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TRANSCRIPT
Characterization of MtfA, a novel regulatory output signal protein of the 1
glucose-phosphotransferase system in E. coli K-12 2
3
Anna-Katharina Göhler1, Ariane Staab1, Elisabeth Gabor, Karina Homann, 4
Elisabeth Klang, Anne Kosfeld, Janna-Eleni Muus, Jana Selina Wulftange, 5
and Knut Jahreis* 6
7
Department of Biology and Chemistry, University of Osnabrück, 8
D-49069 Osnabrück, Germany 9
10
11
Key words: glucose transporter, glucose signaling, PTS, Mlc, MtfA, peptidase, regulation, 12
Protein-protein interaction, E. coli K-12 13
14
1who have equally contributed to this paper 15
16
Running title: Regulation of the Glucose-PTS in E. coli 17
18
*Corresponding author: 19
Phone: +49-541-969-2288 20
FAX: +49-541-969-2293 21
E-mail: [email protected] 22
23
Copyright © 2011, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.06387-11 JB Accepts, published online ahead of print on 16 December 2011
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Abstract 24
The glucose phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory 25
and regulatory system. In addition to its central role in glucose uptake, it informs other global 26
regulatory networks about carbohydrate availability and the physiological status of the cell. 27
The expression of the ptsG gene encoding the glucose PTS transporter EIICBGlc is primarily 28
regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of 29
glucose and dephosphorylation of EIICBGlc, Mlc binds to the B-domain of the transporter 30
resulting in de-repression of several Mlc-regulated genes. In addition, Mlc can also be 31
inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. 32
In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by 33
measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the 34
ability of MtfA to inactivate an Mlc super repressor, which cannot be inactivated by EIICBGlc, 35
by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity 36
of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and showed Mlc’s 37
ability to enhance this activity. 38
Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase 39
activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as 40
they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc 41
constitutes a newly identified PTS output signal that responds to changes in environmental 42
conditions. 43
44 45
46
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Introduction 47
Despite the fact that Escherichia coli K-12 has been an important model organism for many 48
decades, the function of more than 20% of all putative open reading frames in its genome 49
remains unknown. Moreover, in many cases, no obvious sequence similarities with previously 50
characterized proteins exist. The mtfA gene (formerly named yeeI), a monocistronic gene 51
located at 44.1 min in the E. coli chromosome, has clearly belonged to this group. Intensive 52
sequence similarity searches revealed that orthologs of MtfA (mnemonic for Mlc-titration-53
factor A) exist in more than 100 proteobacteria of the α-, β-, and γ- subdivisions. In a previous 54
report (2) we were able to demonstrate that E.coli MtfA is a cytoplasmic protein which binds 55
to the carboxy-terminal part of the Mlc repressor protein (mnemonic for Making-large-56
colonies, gene: dgsA) with a very high affinity. Mlc is one of the global regulators of 57
carbohydrate metabolism in E. coli and is especially involved in the regulation of the ptsG 58
gene expression, which encodes the glucose transporter EIICBGlc. The EIICBGlc together with 59
the cytoplasmic protein EIIAGlc, encoded by the crr gene (as part of the ptsHIcrr operon), 60
forms the glucose-specific phosphoenolpyruvate (PEP) –dependent 61
carbohydrate:phosphotransferase system (glucose-PTS). Both proteins take part in a 62
phosphorylation cascade, which begins with an autophosphorylation reaction of the so called 63
Enzyme I (EI, gene ptsI) at the expense of PEP. The phosphate group is subsequently 64
transferred from EI to the phosphohistidine carrier protein HPr (gene ptsH), then to the sugar 65
specific Enzymes II and finally to the carbohydrate (for review see (23)). In addition to its 66
transport and carbohydrate phosphorylation functions, the glucose-PTS in enteric bacteria has 67
a central regulatory role in carbon catabolite repression and inducer exclusion (reviewed in (7, 68
15)). Many recent studies have revealed that the regulation of ptsG expression is very 69
complex and takes place both at the transcriptional and post-transcriptional levels (reviewed 70
in (3, 12)). Additionally, there also exists a direct regulation of transport activity that prevents 71
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intracellular hexose phosphate stress (10, 32). In the absence of glucose, ptsG expression is 72
