aem accepts, published online ahead of print on 13...

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Dissection of the transcriptional regulator GlpR, the promoter 1

elements, and the post-transcriptional processing involved in the 2

fructose-induced activation of the phosphoenolpyruvate-dependent 3

sugar phosphotransferase system in Haloferax mediterranei 4

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Lei Cai1,2, Shuangfeng Cai1,2, Dahe Zhao1, Jinhua Wu1,2, Lei Wang1, 7

Xiaoqing Liu1,2, Ming Li1,2, Jing Hou1,2, Jian Zhou1, Jingfang Liu1, Jing 8

Han1, Hua Xiang1,* 9

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1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of 11

Sciences, Beijing 100101, China, 2University of Chinese Academy of Sciences, Beijing 100049, 12

China 13

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*To whom correspondence should be addressed. Tel: +86 10 6480 7472; Fax: +86 10 6480 7472; 16

E-mail: [email protected] 17

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Running Title: PTS regulation in haloarchaea 20

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AEM Accepts, published online ahead of print on 13 December 2013Appl. Environ. Microbiol. doi:10.1128/AEM.03372-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Abstract 23

Among all known archaeal strains, the phosphoenolpyruvate-dependent 24

phosphotransferase system (PTS) for fructose utilization is primarily used by 25

haloarchaea that thrive in hypersaline environments, whereas the molecular details of 26

the regulation of the archaeal PTS under fructose induction remain unclear. In this 27

study, we present a comprehensive examination of the regulatory mechanism of the 28

fructose-PTS (HFX_1559-HFX_1563) in the haloarchaeon Haloferax mediterranei. 29

With gene knockout-complementation, microarray analysis and ChIP-qPCR, we 30

revealed that GlpR (HFX_1565) is the indispensable activator, which specifically 31

binds to the PTS promoter (PPTS) during fructose induction. Further promoter 32

scanning mutation indicated that three sites located at the upstream of the H. 33

mediterranei PPTS, which are conserved in most haloarchaeal PPTS, are found to be 34

involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with 35

different 5’ UTR lengths were observed, and the promoter or 5’ UTR swap 36

experiments indicated that the shorter 5’ UTR was most likely generated from the 37

longer one. Notably, the translation efficiency of the transcript with this shorter 5’ 38

UTR was significantly higher, and the ratio of T8 (with the shorter 5’ UTR) to T17 39

increased during fructose induction, implying that a post-transcriptional mechanism is 40

also involved in PTS activation. With these insights into the molecular regulation of 41

the haloarchaeal PTS, we have proposed a working model of haloarchaea in response 42

to environmental fructose. 43

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Introduction 45

The phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS) 46

uses PEP as the phosphoryl donor to phosphorylate sugars for transport into cells 47

(1-2). A typical PTS contains five proteins, PtsI (or EI), HPr, PtsA, PtsB and PtsC. 48

Phosphotransfer from PEP to sugar is mediated by these five proteins in a cascade, 49

and the PtsC component at the end of the cascade couples phosphorylation with the 50

translocation of the specific sugars (3-4). The PTS is an important apparatus for sugar 51

uptake and degradation in bacteria, and most bacteria are shown to possess at least 52

one complete PTS (4). The PTS of bacteria can sense the primary metabolic or 53

environmental signal and turn on the uptake system (5-6). In response to the 54

environmental signal, the derivatives of sugars always serve as positive or negative 55

effectors, while the global or specific transcriptional regulators, in cooperation with 56

cAMP or primary metabolites, are involved in the complicated regulation of PTS via 57

direct binding to the promoter regions of PTS genes (2, 7). 58

In contrast to bacteria, the research of archaeal PTS has just received attention in 59

the past few years. The first report that archaea have PTS genes was published in 60

2006 based on the genome sequencing of the haloarchaeon Haloquadratum walsbyi 61

(8). Recent studies of the haloarchaeal genome sequences have indicated that many 62

haloarchaea may contain PTS genes (8-11), and 6 out of 24 haloarchaeal genomes 63

have a complete fructose-specific PTS gene cluster, including Haloterrigena 64

turkmenica, Halalkalicoccus jeotgali, Haloarcula hispanica, Haloarcula marismortui, 65

Haloferax volcanii and Haloferax mediterranei (11). Recently, a functional 66


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fructose-specific PTS has been identified in H. volcanii using genetic methods, and 67

fructose is shown to be able to up-regulate the transcription of this PTS gene cluster 68

(12), with the molecular details of the fructose-induced PTS activation remain unclear 69

in archaea. Interestingly, earlier research in H. volcanii indicates that a DeoR family 70

transcriptional regulator, GlpR, represses the expression of fructose and glucose 71

metabolic enzymes (2-keto-3-deoxy-D-gluconate kinase (KDGK) and 72

phosphofructokinase (PFK)) at the transcriptional level in H. volcanii when cells were 73

grown on glycerol (13). In addition, it was reported that glpR is cotranscribed with the 74

downstream phosphofructokinase gene (fruK) (13). The PTS gene cluster is located 75

just adjacent to glpR-fruK. As a putative transcriptional regulator which usually acts 76

as a transcriptional regulator in sugar metabolism in bacteria (14-15), GlpR is 77

probably involved in the transcriptional regulation of the PTS cluster. However, so far 78

as we know, the relationship between the GlpR and the PTS activation has not yet 79

been established in haloarchaea. 80

Recently, the genome of H. mediterranei was completely sequenced by our 81

laboratory (16). A genome-wide in silico analysis showed that the arrangement of 82

fructose metabolism-related genes, including the PTS system in H. mediterranei, is 83

identical to that of H. volcanii. However, in contrast to H. volcanii, H. mediterranei 84

can synthesize biodegradable polymers such as polyhydroxyalkanoates (PHA) from 85

many cheap carbon resources (17-19). Studying the regulation mechanism of PTS in 86

H. mediterranei is useful not only to compare the different gene regulation strategies 87

between bacteria and archaea, but can also result in a deeper understanding of the 88


