plasmids used in this study are described in Table S1. The A. pleuropneumoniae S-8 361

strain and mutant were cultured in Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB) 362

(Becton Dickinson, Franklin Lakes, NJ, USA) or CDM supplemented with 10 μg/ml 363

NAD (50). The BCAAs were deleted from CDM when required. The ∆relA∆spoTApp 364

double mutant (named (p)ppGpp0 mutant) was cultured in the presence of kanamycin (50 365

μg/ml) and erythromycin (5 μg/ml). E. coli β2155 (∆dapA) was cultured in DifcoTM

LB 366

Broth, Miller (Luria-Bertani) supplemented with 1 mM diaminopimelic acid (DAP) 367

(Sigma-Aldrich, St. Louis, MO, USA). Bacteria were treated with arginine hydroxamate 368

(RHX, 0.5 mg/ml) to mimic amino acid starvation by depleting charged arginine-tRNAs, 369

or with decoyinine (Dec, 50 μg/ml) to inhibit the GTP biosynthesis, or with guanosine 370

(Guo, 5mM) to increase intracellular GTP pools via the salvage pathway. All bacteria 371

were cultured at 37°C. 372

Construction of (p)ppGpp0 mutant. Deletion of the relA and spoT genes in A. 373

pleuropneumoniae was performed as described previously (50, 51). Firstly, the relA gene 374

was replaced by a kanamycin resistant gene to construct ∆relAApp mutant, followed by 375

spoT gene was replacement of the spoT gene with an erythromycin resistance gene. The 376

final ∆relA∆spoTApp mutant was named (p)ppGpp0 mutant. 377

Detection of intracellular (p)ppGpp. For proving (p)ppGpp0 mutant had lost the 378

ability to synthesize the (p)ppGpp, the WT and (p)ppGpp0

mutant were streaked onto 379

TSA plates, and a single colony was used to inoculate TSB which was grown for 12 h. 380

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Subsequently, the culture was diluted to an OD600 of 0.2 with fresh TSB, and transferred 381

to fresh TSB for additional 2 h. Then, 1 ml of culture (WT and (p)ppGpp0 mutant) were 382

pelleted by centrifugation at 10,000 g for 5 min, and washed once with 1 ml MOPS and 383

re-suspended in 250 μl MOPS, [32

P]-H3PO4 (Perkin Elmer) was added to 100 μCi/ml, and 384

the culture labeled for 1 h at 37°C. For the BCAA deprivation assay, the WT was culture 385

in CDM overnight and transferred to fresh CDM for additional 2 h. After that, 1 ml of 386

culture was pelleted and washed once with 1 ml normal CDM, and then the pelleted 387

strains were re-suspended in MOPS as control, CDM and CDM with single BCAA 388

deleted medium, and incubate with 100 μCi/ml [32

P]-H3PO4 for another 1 h at 37°C. The 389

intracellular (p)ppGpp was extracted and detected as performed previously (33, 52). 390

Briefly, 50 μl labeled culture were mixed with an equal volume of 2 M formic acid and 391

placed on ice for at least 15 min. The mixture was centrifuged for 5 min at 16,000 g, and 392

3 μl of the supernatant were spotted directly onto polyethyleneimine (PEI) cellulose thin-393

layer chromatography plates (Sigma), dried, and developed in 1.5 M KH2PO4 for 2.5 h. 394

Nucleotides were visualized by autoradiography. 395

Growth experiments. The growth curve experiments were performed in CDM and 396

CDM-BCAA medium. The initial optical density at 600 nm (OD600) of all cultures was 397

set at 0.02. Growth of three biological replicates for each strain was measured. The 398

cultures were incubated at 37°C, and aliquots were regularly withdrawn every 1 hour and 399

OD600 measured using an Eppendorf spectrophotometer (Hamburg, Germany). All the 400

growth curves were presented as log2(OD600) vs time (53). The μmax was got by 401

comparing the slope (μ) of different adjacent time point. Colony forming units (CFU) 402

were determined by counting serial dilutions on TSA plates that had been incubated 403

overnight. 404

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Scanning electron microscopy. The pellets of WT and the (p)ppGpp0 mutant were 405

harvested by centrifugation at 10,000 g after growth for 12 h at 37 °C, and scanning 406

electron microscopy performed as described previously (33). 407

Galleria mellonella infection. Assessment of the comparative virulence of WT and 408

(p)ppGpp0 mutant strains for G. mellonella larvae was performed as described previously 409

(7). Briefly, groups of 10 larvae, ranging from 200 to 300 mg in weight were randomly 410

chosen and injected with 10 μl dilutions (1x106~1x10

9 CFU). Groups injected with PBS 411

buffer were used as negative controls. After injection, larvae were kept at 37°C, and 412

survival was recorded at selected intervals. Experiments were performed independently 413

three times with similar results. 414

RNA-sequencing analysis. The WT and (p)ppGpp0 mutant were grown to log-phase 415

(4 h) in CDM and single BCAA deleted medium. The cells were collected at 4°C, and 416

washed twice with precooled PBS buffer, and the pellets were frozen in liquid nitrogen. 417