repressed by Mlc. In the presence of glucose, dephosphorylated EIICBGlc, which is generated 73
during glucose uptake, binds Mlc and sequesters the repressor away from its DNA-binding 74
sites (13, 16, 28, 34) (review in (3, 21)). Hence, in contrast to other regulatory systems, there 75
is no intracellular low-molecular weight inducer for Mlc. The EIICBGlc/Mlc system is 76
considered to be an important glucose sensor in enteric bacteria (13, 34), since Mlc is also 77
involved in the glucose dependent regulation of the ptsHIcrr operon (20, 29) , the malT gene 78
for transcriptional activation of the maltose regulon (6) and the manXYZ operon encoding the 79
EII proteins of the mannose-PTS (18). 80
Although MtfA has been shown to interact with Mlc and to work as an anti-repressor, its true 81
function has not been elucidated yet. We were interested in determining what the cellular role 82
of MtfA was. Thus, in the first part of this study we further characterised the specific 83
interaction between MtfA and Mlc. By generating a collection of MtfA mutants, we identified 84
the Mlc-binding domain within this highly conserved protein. Furthermore, we characterized 85
the interaction between MtfA and the Mlc*H86R super repressor, which is no longer capable 86
of interacting with EIICBGlc (27). This allowed us to elucidate differences between the 87
inactivation modes of Mlc by membrane-sequestration through EIICBGlc on the one hand, and 88
the cytoplasmic titration by MtfA on the other. 89
Recently, the crystal structure of the MtfA homologue from Klebsiella pneumoniae was 90
characterized (DOI:10.2210/pdb3khi/pdb). Interestingly, the structure revealed similarities to 91
the zinc-dependent metalloprotease Lethal Factor (LF) from Bacillus anthracis as well as to 92
other metalloproteases of the gluzincin clan. Hence, the second goal of this study was to 93
characterize the peptidase activity of MtfA and address the influence of Mlc on MtfA’s 94
proteolytic activity. We present a model that postulates MtfA to be a glucose-regulated 95
peptidase, whose activity is regulated by binding to Mlc available in the cytoplasm, which in 96
turn has been released from EIICBGlc during times when no glucose is taken up. Finally, we 97
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speculate about putative biologically relevant MtfA targets, which may have a function in 98
growth transition under changing environmental conditions. 99
100
Materials and Methods 101
Media and growth conditions. Cells were grown routinely either in Lennox broth without 102
glucose and calcium ions (LBo), or in 2xTY medium as described in (1). Antibiotics were 103
used at the following concentrations: tetracycline (Tc), 10 mg/ l; ampicillin (Ap), 50 mg/ l; 104
chloramphenicol (Cm) 25 mg/ l, respectively. 105
106
Bacterial strains and plasmids. All strains used were E. coli K-12 derivatives. Table 1 list 107
the genotypes and sources of the relevant bacterial strains. Informations on the plasmids and 108
oligonucleotides are given in the supplementary materials. P1-transduction was performed as 109
described previously (2). 110
111
Isolation of chromosomal and plasmid DNA, restriction analysis, PCR and DNA-112
sequencing. All manipulations of chromosomal or recombinant DNA were carried out using 113
standard procedures as described previously (1). Plasmid DNA was prepared using QIAprep® 114
Spin Miniprep Kit (Qiagen, Hilden, Germany). Restriction enzymes were purchased from 115
New England Biolabs (Schwalbach, Germany) or Fermentas (St. Leon-Rot, Germany) and 116
used according to supplier recommendations. Oligonucleotides for PCR were purchased from 117
Thermo Fisher Scientific (Ulm, Germany). DNA sequencing was commissioned to Scientific 118
Research and Development (Bad Homburg, Germany). Alternatively, DNA was sequenced 119
with the ABI3100Avant (Applied Biosystems, Foster City CA, USA) at the Center for 120
Medical Genetics in Osnabrück. Polymerase chain reactions (PCR) were performed as 121
described by (24) using TaKaRa DNA polymerase from Lonza (Köln, Germany). 122
123
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Construction of pTM30mlchis. To purify Mlc, we used PCR to amplify the corresponding 124
dgsA gene using the oligonucleotides MlcBamHI and MlcHindIII (see supplementary 125
informations) and genomic DNA of E. coli K-12. Thus, BamHI and a HindIII restriction sites 126
were inserted into the PCR product. Afterwards the PCR product was cloned in the pTM30 127
expression vector. An amino-terminal “his-tag” was added to the protein using his-tag 128
encoding, complementary oligonucleotides MlcHis+ and MlcHis. 10µg of each 129
oligonucleotide was dissolved in 10µl of sterile water. After the addition of 2µl of annealing 130
buffer (GE Healthcare, München, Germany) the mixture was heated to 95°C, and incubated at 131