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carbon sensing and utilization of this specific haloarchaeal PHA producer. In the 89

present study, a comprehensive investigation of the regulatory mechanism of the PTS 90

was performed in H. mediterranei. We demonstrated that GlpR is an indispensable 91

activator of the PTS gene cluster upon fructose induction via direct binding to the PTS 92

promoter region (PPTS). Interestingly, we also revealed an additional 93

post-transcriptional mechanism which could increase the translation efficiency of PTS 94

transcripts. Together, our results helped elucidate the complex and delicate 95

mechanisms of fructose-PTS regulation in the domain of archaea. 96

97

Materials and Methods 98

Strains and growth conditions. The strains used in this study are listed in Table S1. 99

Escherichia coli JM109 was used as the host for the cloning experiments (Novagen, 100

Madison, WI, USA) and was grown in Luria-Bertani medium at 37°C (20). Unless 101

otherwise noted, H. mediterranei DF50 (21) and the gene knockout mutants were 102

cultivated at 37°C in nutrient-rich AS-168L medium (22), and H. mediterranei strains 103

harboring expression plasmids were cultivated in AS-168SYL medium (AS-168L 104

without yeast extract) (22). The chemically defined medium (CDM, consisting of the 105

followings (per liter): 150 g NaCl, 20 g MgSO4 · 7H2O, 2 g KCl, 50 mg FeSO4 · 7H2O, 106

0.36 mg MnCl2 · 4H2O, 5 g NH4Cl, 15 g PIPES, pH 7.2) with different concentrations 107

of fructose or glucose was used to verify the utilization of the carbon source by H. 108

mediterranei mutant strains. When required, ampicillin, uracil and 5-FOA were added 109

to the media at a final concentration of 100 mg/L, 50 mg/L and 250 mg/L, 110


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respectively. 111

Gene knockout and complementation. In-frame deletion and complementation 112

strains were generated according to previously published protocols (21, 23). All the 113

primers used in this study are listed in Table S2, and the plasmids are listed in Table 114

S1. The transformation of H. mediterranei was performed according to the 115

polyethylene glycol-mediated spheroplast transformation method (24). The plasmid 116

sequences and mutant strains were verified by PCR and DNA sequencing. 117

RNA extraction, qRT-PCR and circularized RNA (CR)-RT-PCR. H. 118

mediterranei DF50 cells and the gene knockout mutants were cultured at 37°C in 119

AS-168L medium. When the OD600 reached 1.5, glucose or fructose was added to the 120

medium to a final concentration of 50 mM, and the cells were incubated for 45 min. 121

The sugar-induced cells (3 ml) were then immediately collected for RNA extraction 122

using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the 123

manufacturer’s instructions. An equal volume of TBSL buffer (consisting of the 124

followings (per liter): 150 g NaCl, 20 g KCl, 5 g MgSO4 · 7H2O, 100 mM Tris-HCl, 125

pH 7.1) (22) was added to the cells in the control group. To remove DNA 126

contamination, DNase I (Promega, Madison, WI, USA) digestion was performed on 1 127

μg of diluted RNA. 128

The specific primer pairs in Table S2 of the target DNA regions and suitable 129

concentrations of the cDNA templates or genomic DNA were used for quantitative 130

PCR (qPCR). The amplification and detection of target regions were performed on a 131

Rotor-Gene® Q real-time cycler (Qiagen, Valencia, CA) under a standard three-step 132


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PCR procedure [including initial denaturation at 95 °C for 10 min followed by 40 133

cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and synthesis at 134

72 °C for 30 s. A melting curve was generated by linear heating from 70°C to 95°C 135

over 25 minutes]. For the synthesis of the cDNA, 200 ng of DNase I-treated total 136

RNA was reverse transcribed via random hexamer primers by using the Moloney 137

murine leukemia virus RTase (M-MLV-RT; Promega, Madison, WI, USA). DNase 138

I-treated RNA (without reverse transcription) was used to check for genomic DNA 139

contamination. 140

CR-RT-PCR (25-26) was used to determine the 5’ UTR of the PTS gene cluster. 141

RNA circularization was carried out as described (25). Self-ligated RNA was reverse 142

transcribed via random hexamer primers as described above. The cDNA was first 143

amplified with a gene-specific primer pair, cRT1F and cRT1R, and a second PCR was 144

performed to enhance the specificity by using an inner primer pair, cRT2F-cRT2R. 145

The products of the second PCR were cloned into the TA vector pUCm-T (Sangon 146

Biotech, Shanghai, China) according to the standard procedures, and 15 clones from 147

each RNA sample were analyzed by sequencing. 148

Constructs used for the transformation of H. mediterranei. For analysis of the 149

promoter activity in vivo, a plasmid-based transcriptional reporter system using a 150

soluble-modified red-shifted GFP (smRSGFP) (27) was constructed as previously 151

described (28). All the plasmids used to transform H. mediterranei cells were derived 152

from pWL502, and the details of their constructions are shown in the “Supplemental 153

material”. The plasmids pL117, pPR and pPF were used to analyze the wild-type 154


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promoter activity of PTS (PPTS), glpR (PglpR) and fba (Pfba), respectively. To dissect the 155

regulatory region of the PPTS, the 5’ flanking deletion mutants of the PPTS (named 156

pL93, pL77, pL56, pL24 and pGFP-0) and the scanning site-directed mutants (from 157

pM+1216 to pM7975) were transformed into H. mediterranei DF50 to detect the 158

mutated promoter activity. The activities of GlpR and myc-tagged GlpR were 159

determined by introducing the plasmids pL117CR and pL117Rm into the H. 160

mediterranei DF50 ΔglpR strain, respectively. In the promoter or 5’ UTR swap 161

experiment, pHSP, pUTR-M, pT8 and pT17 were generated by constructing fusions 162

between the promoter or 5’ UTR regions of the hsp5 (amplified from the plasmid 163

pSCM307 (29)) and PTS gene cluster. 164

Analysis of the smRSGFP fusion reporter system. The fluorescence intensity 165

of each smRSGFP-fusion reporter plasmid-harboring strain was measured using a 166

Synergy H4 hybrid microplate reader (BioTek Instruments Inc; Winooski, VT, USA). 167

The excitation wavelength was set to 488 nm, and the emission wavelength was 509 168

nm (27). All strains were incubated at 37°C in AS-168SYL medium until the OD600 169

reached 1.5; 90 μL of each culture was then transferred to the black polystyrene 170