Subsequently, the samples were sent to Beijing Genomics Institute (BGI)-Shenzhen in 418

China (http://www.genomics.cn/index) in dry ice for sequencing. The RNeasy kit 419

(Qiagen) and Ribo-Zero™ rRNA Removal Kit (EPICENTRE Biotechnologies) was used 420

to isolate RNA and remove rRNA. The Nanodrop ND-1000 spectrophotometer 421

(NanoDrop,Wilmington, DE, USA) was used to quantify RNA. Total 20 μg of RNAs for 422

both WT and (p)ppGpp0 mutant were pooled for cDNA library construction. The cDNA 423

libraries were constructed according to Illumina’s protocols and sequenced using the 424

Illumina HiSeq 2000 platform. This experiment was done on three biological replicates. 425

The raw RNA-seq data have been deposited in ArrayExpress (EBI, 426

https://www.ebi.ac.uk/arrayexpress/) and are accessible through accession number E-427

MTAB-8514. 428

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Differential expression analysis. The raw sequence reads were filtered using the 429

Illumina pipeline. Firstly, the reads with adaptor contamination and low-quality or only 430

one copy were excluded from the analysis. And then, the remaining clean reads were 431

mapped to the reference sequence of A. pleuropneumoniae S8 (Genbank accession No. 432

ALYN00000000.1). The number of reads for each genes was determined and normalized 433

between the libraries, and then the ratio of reads between WT and (p)ppGpp0 mutant was 434

calculated. The differentially expressed genes which have a false discovery rate (FDR) 435

threshold of 0.01 were identified as previously described (54, 55). The value of log2Ratio 436

≥1 and FDR≤0.001 was set as threshold for significant differences in gene expression. 437

Gene annotation was done according to A. pleuropneumoniae S8 reference sequence 438

(Genbank accession No. CP001091.1) and A. pleuropneumoniae serovar 7 strain AP76 439

reference sequence (Genbank accession No. CP001091.1) using the BlastN program 440

(http://blast.ncbi.nlm.nih.gov/). The GO annotations were done by the Blast2GO program 441

(http://www.geneontology.org). Pathway assignments were done according to the Kyoto 442

Encyclopedia of Genes and Genomes pathway database (http://www.genome.jp/kegg). 443

Promoter sequence motif analysis. The homologous sequence and operon of the 444

target genes involved in the purine biosynthesis pathway were searched by ProOpDB 445

(56). The co-linearity relationship between of A. pleuropneumoniae was analyzed by 446

Mauve to get the operon distribution of target genes 447

(http://darlinglab.org/mauve/mauve.html). The target genes were Blasted according to the 448

reference genome sequence by Diamond to get the information about gene ID and 449

arrangement location (57). Finally, promoter predictions were done according to the 450

sequence which had been extracted from the 500 bp upstream of the first gene of the 451

operon of target genes (58). 452

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H2O2 measurements. The production of H2O2 was measured using an H2O2-453

peroxidase assay kit (Life Technologies, A22188) (39). The WT and (p)ppGpp0 mutant 454

were grown to stationary phase (8 h) in different medium, harvested by centrifugation at 455

4°C, and washed twice by assay buffer (50 mM Tris-HCl at pH 7.4), and the pellets were 456

resuspended in the assay buffer. The final reaction included equal volumes of cell 457

suspension and reaction mix (20 μM Amplex UltraRed, and 0.2 U ml-1

horseradish 458

peroxidase) in 96-well microtiter plates at 37°C for 30 min. The absorbance was read at 459

560 nm by a BioTek ELx808 spectrophotometer. To normalize absorbance by CFU, the 460

cells aliquots from different cultures were serially diluted and plated onto TSA for colony 461

counting. 462

Statistical analysis. All statistical analyses were conducted with the GraphPad Prism 463

5 software. All data were collected from three biological replicates and expressed as the 464

arithmetic mean +/- standard deviation (SD). 465

466

467

ACKNOWLEDGMENTS 468

This work was funded by National Natural Science Foundation of China (No. 31672575, 469

31772757), Heilongjiang Province Natural Science Foundation (No.C2017078). Prof 470

Paul Langford was supported by the UK Biotechnology and Biological Sciences 471

Research Council (BB/S019901/1 and BB/K020765/1). We thank Dr. Zhai lei (China 472

National Research Institute of Food & Fermentation industries) for technical assistance 473

with the SEM experiments. The funders had no role in study design, data collection and 474

analysis, decision to publish, or preparation of the manuscript. 475

476

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477

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Genome Res 7:986-95. 639

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genes using false discovery rate controlling procedures. Bioinformatics 19:368-641