RT in a 1l beaker with 95°C water until the water temperature reached 40°C. We then opened 132
the vector pTM30mlc with BamHI and ligated the previously annealed oligonucleotides with 133
this vector to generate the vector pTM30mlchis. Additionally, for the MtfA protease activity 134
assay we amplified the dgsA gene using the oligonucleotides Mlchis-tev and MlcHindIII (see 135
supplementary informations) and ligated it into the pTM30 vector to generate the plasmid 136
pTM30mlchis-tev. This plasmid encoded an amino-terminal his-tagged dgsA gene as 137
pTM30mlchis, and a TEV-protease cleavage site between the tag and protein. We treated the 138
purified protein with TEV-protease (AcTEV ProteaseTM, Invitrogen, Darmstadt, Germany) 139
according to the manufactures’ instructions. This resulted in an untagged Mlc protein with an 140
additional glycine in front of the native amino acid sequence. 141
142
Site-directed mutagenesis. We generated defined mutations in the mtfA gene using the 143
pTMByeeI-S plasmid (2) and the Quick Change® II Site-Directed Mutagenesis Kit according 144
to standard protocol (Stratagene; Amsterdam, The Netherlands). In addition, we introduced 145
mutations into the dgsA gene with the same kit and using the pTM30mlchis plasmid as a 146
template. The oligonucleotides used are listed in the supplementary informations. 147
148
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Construction of strains JSW1 and JSW2. To characterize the interaction between MtfA and 149
Mlc, we inserted a single copy of the dgsA wild-type gene or the dgsA* derivative encoding 150
the Mlc*H86R super repressor mutation (in combination with a cat gene for a 151
chloramphenicol resistance selection marker) into the chromosome of JEK5 at the pheU site 152
via site-directed recombination. In doing so, we applied the RecA-independent recombination 153
mechanism of the conjugative transposon CTnscr94 ((11); exact procedure described 154
elsewhere). PCR was used to confirm correct integration of the desired gene copy. 155
156
Western blot analyses and β-galactosidase assays. Bacterial cells harbouring pTMByeeI-S 157
as described in (2) grown in LBo medium were harvested at an optical density at 650 nm 158
(OD650) of 1 by centrifugation and resuspended in 100µl sterile water and 100µl of SDS-159
PAGE loading buffer (125 mM Tris-HCl (pH 6.8), 2 % sodium dodecyl sulphate (SDS), 10 % 160
glycerol, 5 % β-mercaptoethanol, 0.01 % bromophenol blue). If not described otherwise, 161
samples were heated at 95°C for 10min. 15 µl of total cellular proteins were separated by 162
electrophoresis on 0.1 % SDS-containing 15 % polyacrylamide gels and transferred to a 163
Nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). For the detection of MtfA 164
protein derivatives, we used a penta-his antibody (Qiagen, Hilden, Germany) except in the 165
case of the two hybrid studies, where we used a polyclonal anti-MtfA antibody. Detection of 166
antibody-binding was performed using infrared-labeled second antibodies (LI-COR 167
Biosciences, Bad Homburg, Germany). Visualization and quantification were done using an 168
Odyssey infrared imager (LI-COR Biosciences). All β-galactosidase assays were performed 169
as previously described (2). 170
171
Bacterial two-hybrid studies. Bacterial two-hybrid studies were performed as described (8). 172
The genes mtfA, mtfAΔ1-83 and dgsA were amplified by PCR. PCR products were cloned into 173
the pGEM®-T vector (Promega, Mannheim, Germany) and subsequently transferred into the 174
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vectors pMS604 or pDL804 (8) using a combination of the restriction enzymes BstEII /XhoI 175
or XhoI/BglII, or in the case of pMM4, PstI /XhoI, respectively. Oligonucleotides were used 176
in PCR as follows: pMM2: BTH7 and BTH8, pDM2: BTH1 and BTH3, pMM3: MlcPX+/-, 177
pDM3: MlcXB+/- and for pMM4: Ymlcpms+/-. All oligonucleotide sequences are listed in 178
the supplementary informations. 179
180
Protein purification. For purification of MtfA-His5, cells harbouring pTMByeeI-S or 181
derivatives were grown in LBo with ampicillin and 500µM IPTG. When cultures reached an 182
OD650 = 3, cells were harvested by centrifugation. Cells were subsequently resuspended in 183
buffer A (300mM NaCl, 2mM ß-mercaptoethanol, 100mM KPi and 10% glycerol pH 8.0) and 184
disrupted by sonication using a Branson Sonifier W-250D (Danbury, Conn., USA). Cell 185
debris was removed by low speed centrifugation (14000rpm). We then performed affinity 186
chromatography using the ÄKTA-FPLC and “His-TrapTM FF” columns (GE Healthcare). 187
Elution was performed with the same buffer as above with additional 300mM Imidazol. 188
Subsequently, we obtained the samples and dialysed them against 20mM Tris-buffer, pH 8.0. 189
Finally, we stored them at 4°C or supplemented them with 50% glycerol and stored them at –190