96-well plates (Costar-3916; Corning, NY, USA). In most cases, 10 μL of a fructose 171

or glucose stock solution was added to the designated induction wells on the 96-well 172

plates to a final concentration of 50 mM. For the metabolic intermediate F-1-P 173

(fructose-1-phosphate) induction tests, the final concentrations of F-1-P and fructose 174

were reduced to 2 mM. In each assay, 10 μL of TBSL buffer was used as a negative 175

control for basal fluorescence intensity. All of the plates were incubated at 37°C for 8 176


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hours before they were measured. 177

Primer extension. The H. mediterranei strains harboring different plasmids were 178

used in the primer extension assay with a specific primer based on the sequence of gfp. 179

Primer extension reactions were performed using 5 μg of total RNA and 3 pmol of the 180

5'-biotin-labeled primer gfpRbio (Table S2) with the reverse transcription protocol 181

described above. The extension products were analyzed on an 8% acrylamide 182

sequencing gel. A Chemiluminescent Nucleic Acid Detection Module Kit 183

(Pierce-Thermo Scientific, Rockford, IL) was used for biotin detection. 184

Immunoprecipitation. The interaction between GlpR and the promoter region 185

was analyzed by the ChIP assay. A Myc-tagged GlpR was expressed using the pRm 186

plasmid (Table S1) in the H. mediterranei ΔglpR. Cells were harvested at the 187

mid-logarithmic phase (OD600=1.5) with or without fructose induction. The ChIP 188

experiments were performed according to the previously described protocol (30-31). 189

The enrichment of genomic fragments was analyzed by qPCR with the input DNA 190

samples as controls. The primers used are listed in Table S2. The PCR and 191

thermocycling conditions were the same as described above for qRT-PCR. Each ChIP 192

assay had five biological replicates. In each ChIP sample, the enrichment of 193

GlpR-Myc interacting with each locus was calculated compared to the input sample 194

using relative quantitation. 195

Microarray assay and deep sequencing. The RNA samples used for qRT-PCR 196

from DF50 cells with or without fructose induction were subjected to microarray 197

analysis. Oligonucleotide microarrays (8×15K) were designed and manufactured by 198


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Capital Bio and Agilent Technologies (http://www.agilent.com) based on the whole 199

genomic sequence of H. mediterranei. The microarray was carried out as previously 200

described (32). Each assay was repeated three times. The resulting data were analyzed 201

by Significance Analysis of Microarrays (SAM) software version 2.23b (33). The 202

same protocols used for the microarray assays were also performed on the total RNA 203

samples from the mutant ΔglpR strain with or without fructose induction. The deep 204

sequencing of the transcriptome of H. mediterranei was performed on HiSeq 205

Sequencing Systems (Illumina HiSeq 2000) at the Beijing Institute of Genomics of 206

Chinese Academy of Sciences. 207

Accession numbers. The microarray data are deposited in the NCBI GEO 208

library under the accession number GSE41134. 209

Prediction of RNA secondary structures. The program ‘Sfold’ (34-35) was 210

used for the prediction of putative secondary structures of RNA 211

(http://sfold.wadsworth.org). 212

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Results 214

GlpR is essential for the activation of the PTS promoter by fructose. In H. 215

mediterranei, the PTS genes (HFX_1559-HFX_1563) were organized in an operon 216

corresponding to a polycistronic transcript (Fig. 1A). The functional involvement of 217

the PTS in fructose utilization was confirmed via genetic methods that were used in H. 218

volcanii (21) (Fig. S1 and S2). The DNA sequence of the intergenic region between 219

fruK and ptsC was listed in Fig. 1B. Deep sequencing of the total RNA of H. 220


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mediterranei DF50 indicated that there were two transcripts with a 17-nt or 8-nt 5' 221

untranslated region (UTR), respectively. The start site (A) (+1) of the longer transcript 222

(T17) is 9-nt away from the start site (G) (+10) of the shorter transcript (T8) (Fig. 1B 223

and 1C), and these start sites were confirmed by CR-RT-PCR. However, only one 224

typical promoter containing a putative TFIIB-recognition element (BRE) (-36 225

GAAAGG -31) and a putative TATA box (-30 ATTTTT -25) was found (Fig. 1B). 226

In silico analysis showed that the DeoR family transcriptional regulator, GlpR, is 227

highly conserved in haloarchaea (>60% identity), but it is more distantly related to its 228

homologs in other archaea and bacteria (<45% identity) in comparison. The protein 229

sequence multiple alignment analysis by BLASTP indicated that approximately 70 of 230

the 255 amino acids in the N-terminus of GlpR form a putative HTH motif, and the 231

remaining amino acids near the C-terminus form a DeoR-type regulator C-terminal 232

sensor domain. To determine whether the transcription of the PTS gene cluster is 233

regulated by GlpR, the H. mediterranei DF50 and the ΔglpR strain were analyzed by 234

microarray assays with or without fructose induction (GEO accession number: 235

GSE41134). The transcriptional fold changes of the PTS gene cluster and neighboring 236

genes (HFX_1558-1565) are listed in Table 1. It was shown that the PTS gene cluster 237

and the glpR-fruK operon, which were highly up-regulated by fructose in H. 238

mediterranei DF50, were not inducible in the ΔglpR strain. In addition, the 239

transcription of fba was not significantly changed in either group (Table 1). These 240

results strongly suggested that GlpR is an indispensable regulator in fructose-induced 241

PTS activation in H. mediterranei. 242


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To confirm the regulation of PTS by GlpR in vivo and to conveniently investigate 243

the PTS promoter, the ΔglpR strain harboring different GFP-based reporter plasmids 244

was investigated; the DF50 strain served as a positive control. It was shown that 245

whether induced by fructose or not, the fluorescence intensity of the ΔglpR strain 246

harboring pL117, which expressed smRSGFP under the wild-type PTS promoter (134 247

bp upstream of translation start codon of ptsC), was much closer to the basal 248

fluorescence intensity of the DF50 harboring pL117 (without fructose induction) (Fig. 249

2). The ΔglpR strain harboring pL117CR showed that the activation of the PPTS by 250

fructose was restored through the expression of GlpR (using its native promoter, PglpR) 251

in the ΔglpR strain (Fig. 2). Furthermore, the transcriptional activity of PglpR was also 252

analyzed via the reporter plasmid pPR, which expresses smRSGFP using PglpR. The 253

fluorescence intensity of the PglpR-fusion reporter system increased slightly 254