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Prokaryotic Operon DataBase. Nucleic Acids Res 40:D627-31. 644

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DIAMOND. Nat Methods 12:59-60. 646

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Metagenomic Sequences, p p. 61-78, In Metagenomics and its Applications in 648

Agriculture, Biomedicine and Environmental Studies (Ed RW Li). Nova Science 649

Publishers. 650

651

FIG 1 Synthesis of (p)ppGpp and growth profiles of the WT and (p)ppGpp0 grown in 652

rich medium. (A) Accumulation of (p)ppGpp in WT and (p)ppGpp0. Cells were labeled 653

with H3[32

P]O4 in MOPS under starvation conditions, the formic acid extracts of the cells 654

were subjected to TLC analysis as described in Materials and Methods. (B) Growth 655

curves of WT and (p)ppGpp0. Growth was monitored by OD600 at various time points. 656

(C) Cell viability of the WT and (p)ppGpp0. Overnight cultures were serial diluted with 657

fresh medium and spotted onto the TSA plate. (D) (p)ppGpp0 failed to survive sudden 658

starvation. WT and (p)ppGpp0 were treated with 0.5 mg/ml RHX for the indicated time 659

and plated on TSA. Percent survival was calculated by counting the number of colonies 660

and normalized to T=0. All data were shown as arithmetic means and standard deviations 661

from three replicates. 662

663

FIG 2 Growth and accumulation of (p)ppGpp in WT and (p)ppGpp0 in different growth 664

conditions (A, B) Growth curves of WT and (p)ppGpp0 in CDM and CDM-BCAA. The 665

value of OD600 was monitored at various time points. (C, D) The viability of WT and 666

(p)ppGpp0 in CDM and CDM-BCAA. Overnight cultures were serial diluted by fresh 667

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medium and spotted onto the TSA plate. (E) Determination of the production of 668

(p)ppGpp by WT in CDM and CDM-BCAA medium. Graphs A and B show arithmetic 669

means and standard deviations of results from at least three independent experiments. 670

671

FIG 3 Morphology of A. pleuropneumoniae WT and (p)ppGpp0

mutant grown in 672

different medium determined by scanning electron microscopy. 673

674

FIG 4 Transcriptome analysis of (p)ppGpp-dependent genes during grown in CDM or 675

CDM-BCAAs. (A) Schematic of differentially expressied genes affected by (p)ppGpp in 676

different media. (B) Venn diagrams showing the number of genes that are up-regulated 677

and down-regulated in a (p)ppGpp0 dependent manner in CDM and CDM-BCAA growth 678

conditions. (C) Functional categories of genes differentially expressed in (p)ppGpp0 679

compared with the WT in the absence of BCAA. The number of genes whose expression 680

is differentially expressed in (p)ppGpp0 compared with WT are presented according to 681

the functions assigned by the browser of CLRNA-Seq data analysis software. 682

683

FIG 5 Schematic of the pathways affected by (p)ppGpp. Starvation-induced changes in 684

the purine biosynthesis pathway. The purF, purD, purN, purL, purM, purK, purC, purE, 685

purH, purB, guaB, hprT, ndK and gmK are genes involved in the purine de novo and 686

salvage biosynthesis pathways. The genes with significant changes are colored: red 687

indicates more than two-fold up-regulation, grey indicates no detectable changes in 688

transcript level. 689

690

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Fig 6 Sequence characteristics of promoters regulated by transcript factor. Consensus 691

promoter elements (A). Sequence conservation in the -35 element (B, C), extended −10 692

region (D, E), −10 element (F, G), discriminator element (H, I), TSS region (J, K) and -693

10/-35 spacer length (L). Promoters of genes involved in purine biosynthesis pathway 694

were aligned to create sequence logos (B, D, F, H, J) and histograms of base distributions 695

(C, E, G, J, I, K, L) for each position. 696

697

FIG 7 Enhanced H2O2 production by the (p)ppGpp0

mutant. The cells of WT and 698

(p)ppGpp0

grown in CDM and CDM-BCAA were harvested and washed in PBS buffer. 699

The washed cell suspensions and medium were mixed with an equal volume of buffer to 700

determine cytoplasmic H2O2 production according to the instructions of H2O2-peroxidase 701

assay kit. 702

703

FIG 8 Growth, viability and H2O2 accumulation affected by adding decoyinine and 704

guanosine. Cells were grown to early log phase (2 h) in CDM medium and supplemented 705

with 5 mM guanosine and 50 μg/ml decoyinine. (A, B) The growth curves of WT and 706

(p)ppGpp0

were determined by monitoring the value of OD600 at various time points. (C, 707

D) The viability of WT and (p)ppGpp0

were determined by counting the CFU at various 708

time points. (E) The accumulation of H2O2 was determined by using the H2O2-peroxidase 709

assay kit. 710

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