20°C. 191
For the purification of Mlc, cells harbouring pTM30mlchis were grown in 2xTY or LBo with 192
ampicillin until an OD650= 0.5, induced with 500µM IPTG and harvested at an OD650= 2. 193
Harvesting and disruption were performed as described previously, using a 50mM NaPi pH 194
8.0, 300mM NaCl and 20mM imdazole buffer. For Mlc purification, it was also suitable to 195
use affinity chromatography as described for MtfA. Elution was performed with a buffer 196
containing 50mM NaPi pH 8.0, 300mM NaCl, 500mM imidazol, 200µM ZnCl2. Afterwards 197
the sample was dialysed against Hepes-buffer (100mM Hepes pH 9.0, 25mM Na-glutamate, 198
1mM dithiothreitol) and stored at 4°C. 199
200
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Electrophoretic mobility shift assays. For the electrophoretic mobility shift assays (EMSA) 201
the fluorescence labelled ptsG-operator fragment according to (19) encoded by the 202
complementary oligonucleotides ptsG01 (5´ labelled with DY782) and ptsG02 (5´ labelled with 203
DY682) was annealed as described for the Mlc “his-tag”. 10µg BSA, 0.5µg Herring sperm-204
DNA as heterologous DNA, 100nM ZnCl2 and 500nM MgCl2 were added to 1ng of the 205
operator-fragment (4nM final concentration in the assay). The stated amounts of Mlc and 206
MtfA were incubated for 10min at RT and afterwards added to the mixture, which was then 207
brought to a final volume of 20µl using Hepes buffer containing 1µg/µl BSA. Samples were 208
incubated for an additional 10min at RT and loaded on a 5% acrylamid-bisacrylamide gel. We 209
performed electrophoresis at a constant voltage of 150V and detection using the “Odyssey 210
Infrared Imaging System” (LI-COR Biosciences). 211
212
Protease activity assays. The Protease Assay Kit from Calbiochem (Merck, Darmstadt, 213
Germany) was used to determine endoprotease activity of purified MtfA according to the 214
manufactures’ instructions. We determined the amino peptidase activities of MtfA by 215
measuring the hydrolysis of L-alanine 4-nitroanilide, L-arginine 4-nitroanilide, L-proline 4-216
nitroanilide, L-valine 4-nitroanilide or L-alanine-L-alanine-L-alanine-4-nitronalide (Sigma, 217
Taufkirchen, Germany) and performed an endpoint measurement of the chromophoric end 218
product 4-nitroanilide using spectral photometric determination of the absorbance at 390 nm 219
(Shimadzu UV mini 1240 photometer, molar extinction coefficient 11,5x106 cm² mol-1). The 220
assay was carried out in semi micro-cuvettes (Sarstedt, Nümbrecht, Germany). We added 100 221
µg of purified protein to 500 µl of 1 mM L-amino 4-nitroanilide. The samples were then filled 222
to a final volume of 1 ml with Tris-buffer pH 8.0 and incubated at 37°C for 4 hours. The 223
absorbance was measured against a blank sample without protein (500 µl L-alanine 4-224
nitroanilide with 500 µl Tris-buffer) at 390nm. In case of hippuryl-L-phenylalanine and 225
hippuryl-L-arginine (Sigma) absorbance was determined at 254 nm. We conducted a 226
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subsequent endpoint measurement, determined the extinction difference ΔE and calculated the 227
specific activity in U/mg protein of each preparation. All specific activities of purified 228
proteins were compared to a fresh sample of the wild type protein. If indicated, we added the 229
inhibitors AEBSF (Applichem, Darmstadt, Germany), EDTA (Roth, Karlsruhe, Germany) or 230
phenantroline (Sigma) in denoted concentrations to the assay. 231
232
Results 233
Multiple alignments to identify highly conserved amino acids. MtfA is a highly conserved 234
protein that is not only restricted to the group of enterobacteria, but can also be found in the 235
subdivisions of α-, ß-, and other γ -proteobacteria as well as in cyanobacteria. In order to 236
further characterize the interaction between MtfA and Mlc, we created a multiple alignment 237
of various MtfA proteins to identify highly conserved amino acid residues which might be of 238
functional importance (Fig.1). In this alignment, MtfA homologs from ten different bacteria 239
and covering a broad spectrum of identity were used (39 to 89% identical amino acid 240
residues). All of these bacteria harbor an Mlc-homolog protein. Twenty-five amino acid 241
residues were identified as absolutely invariant, and another 32 residues were highly 242
conserved. One important conserved motif is composed of the sequence 149HExxH153 which 243
characterizes zinc-dependent metalloproteases. Interestingly, many invariant amino acid 244
residues were clustered at the carboxy-terminal end of MtfA, indicating another important 245
region of the protein. Based on this multiple alignment, a collection of highly conserved 246
amino acid residues were separately substituted by site-directed mutagenesis (marked in 247
grey). 248
249