(approximately 1.5-fold) when DF50 cells were induced by fructose but did not 255

change when ΔglpR cells were tested (Fig. 2). In contrast, regardless of fructose 256

induction, the fluorescence intensity was similar between DF50(pPF) and ΔglpR(pPF) 257

transformants (both expressing smRSGFP with the Pfba promoter) (Fig. 2). These 258

results confirmed that GlpR is essential for the fructose-induced transcriptional 259

activation of the PTS and the glpR-fruK gene clusters, and it may act as a positive 260

regulator for fructose-induced PTS expression. This finding is quite interesting, as 261

GlpR has been previously identified as a global repressor that inhibits the activities of 262

KDGK and PFK in H. volcanii when cells were cultured in a glycerol-based medium 263

(13). The different functions of GlpR are likely attributable to the different carbon 264


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sources (fructose vs. glycerol) being used, and/or on different promoters. 265

GlpR binds directly to the PPTS during the fructose induction. To determine 266

whether the activation of gene expression by GlpR occurs via a direct binding to the 267

promoter sequence, a ChIP assay coupled with qPCR analysis was performed in the 268

ΔglpR strain harboring pRm (a Myc-tagged GlpR expression plasmid) with or without 269

fructose induction. The recombinant GlpR-Myc (expressed by pL117Rm) was 270

revealed to be able to restore the activity of wild-type GlpR in the ΔglpR strain (data 271

not shown). Three DNA loci (FPTS, Fiic, and FphaE) were investigated for their 272

interaction with GlpR, with the F16S locus (a fragment of the 16S rRNA gene) used as 273

an internal control for data normalization. The FPTS (119 bp) represented the fragment 274

of the PPTS region (-92 to +27 bp upstream of the PTS gene cluster), Fiic (125 bp) 275

represented the intragenic region of ptsC located approximately 550 bp to 650 bp 276

downstream of FPTS. The FphaE (189 bp) locus containing the promoter region of phaE 277

(which encodes a subunit of polyhydroxyalkanoate synthase in H. mediterranei (19)) 278

was tested as a negative control because the expression level of phaE did not change 279

in the microarray experiment when the cells were treated with fructose (data not 280

shown). After the induction by fructose, the FPTS locus exhibited a 2-fold enrichment 281

of binding to GlpR-Myc over the negative control locus FphaE, but the enrichment of 282

the FPTS and FphaE loci were similar to each other in the absence of fructose induction 283

(Fig. 3). As expected, the fold enrichment of the Fiic locus remained unchanged and 284

was similar to that of locus FphaE, with or without fructose induction (Fig. 3). These 285

results demonstrated that GlpR could directly bind to the PPTS when H. mediterranei 286


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cells are treated with fructose, but the interaction between GlpR and PPTS was not 287

apparent without fructose induction. The significantly increasing binding ability 288

between GlpR and PPTS under fructose induction indicated again that GlpR is an 289

activator of PTS transcription. 290

Three regions within the PPTS account for the fructose induction. The above 291

results demonstrated that GlpR acts as a positive regulator for the induction of the 292

PTS gene cluster by fructose via direct binding to the PPTS. To experimentally analyze 293

the cis-acting elements of the PPTS, 5’ flanking deletion and site-directed mutagenesis 294

analysis of the PPTS were carried out based on the smRSGFP-fusion reporter system of 295

plasmid pL117. The fluorescence intensity of H. mediterranei DF50 transformants 296

harboring deletions or site-directed mutagenesis constructs were measured with or 297

without fructose induction (Fig. 4). 298

The wild-type PTS promoter in pL117 and the deletion mutants in pL93, pL77 299

and pL56 exhibited similar basal transcription activities when cells were grown in 300

AS-168SYL medium (data not shown). However, in the presence of fructose, the 301

pL56 mutant completely lost the ability to respond to the fructose induction. The 302

pL93 and pL77 mutants showed a 2- to 3-fold increase in fluorescence intensity after 303

the fructose induction. When the putative BRE and TATA box of PPTS were deleted 304

(pL24), the transcription activities of both the basal and fructose-inducing conditions 305

were almost undetectable (Fig. 4A). These results revealed that the pL77 mutant still 306

contains the main cis-acting elements that respond to the fructose induction. Thus, the 307

promoter region from -79 bp to +16 bp of pL117 was analyzed using site-directed 308


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scanning mutagenesis to pinpoint the essential regions that account for the fructose 309

induction. The resulting constructs were named pM7975 to pM+1216, in which the 310

numbers indicate the mutation region. For example, pM7975 indicates mutagenesis 311

from -79 bp to -75 bp relative to the TSS of the PPTS, and pM+1216 indicates 312

mutagenesis from +12 bp to +16 bp. These plasmids were transformed into H. 313

mediterranei DF50 to generate the reporter strains M7975 to M+1216 for the 314

detection of fluorescence intensity. 315

It was observed that mutations in the putative BRE (-36 to -31 bp) and TATA box 316

(-30 to -25 bp) regions (M3935, M3430 and M2925) and the -10 region (M1410) led 317

to a complete loss of transcriptional activity. Only three mutants, M7975, M6864 and 318

M2420, have the same response to fructose induction as the pL117 transformant, and 319

a 1.5- to 2-fold induction was detected in mutants M6360, M5553, M4440 and M1915 320

(Fig. 4B). It is noteworthy that the basal fluorescence intensity of M1915 rose to a 321

very high level (19 times that of DF50 harboring pL117). However, the fluorescence 322

intensity of M1915 was still induced by fructose, and thus the region from -19 to -15 323

bp might not be directly involved in fructose activation. The GlpR seemed unrelated 324

to any inhibition at the region from -19 to -15, since the fluorescence intensity of the 325

ΔglpR strain harboring pL117 (with or without fructose induction) was similar to the 326

basal intensity of DF50 cells harboring pL117 (without fructose induction) (Fig. 2). 327

These results showed that the knockout of the GlpR can not enhance the PPTS activity 328

to as high of a level as that detected in the PPTS mutated plasmid pM1915. We 329

speculate that either the site from -19 to -15 bp is required for the binding of an 330


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unknown inhibitor or the mutation from -19 to -15 bp changes the promoter 331

architecture, both of which may lead to a higher activity of the mutated promoter 332

under basal conditions. 333

Notably, the fructose induction did not significantly change the transcriptional 334

levels of the mutants M7469, M5957, M5755 and M5351 to M4745 (the fold change 335

for each was no more than 1.1), indicating that the corresponding regions in these 336

mutants are important for the fructose-induced up-regulation of the PTS. These results 337

revealed that the promoter regions I (-74 bp to -69 bp), II (-59 bp to -56 bp), and III 338

(-52 bp to -45 bp) are particularly important for the fructose induction in H. 339

mediterranei, and mutations in these three regions made the PPTS lose its ability to 340

respond to the fructose induction (Fig. 4B). This phenomenon of which was similar to 341

that was observed in the ΔglpR mutant strain harboring pL117 (Fig. 2), indicating that 342

the regions I, II, and III were essential for the cellular responses to fructose induction 343

and were likely to be the binding sites of GlpR under fructose induction. Interestingly, 344

after analyzing the PPTS of all six haloarchaea that possess the PTS gene cluster, an 345

conserved 8-bp motif, which overlapped with seven base pairs of region III, was 346

identified (Fig. 5). In addition, a palindromic DNA sequence pattern belonging to 347

regions I and II, named motif P (short for ‘palindromic’) in this study, was also at the 348

upstream of the 8-bp motif (Fig. 5), implying that the regulatory mechanism of PTS 349

revealed in H. mediterranei may be shared by other haloarchaea. 350

It is noteworthy that the basal fluorescence intensity of mutant M+911 was more 351

than 2.4 folds increasing to that of the pL117 transformant, and both the basal and 352


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induced fluorescence intensities in the mutants M+38 and M+1216 decreased to a 353

very low level in these strains (Fig. 4B). These results suggest that the mutations in 354

the 5’ UTR altered either the mRNA stability or the translation efficiency of the PTS 355

gene transcripts. 356

Generation of the PTS transcript with the shorter 5’ UTR due to 357

post-transcriptional processing. The results of scanning mutagenesis showed that 358

only one TATA box, BRE and -10 elements were identified in the PTS operon (Fig. 359

4B), which indicated that there is only one promoter for PTS transcription. However, 360

two transcripts with 5’ UTRs of different lengths (17 nt and 8 nt) were observed 361

among the PTS transcripts (Fig. 1B and 1C), and the 5’ UTR of the PTS gene cluster 362

was found to be important for their expression (Fig. 4B). To investigate how the two 363

transcripts containing 5’ UTRs of different lengths were produced and whether they 364

were involved in the PTS activation, the feature and function of this 5’ UTR region 365

were further analyzed. 366

First, RNA folding and the general features of the 17-nt 5’ UTR were predicted 367

using ‘Sfold’ software, and a stem-loop structure was indicated. The start site (G) 368

(+10) of the 8-nt 5’ UTR was located at the loop region (Fig. 6A). DF50 strains 369

harboring different plasmids were used to conveniently characterize the 5’ UTR at 370

both the transcriptional and translational levels (Fig. 6B). In a primer extension assay, 371

when the nucleotides in the +3 to +11 bp region (M+911 and M+38) of the 5’ UTR 372

were mutated, the small extension product was hardly detectable (Fig. 6B). Therefore, 373

the production of the shorter gfp transcripts likely depends on the sequence or 374


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structure of the 5’ UTR (same as the 5’ UTR of PTS transcripts) of the mRNA. 375

To test this hypothesis, four other promoter- or 5’ UTR-swapped constructs were 376

generated as shown in Fig. 7A. The plasmids, pT8, pT17, and pHSP contained the 377

hsp5 promoter from Halobacterium sp. NRC-1 (Phsp5) (36) and different 5’ UTR 378

regions either from PTS (pT8 and pT17) or hsp5 (pHSP), respectively, whereas 379

pUTR-M (containing the PTS promoter and the 5’UTR of hsp5) was constructed in a 380

manner similar to that of pHSP by only replacing the Phsp5 with the PPTS. The results 381

showed that the two constructs that contained the 17-nt 5’ UTR, pT17 and the positive 382

control (PC) pL117 could both produce two transcripts (corresponding to T8 and T17 of 383

the PTS transcripts, and named T8G and T17G thereafter for the gfp transcripts) with 384

different 5’ UTRs (Fig. 7B), despite the fact that the transcription of these constructs 385

was controlled by different promoters (Phsp5 and PPTS, respectively). On the contrary, 386

only one transcript was generated from pHSP or pT8 as expected (Fig. 7B), and the 387

PTS promoter (PPTS) combined with the hsp5 5’ UTR sequence (pUTR-M) was also 388

unable to generate the shorter transcript. These results indicated that the production of 389

the transcript with shorter 5’ UTR was only related to the sequence of the longer 5’ 390

UTR, but not to the promoter, and therefore indicated a potential post-transcriptional 391

processing of PTS transcripts. 392

Physiological significance of the generation of the shorter 5’ UTR. To 393

understand the physiological significance of the production of two transcripts 394

containing 5’ UTRs of different lengths, the translation efficiency of these two 395

transcripts was investigated. The relative amounts of gfp transcripts (detected by 396


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qRT-PCR) and their translation activity (measured by fluorescence intensity) were 397

determined using DF50 strains harboring pT8 or pT17 (Fig. 8A). There was no 398

significant difference in the amount of gfp transcripts between DF50 strains harboring 399

pT8 or pT17 as evaluated by the Student’s t test, but the GFP expression from the 400

shorter transcript in the DF50 strain harboring pT8 was over 6-fold higher than the 401

GFP expression from the mixture of two transcripts in the DF50 strain harboring pT17 402