Substitution of conserved amino acids and determination of protein stability. To further 250
analyse the interaction between MtfA and the repressor protein Mlc, we used site-directed 251
mutagenesis to define mutants of MtfA. In order to identify specific effects of single amino 252
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acid residues, we created substitutions with chemically related residues. Western blot analysis 253
was used to test protein stability of all MtfA derivatives. In almost all cases a signal was 254
detected at the expected size of 32 kDa. Only the two derivatives with the substitutions I148E 255
(in contrast to I148Y) and A215D (in contrast to A215S) exhibited massive protein decay 256
(data not shown). Thus, compared to wild type, the majority of MtfA derivatives provided an 257
equally stable protein, which could then be further characterized. 258
259
Titration of Mlc by MtfA and its derivatives in vivo. The intensity of the interaction 260
between Mlc and MtfA wild type or its derivatives was tested in vivo using a quantitative 261
measure of the β-galactosidase activity of a single copy ptsGop-lacZ fusion (34). The test 262
system is shown in figure 2. We introduced plasmids encoding MtfA or its derivatives into the 263
indicated strain background (e.g. dgsA+, deletions of ptsG and lacZ), induced mtfA expression 264
and measured the strength of Mlc titration by determining ß-galactosidase activities. The β-265
galactosidase activity of completely induced MtfA wild type was always used as a control and 266
set to 100%, basal expression levels were determined using the empty expression vector 267
pTM30 (shown by grey columns in Fig. 3). 25 of the 59 MtfA derivatives tested exhibited no 268
or only minor reduction in Mlc titration (at least 80% wild type activity, white columns in 269
Fig.3). However, 34 different MtfA mutants exhibited a significantly reduced Mlc titration (as 270
indicated by black columns). Within the amino-terminal part of the MtfA, only the two 271
substitutions L40E and A70D led to decreased interaction with Mlc. In contrast, a 272
replacement of these two residues by other neutral amino acids (L40A or L40T and A70S, 273
respectively) had almost no effect, indicating that negative charges at these positions either 274
interfere with the protein-protein interaction or with the overall conformation of the protein. 275
Within the carboxy-terminal part of MtfA, many different amino acid substitutions led to 276
substantial changes in the interactions with Mlc. In particular, the two regions, from position 277
132 to 149 and position 214 to 243, seemed to be essential for the binding of Mlc. Due to 278
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PCR-artifacts; we found eight derivatives with an additional mutation next to the substitution 279
of interest. In cases where strong effects were present, we reintroduced the conserved amino 280
acid residue to study the impacts of each single substitution. In the case of the double mutant 281
D156N/D161E, the latter mutation suppressed the phenotype of the first. In the double mutant 282
Y205S/A207D, the decreasing interaction with Mlc appeared to be due to a cumulative effect, 283
since each single mutation exhibited higher ß-galactosidase activity. In the case of the double 284
mutant V216L/L217I the decrease in interaction seems to be caused by the latter change, 285
because the single V216L mutation had almost no effect. However, changing V216 to an 286
aspartate residue again reduced the activity to less than 50%, indicating that both residues 287
may be of importance. For all other double mutants, the second substitution did not change 288
the result of the single substitution of the conserved amino acid. 289
290
The carboxy-terminus of MtfA binds Mlc in a bacterial-two-hybrid assay. Given the 291
results from the in vivo titration assays, the carboxy-terminal part of MtfA appears to be 292
responsible for the binding of Mlc. To verify this hypothesis, we used a bacterial-two-hybrid 293
assay (8). This assay employed a chromosomal lacZ gene under the control of a hybrid lexA 294
operator-promoter region in the E. coli test strain SU202. One half of this artificial operator is 295
recognized by the helix-turn-helix DNA-binding domain of the LexA wild type repressor, the 296
other half by a LexA* mutant. Both DNA-binding domains are encoded by compatible 297
plasmids and can be coupled to different proteins of interest. Expression of these hybrid genes 298
can be induced by IPTG. Only in the case of crosstalk between the two hybrid proteins, the 299
two different DNA-binding domains of LexA and LexA* come together to repress lacZ 300
expression as was seen in the two control plasmids pMS604 (encoding a LexA-Fos-hybrid 301
protein) and pDL804 (encoding a LexA*-Jun-hybrid protein). As shown in figure 4, when 302