(Fig. 8A). These results indicated that the translation efficiency of the shorter 403

transcript was much higher than that of the longer transcript. 404

Further research was performed to find a correlation between the fructose 405

induction and the production of the two transcripts. The primer extension products of 406

the DF50 and ΔglpR strains harboring pL117 with or without fructose induction 407

were analyzed. The results showed that the amount of the two transcripts increased 408

as a result of the fructose induction in the DF50 strain, and the ratio of the shorter 409

transcript to that of the longer transcript was also increased (Fig. 8B). To confirm 410

this result quantitatively, CR-RT-PCR was performed on the DF50 and ΔglpR strains 411

with or without fructose induction. The ratio of clone counts of the PTS transcript 412

with the 8-nt 5’UTR to that of the 17-nt 5’ UTR was doubled (from 14% to 31%) 413

when DF50 cells were induced by fructose (Table 2). The ratios of T8 to T17 counts 414

in the ΔglpR strain with or without induction were similar to each other and much 415

closer to the non-induced ratio in the DF50 strain (Table 2). These results indicate 416

that the translation efficiency of the PTS genes would be enhanced when the cells 417

were induced by fructose due to the increased proportion of the shorter transcript. 418


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F-1-P may acts as a positive intracellular effector. During PTS regulation, the 419

derivatives of sugars always serve as positive or negative effectors to enhance or 420

repress the activity of regulators. F-1-P was shown to act as an important intracellular 421

effector for the transcriptional regulation of PTS in many bacteria (2, 14-15, 37). To 422

investigate the function of F-1-P during PTS regulation, the smRSGFP-fusion reporter 423

assay was performed with the pL117 transformant of H. mediterranei. In the fruK 424

knockout mutant containing pL117, whether induced by fructose or not, the 425

fluorescence intensity both increased to a very high level (more than 20 times higher 426

than that in DF50) and the induced expression of PTS by fructose disappeared (Table 427

3). In H. volcanii, it has been revealed that fructose is transported through PTS, which 428

would generate F-1-P, after which it is further phosphorylated by 1-PFK (encoded by 429

fruK) (12). The high level of gfp expression in H. mediterranei ΔfruK may be caused 430

by the accumulation of F-1-P when 1-PFK is inactivated, which implies that F-1-P 431

may enhance the expression of PTS as an intracellular effector as observed in bacteria. 432

This hypothesis is also supported by detecting the fluorescence intensity of H. 433

mediterranei DF50 cells harboring pL117 when adding F-1-P to the culture medium 434

(Fig. S3). Either Fructose or F-1-P induction significantly increased the fluorescence 435

intensity, whereas the other derivative of fructose, fructose-1,6-bisphosphate 436

(F-1,6-2P), decreased the fluorescence intensity (Fig. S3). 437

438

Discussion 439

During the fructose induction, GlpR is shown to be an indispensable activator for 440


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the up-regulation of the fructose-specific PTS gene cluster (Table 1, Fig. 2) through 441

the direct binding to PPTS in H. mediterranei (Fig. 3). Therefore, GlpR is essential for 442

the cellular responses to the fructose induction. Interestingly, in H. volcanii, GlpR has 443

been shown to be a global regulator by repressing the transcription of the key 444

enzymes, including KDGK and PFK, when using glycerol as the carbon resource (13). 445

It seemed that the function of GlpR was decided by the environmental carbon sources. 446

Through the regulator GlpR, glycerol represses sugar metabolism and fructose 447

activates PTS expression. Previous reports suggest that the DeoR-type proteins always 448

contain several highly conserved regions, one of which is the second helix of the 449

helix-turn-helix (HTH) DNA-binding motif in the N-terminus (38). The other 450

conserved regions are involved in oligomerization or inducer binding (in many cases, 451

the inducer is a phosphorylated sugar). As one of the DeoR-type proteins, it makes 452

sense that GlpR could be activated through the interaction of its C-terminal sensor 453

domain with the fructose effector F-1-P in H. mediterranei. The activated GlpR (or 454

GlpR accompanied by unknown regulators) could then bind to the promoter region 455

through the N-terminal DNA binding motif to increase the transcriptional activity of 456

PPTS. This mode of action of GlpR was supported by the study of another DeoR-type 457

transcriptional regulator, SugR, in Corynebacterium glutamicum (15, 39-41). 458

Although the molecular details of the activation by GlpR require further investigation, 459

one hypothesis based on other transcription activation models in archaea (42-44) is 460

that the activated GlpR recruits general transcription factors (TFBs and TBPs) to bind 461

to the TATA box or BRE to enhance transcription. 462


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According to a previous report, the possible binding sites of the GlpR as a 463

repressor are located at the BRE or downstream of the TATA box by in silico 464

searching for the inverted repeat sequence (13). However, this kind of binding site 465

that was reported for H. volcanii GlpR was not observed in the PTS promoter region 466

in H. mediterranei. Instead, an upstream sequence (including regions I, II and III) of 467

PPTS was indicated as the possible binding site of GlpR by the mutation scanning 468

experiment (Fig. 4B). This finding showed a positional similarity with the UAS 469

(upstream activator sequence, from -52 bp to -39 bp) of the bop gene in 470

Halobacterium NRC-1 (44-46). The conserved motifs (motif P and the 8-bp motif) in 471

promoter regions I, II and III of the PTS implied that the mechanism of PTS 472

regulation by GlpR is similar in haloarchaea (Fig. 5). 473

It is noteworthy that the shorter PTS transcript would be generated through the 474

post-transcriptional processing in H. mediterranei (Fig. 7). This type of expression 475

seemed to be different from its bacterial counterpart. Multiple TSSs were reported in 476

the PTS genes of E. coli, which resulted from multi-core promoters upstream of the 477

coding sequence of the PTS genes and were influenced by DNA supercoiling and the 478

transcription factor CRP (47), but the translation efficiency of different transcript 479

patterns was not very clear. Further analysis of the 5’ UTR sequences of T8G and T17G 480

indicated that the relatively more efficient translation of T8G was probably due to the 481

presence of the shorter 5’ UTR (Fig. 8A). This hypothesis that the length of 5’ UTR 482

affects the translation efficiency of mRNA has also been reported in other haloarchaea. 483

In Halobacterium salinarum, leaderless mRNAs showed a higher translation activity 484


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than mRNAs with the SD-sequence (48). On the other hand, the predicted stem-loop 485

structure in T17 (Fig. 6A) might also inhibit the reorganization or binding of the 486

ribosome and hence repress the translation. It can be speculated that when induced by 487

fructose, the translation efficiency of the PTS mRNA could be enhanced by increasing 488

the ratio of T8 to T17 (Table 2). The relatively higher proportion of T17 in the 489

non-fructose conditions implied a constitutively low level of expression of the PTS. 490

The fructose-specific PTS of haloarchaea was regarded to be acquired from 491

bacteria by horizontal gene transfer (HGT) during evolution (12, 49). As a “gift from 492

the neighbors”, the haloarchaeal PTS was capable of also “borrowing” the regulatory 493

mechanism from bacteria at the transcriptional level. Furthermore, to acclimate to 494

nutrient fluctuations in a competitive extreme hyper-saline environment, the 495

haloarchaea evolved their own mechanisms to control the PTS at the 496

post-transcriptional and translational levels. Based on the results in this study and 497

previous reports, we propose a working model for the PTS regulation in haloarchaea 498

(Fig. 9). In this model, fructose is transported into the cell and phosphorylated to 499

F-1-P via PTS, and further catalyzed to F-1,6-2P by 1-PFK. GlpR (or a combination 500

of GlpR and other unknown regulators) up-regulates the transcription of this PTS 501

gene cluster after the induction by fructose via direct binding to the PPTS, most 502

probably at the three conserved regions. F-1-P may act as the intracellular inducer, 503

while F-1,6-2P as the negative effector, to be involved in this transcriptional 504

regulation of the PTS gene expression. A post-transcriptional processing of the PTS 505

transcripts at the 5’ UTR, which increases the translational efficiency, is also involved 506


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in the PTS activation upon fructose induction (Fig. 9). 507

In conclusion, the activation at the both transcriptional and translational levels 508

would make the haloarchaeal PTS more efficient in response to environmental 509

fructose. Although the working model has explained the main mechanisms of the PTS 510

regulation in haloarchaea, further study is warranted to determine whether other 511

transcriptional regulators are involved in the regulation of PTS expression and to 512

elucidate the mechanism of post-transcriptional processing. Such studies would help 513

towards a comprehensive understanding of the PTS regulation in haloarchaea. 514

515

516

517

ACKNOWLEDGMENTS 518

We thank Prof. Julie A. Maupin-Furlow (University of Florida, USA) for providing us 519

with the plasmid pJAM1020. This work was supported by grants from the National 520

Natural Science Foundation of China (31330001, 30925001, and 31271334) and the 521

Chinese Academy of Sciences (KSCX2-EW-G-2-4). 522

523

524

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660

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Tables 663

664

TABLE 1 A comparative analysis of the transcriptional levels between H. mediterranei DF50 665

and ΔglpR groups of the most relevant genes (HFX_1558 to HFX_1565) during the fructose 666

induction using a microarray assay (GEO accession numbers: GSE41134) 667

668

Gene ID Annotation

DF50 ΔglpR

Fold

Change

q-value

(%)

Fold

Change

q-value

(%)

HFX_1558 Fructose-1,6-bisphosphate aldolase 0.82±0.14 4.23 0.86±0.11 10.58

HFX_1559 PTS IIB component 29.74±7.31 0 0.92±0.02 12.77

HFX_1560 PTS enzyme I 34.97±14.17 0 0.88±0.03 7.85

HFX_1561 PTS protein HPr 31.3±12.77 0 0.85±0.17 13.33

HFX_1562 PTS IIA component 40.37±16.45 0 0.94±0 20.2

HFX_1563 PTS IIC component 31.97±4.33 0 1.11±0.08 17.89

HFX_1564 1-phosphofructokinase 17.33±5.74 0 0.91±0.12 21.9

HFX_1565 GlpR family regulator 21.07±2.42 0 - -

669

670

671

TABLE 2 The statistical results of shorter and longer PTS transcripts in DF50 and 672

ΔglpR strains with (+) or without (-) fructose induction. 673

674

Transcript

DF50 ΔglpR

－＋－＋

8-nt counts 5 8 2 2

17-nt counts 36 26 14 20

Ratio

(8-nt: 17-nt) 14% 31% 14% 10%

675


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676

677

TABLE 3 Expression of the smRSGFP-fusion reporter gene in H. mediterranei 678

strains with or without fructose induction*. 679

Expt. no. Relevant host

genotype

Relevant

plasmid

Fluorescence intensity Fold

change Non-

induced

Fructose

induced

1 DF50 pL117

896±158 5052±753 5.64

2 fruK- 22108±570 19652±612 0.89

* At least three independent experiments were carried out, and each experiment consisted of three replicates. 680

681

682

683

684


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Figures 685

FIG 1. Map of the PTS gene cluster, the promoter sequence and the analysis of PTS 686

transcripts. (A) Genetic organization of the H. mediterranei PTS gene cluster and 687

neighboring genes. glpR (HFX_1565) encodes a protein homologous to a DeoR 688

family transcriptional regulator, and fruK (HFX_1564) encodes the 689

1-phosphofructokinase; ptsC, ptsA, hpr, ptsI, and ptsB (HFX_1563-1559) encode a 690

complete PTS; and fba (HFX_1558) is a fructose-1,6-bisphosphate aldolase. The 691

primers used for CR-RT-PCR are indicated with arrows. (B) The promoter sequence 692

of the PTS gene cluster is shown. The stop codon TAA of the upstream gene fruK and 693

the start codon ATG of ptsC are boxed. The start site (indicated by an arrow) of the 694

two transcripts containing different length 5’ UTRs was determined by CR-RT-PCR. 695

The putative TATA box and BRE are indicated by single and double underlines, 696

respectively. (C) The statistical results of mRNA deep sequencing. The counts (Y-axis) 697

of corresponding nucleotides (X-axis) of 24-bp sequence of the PPTS are presented. 698

The positions with the most significant increases in sequencing counts are marked by 699

arrows, which indicate the two start sites of PTS transcripts. 700

701

FIG 2. The GFP expression profiles of the DF50 and ΔglpR strains harboring 702

smRSGFP-based reporter plasmids with or without fructose induction. The plasmids 703

pL117, pPR and pPF were constructed to express smRSGFP with the promoter PPTS, 704

PglpR and Pfba, respectively. Another construct, pL117CR was transformed into the 705

ΔglpR strain to expresses GlpR (using its native promoter, PglpR) and smRSGFP (using 706