fused to the LexA-DNA-binding domain, both full length MtfA and MtfAΔ1-83, which is 303
missing the amino-terminal part of the protein, are able to interact with Mlc fused to LexA*. 304
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These results clearly demonstrate that the carboxy-terminal part of MtfA is sufficient for Mlc 305
binding. Furthermore, as an internal control, we fused Mlc to both different DNA-binding 306
domains of the LexA repressor. Expression of these two hybrid proteins resulted in a low β-307
galactosidase activity after induction, confirming the previously published dimerization 308
(tetramerization) of Mlc (26). Cells with only one of the two LexA-Mlc hybrid proteins 309
always showed a very high β-galactosidase activity, indicating a high specificity of the 310
different LexA DNA-binding domains for their cognate operator sites (data not shown). 311
312
Gel shift assays confirm Mlc titration by MtfA. Our in vivo results indicated that the 313
interaction between MtfA and Mlc prevents binding of the repressor to the ptsG-operator. In 314
order to further characterize this effect, we employed electrophoretic mobility shift assays 315
(EMSA) with purified Mlc and MtfA proteins. Addition of Mlc to a ptsGop fragment led to the 316
formation of a repressor-operator complex and thus a mobility shift (Fig.5a). In contrast, 317
addition of MtfA to the labeled operator-fragment had no effect, indicating that MtfA has no 318
DNA-binding properties. Incubation of Mlc and MtfA prior to incubation with the DNA 319
reduced the amount of the repressor-operator complex. Increasing amounts of MtfA led to an 320
increasing fraction of free ptsGop-DNA. A super complex of DNA, Mlc and MtfA was never 321
observed. 322
323
MtfA binds to the super repressor Mlc*H86R in vivo and in vitro. In order to distinguish 324
between the two currently known inactivating mechanisms for Mlc, we used an Mlc super 325
repressor. This Mlc* derivative carries an amino acid substitution of histidine 86 to arginine 326
and is unable to bind the unphosphorylated B domain of the EIICBGlc (27). Thus, in contrast 327
to the wild type, the super repressor cannot be inactivated by EIICBGlc. The in vivo interaction 328
between the super repressor Mlc*H86R and MtfA was investigated using a similar system as 329
presented in figure 2. In the case of the Mlc wild type in strain LZ110, overexpression of 330
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MtfA caused a high β-galactosidase activity of the single copy ptsGop-lacZ fusion as expected 331
(Fig. 6). In contrast, the introduction of a dgsA deletion into this strain background (resulting 332
in JEK5) led to a constitutive ß-galactosidase activity. Interestingly, overexpression of MtfA 333
caused a significant increase of ptsG expression in the absence of Mlc. Since MtfA does not 334
directly bind to the ptsG operator as shown in figure 5, this might indicate some yet unknow 335
interaction of MtfA with a different regulator of ptsG expression. Subsequently, we 336
reintroduced into JEK5 a single copy of either a wild type dgsA+ (giving strain JSW1), or the 337
dgsA* allele encoding the Mlc*H86R super repressor (giving strain JSW2). Both repressors 338
strongly reduced the ptsGop-lacZ expression. Remarkably, JSW2 harbouring the super 339
repressor Mlc*H86R exhibited almost no basal ß-galactosidase activity. Induction of plasmid-340
encoded mtfA+ in both strain backgrounds resulted in high expression of the ptsGop-lacZ 341
fusion, meaning that both wild type Mlc and Mlc*H86R can be bound and inactivated by 342
MtfA. However, corresponding to the very low basal expression level in the presence of the 343
super repressor, ß-galactosidase activity in JSW2 after induction of mtfA+ were always lower 344
compared to the wild type. 345
To further characterize the MtfA interaction with Mlc*H86R in vitro we purified the super 346
repressor and used it in an EMSA experiment with the ptsGop-fragment. In accordance to the 347
in vivo titration data and the results from the band shift assays with the wild type repressor, 348
addition of MtfA to the complex of Mlc*H86R with the ptsGop-fragment led to a release of 349
the operator DNA (Fig. 5b). This seemed to indicate that the inactivation mechanisms of Mlc 350
by unphosphorylated EIIBGlc and MtfA differ significantly. 351
352
MtfA shows amino-peptidase activity. The MtfA crystal structure from Klebsiella 353
pneumoniae was recently characterized (DOI:10.2210/pdb3khi/pdb). 76% of the amino acids 354
are shared by MtfA of K. pneumoniae and E. coli MtfA. The analysis of the overall MtfA 355
architecture revealed structural similarities to the zinc-dependent metalloprotease Lethal 356
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Factor (LF) of Bacillus anthracis or to the Mop protein, which is involved in virulence of 357
Vibrio cholera (Qingping Xu, Anna Göhler et al., in preparation). Therefore, we tried to 358
characterize the putative protease activity of E. coli MtfA. As the natural target and the 359
substrate specificity of MtfA are currently unknown, we made use of several relatively 360
unspecific and artificial protease activity assays, which monitor the cleavage of peptide 361
substrates fused to 4-nitroanilide. Originally, a 4-nitroanilide substrate was used to 362
characterize the in vitro activity of LF from B. anthracis (30). Various different L-amino 4-363
nitroanilide derivatives were chosen to look for amino-peptidase activity as indicated. The 364
greatest activity and specificity was observed for L-alanine fused to 4-nitroanilide as shown in 365
figure 7. Other artificial substrates (arginine, proline or valine fused to 4-nitroanilide, 366
respectively) were cleaved off with significantly lower activities. Moreover, L-glutamic-acid-367
4-nitroanilide as an acidic amino acid and substrates larger in size like L-alanine-L-alanine-L-368
alanine-4-nitronalide were not cleaved at all (data not shown). Subsequently, hippuryl-L-369
phenylalanine and hippuryl-L-arginine were used to test for carboxy-peptidase activity. Again, 370
only very low activities were obtained for these artificial substrates. Furthermore, we tested 371
FTC-casein, a substrate for many endo-peptidases regarding proteolytic conversion by MtfA. 372
In contrast to the artificial amino-peptidase substrates we did not detect any protease activity 373
after incubation of FTC-casein with purified MtfA (data not shown). These results suggest 374
that MtfA is an amino- rather than a carboxy- or endo-peptidase. Since the highest efficiency 375
of MtfA activity was monitored by cleaving off an amino acid from the artificial substrate L-376
alanine-p-nitroanilide, this substrate was used to further characterize the peptidase activity of 377
MtfA. Subsequently, we tested several protease inhibitors. Addition of the chelators EDTA or 378
phenantroline significantly reduced the peptidase activity of MtfA, whereas the addition of 379
other protease inhibitors (e.g. AEBSF) had much less effect. This finding is consistent with 380
the idea that MtfA is a zinc-dependent protease. Addition of higher amounts of EDTA or 381
phenantroline caused an irreversible denaturation of MtfA, meaning that zinc-ions are 382
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absolutely required to stabilize the protein structure and keep it in solution. Furthermore, lack 383
of complete inhibition by the weaker chelator EDTA was previously also observed with other 384
HExxH metalloproteases, such as PepN (4). 385
386
Amino-peptidase activities of MtfA mutants. In the next step we examined the collection of 387
MtfA mutants for their amino-peptidase activities. All mutants were purified and tested for 388
their ability to cleave off the alanine residue from the L-alanine-p-nitroanilide substrate. 389
Freshly prepared, wild type MtfA protein was always taken as a control and set to 100%. The 390
results for all MtfA derivatives are given in figure 8. Mutants with substitutions at positions 391
H149, E150, H153 (HExxH motif) or E212 displayed clearly reduced protease activities 392
probably due to the fact that the coordination of the zinc-ion, the overall conformation of the 393
protein and thus the catalytic activity were affected. With respect to the histidine residues 149 394
and 153 and their involvement in zinc-coordination, a substitution for lysine led to a stronger 395
effect than a replacement by glutamine. A triple mutant with replacements (by alanine) of all 396
three functionally important residues of the HExxH motif was insoluble and could not further 397
be analysed (data not shown). Substantial changes in the proteolytic activities were also 398
observed for mutants with substitutions of the two glutamate residues at positions 150 and 399
212. On the basis of the MtfAKpn crystal structure one would expect these residues to be 400
involved in catalysis. The substitution E150Q influenced the protease activity most 401
significantly. In contrast, as shown in figure 3, the replacement of this residue with aspartic 402
acid or glutamine, respectively, did not affect Mlc binding in vivo. Moreover, as illustrated in 403
figures 5c and 5d, the purified MtfAE150D or MtfAE150Q proteins are still capable of 404
causing a release of the ptsGop -fragment in a band shift assay with purified Mlc in vitro. 405
These results demonstrate that the two MtfA functions of Mlc-binding and proteolysis are 406
clearly distinct from each other. Other substitutions that led to reduced proteolytic activities, 407
but retained Mlc binding include Y94S and P225S. MtfA derivatives with the substitutions 408