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PPTS). The fluorescence intensity was detected as described. Fructose was added into 707

the cell cultures to a final concentration of 50 mM (Fru+). TBSL buffer was used as a 708

negative control (Fru-). At least three independent experiments were carried out, and 709

each experiment consisted of three replicates. 710

711

FIG 3. ChIP-qPCR data suggest that GlpR binds to DNA directly. The relative 712

enrichment ratio of a 119-bp region of the PPTS (FPTS) immunoprecipitated (IP) by 713

GlpR-Myc compared to randomly sheared chromosomal DNA (input samples) using a 714

145-bp region in 16S rRNA (F16S) as an internal control to normalize the data. 715

Enrichments are also compared for a 125-bp coding region (Fiic) of the ptsC gene, and 716

a 189-bp promoter region (FphaE) of phaE, that was not regulated by the fructose 717

induction in the microarray data. Cell samples for the ChIP assay were harvested 718

under growth conditions with (Fru) or without (CK) fructose induction. 719

720

FIG 4. Deletion analysis and site-directed mutagenesis of the PPTS region. (A) 721

Schematic representations (not to scale) of constructs pL117, pL93, pL77, pL56 and 722

pL24 are shown. The 117-bp PPTS and truncated 5’ flanking sequence promoter 723

mutants (solid lines, -93-+17 bp to -24-+17 bp) were fused with the smRSgfp reporter 724

gene (gray arrow). (B) Site-directed mutagenesis from -79 to +16 bp of the PPTS. The 725

DNA sequence of wild-type PPTS is shown at the top (pL117). The two transcripts of 726

the reporter gene with 5’ UTR start respectively from +1 and +10, which are the same 727

as what were observed in the PTS transcripts, were identified by CR-RT-PCR. The 728

mutated nucleotides of different mutants (M7975 to M+1216) are shown below the 729


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wild-type promoter sequence. The basal (non-induced) and fructose-induced 730

transcriptional activities of these promoters revealed by fluorescence intensity were 731

detected using a microplate reader. The basal transcriptional activities are expressed 732

as a percentage of the pL117 activity (set to 100%), and the fructose-induced fold 733

changes are calculated using the fluorescence intensity. Mutants that cannot respond 734

to fructose are indicated with an asterisk, and the mutants with a fluorescence 735

intensity that was hardly detectable are marked by a dash. The significant fold change 736

in strains M+38 and M+1216, marked with a rhombus, were caused by the large 737

decreases in the basal fluorescence intensity. At least three independent experiments 738

were carried out, and each experiment consisted of three replicates. 739

740

FIG 5. Multiple alignments of promoter sequences of the PPTS in the haloarchaea 741

which contain at least one complete PTS. Bases marked with asterisks are the 742

sequences of region I, II and III. A palindromic DNA sequence (underlined, motif P) 743

and an 8-bp sequence (boxed) are indicated. These sequences were found to be 744

conserved via the alignment of the PPTS in all promoter regions of haloarchaeal PTS. 745

746

FIG 6. The mutagenesis analysis of the 5’ UTR of PTS transcripts. (A) The in 747

silico-predicted secondary structure of the 17-nt 5’ UTR of mRNA, ΔG°37 = -2.50. 748

The designed mutation regions are marked with lines. (B) The electrophoretic analysis 749

of the primer extension products of gfp in strains M+911, M+38 and M1+2 after the 750

induction by fructose. DF50 cells harboring pGFP-0 was used as a negative control 751

(NC), and DF50 cells harboring pL117 was used as a positive control (PC). The 59-nt 752


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and 41-nt oligonucleotides were used as molecular markers. The non-specific 753

products appearing in the NC are indicated with (a) and (b), and the two primer 754

extension products, which corresponding to the transcript with 17-nt (T17G) or 8-nt5’ 755

UTR (T8G), are indicated with arrows. 756

757

FIG 7. Primer extension assay for identifying the different transcripts generated by 758

Phsp5-directed or PPTS-directed reporter genes. (A) The different sequences of four 759

constructs (pT8, pT17, pHSP and pUTR-M) are shown. The TSS (G) of Phsp5 or the 760

TSS (A) of PPTS in four constructs is marked with asterisks, and the different length 5’ 761

UTR of each construct is underlined. The sequence of the extension primer is boxed, 762

and the expected size of the extension products is indicated in parentheses. The 763

difference between pT8, pT17, and pHSP is the downstream sequence of the TSS (G), 764

which is the 8-nt (pT8) or 17-nt (pT17) 5’ UTR sequences of PTS, or the 5’ UTR 765

sequences of the wild-type hsp5 (pHSP), respectively. The only difference between 766

pHSP and pUTR-M is that the Phsp5 promoter is replaced by the PPTS in pUTR-M. (B) 767

The primer extension products of pT8, pT17, pHSP (T56), and pUTR-M were 768

analyzed by electrophoresis. The primer extension products with same size are 769

indicated with arrows (T56, T17G or T8G). 770

771

FIG 8. Functional characterization of the in vivo generation of the shorter 5’ UTR in 772

H. mediterranei using a reporter gene. (A) The relative level of transcription activity 773

and the translation efficiency of gfp transcripts in DF50 strains harboring pT8 or pT17 774


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were determined via qRT-PCR and a fluorescence reporter assay. The levels of gfp 775

transcription and the fluorescence intensity in strain DF50 harboring pT17 were both 776

assigned a value of 1. At least three independent experiments were carried out, and 777

each experiment consisted of three replicates. The statistical significance of the 778

difference between the DF50 strains harboring pT8 or pT17 was analyzed using the 779

Student’s t test. (B) The electrophoretic analysis of the primer extension products of 780

gfp in DF50(pL117) and ΔglpR(pL117) strains with (+) or without (-) fructose 781

induction. Major transcripts are indicated on the left of the panel as described in Fig. 782

6. 783

784

FIG 9. A working model of the regulation of PTS expression and fructose utilization in 785

H. mediterranei. CM, cytoplasmic membrane; 1-PFK, 1-phosphofructokinase; F-1-P, 786

fructose-1-phosphate; F-1,6-2P, fructose-1,6-bisphosphate; TIC, transcriptional 787

initiation complex. 788


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aem accepts, published online ahead of print on 13...

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