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S142F, D156N, A207D, V216D, or Y243S, respectively, exhibited both reduced enzyme 409
activity and reduced Mlc binding, which might indicate severe changes in structure and 410
function. Furthermore, replacements of the residues isoleucine 148 with glutamic acid and 411
alanine 215 with aspartic acid resulted in protein instability. Thus, the reduced activities of 412
MtfAI148Y and MtfAA215S may also be caused by enhanced protein degradation. 413
414
Mlc influences protease activity. The results described above raised the question as to 415
whether Mlc is a substrate for MtfA or whether it has an influence on its protease activity. 416
Therefore, purified MtfA and Mlc were incubated together in a ratio of 1 to 2 (MtfA is known 417
to form dimers, Mlc to form tetramers) and tested for amino-peptidase activity. As illustrated 418
in figure 9, the addition of amino-terminal his-tagged Mlc to the standard assay led to an 419
increased proteolytic activity. The effect was even more severe with untagged Mlc. Addition 420
of equal amounts of BSA caused no significant effect.The mutant protein MtfAN145D, which 421
displayed a reduced interaction with Mlc in vivo (Fig. 3) but almost wild type proteolytic 422
activity (Fig. 8), was significantly less stimulated by his-tagged Mlc. The presence of some 423
stimulation may have been caused by the in vitro test conditions, which required high 424
amounts of purified protein. Regardless, Mlc can clearly be excluded as a biologically 425
relevant substrate. 426
427
Discussion 428
In Escherichia coli K-12, one of the best studied model organisms, the function of many 429
putative open reading frames remains unknown. These had included mtfA (formerly yeeI), 430
despite the fact that identification of orthologs of the E. coli mtfA gene in more than 100 431
proteobacteria of the α-, β-, and γ- subdivisions have implied an important function of this 432
protein. Using surface plasmon resonance assays we were able to demonstrate in a previous 433
paper that MtfA binds with high affinity to the glucose-repressor Mlc (2). Moreover, we could 434
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demonstrate that MtfA binds specifically to the carboxy-terminal segment of Mlc, which is 435
usually involved in protein tetramerization. In this manner, MtfA amongst other factors seems 436
to play an important role in the regulation of the glucose-PTS. In turn, the glucose-PTS has an 437
important function in the regulation of carbohydrate metabolism in E.coli. Mlc, for example, 438
not only regulates ptsG gene expression, but is also a signal mediator for glucose induction of 439
the ptsHIcrr operon (20, 29, 34), the malT gene for the transcriptional activator of the maltose 440
regulon (6), the manXYZ operon for the mannose-PTS (18) and enzymes involved in 441
glycolysis (5), respectively. Interestingly, DNA-binding activity of Mlc in E. coli is not 442
regulated by a small molecular inducer. Instead, Mlc is inactivated by binding to the 443
dephosphorylated EIIBGlc domain of the EIICBGlc during glucose transport (13, 16, 28, 34). 444
The localization of EIIBGlc at the membrane appears to be essential for this kind of 445
inactivation mechanism (27), since overproduction of the liberated soluble EIIBGlc domain 446
does not lead to Mlc inactivation (13, 34), though unphosphorylated EIIBGlc alone binds to 447
Mlc as shown in surface plasmon resonance assays (16). In contrast, membrane-bound fusion 448
proteins consisting of the EIIBGlc domain in combination with either the lactose permease 449
LacY (27) or the bacteriophage M13 membrane coat protein Gp8 (26) are perfectly able to 450
sequester Mlc. Thus, binding of EIIBGlc to Mlc might either not cause any conformational 451
change of the repressor, or there might be no similar conformational change as caused by 452
MtfA binding. Instead by anchoring the Mlc tetramer to the membrane via EIICBGlc, the 453
structural flexibility of Mlc monomers within the complex becomes restricted, which seems to 454
be responsible for the loss of its DNA binding activity (17). Crystal structure analysis of E. 455
coli Mlc (25) revealed the existence of three functional domains: the amino-terminal part 456
containing the helix-turn-helix DNA-binding motif (D-domain), an oligomerization domain 457
from residues 195-380 (O-domain) and an inner EIIBGlc binding domain (E-domain). There 458
appears to be only weak interactions between the three domains and in particular the O-459
domain is separated from the residual protein by a highly flexible hinge. However, structural 460
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rearrangements of the O-domain remotely affect the conformation of the DNA-binding 461
domain by an unknown mechanism (17). In contrast to EIIBGlc, inactivation of Mlc by MtfA 462
occurs in the cytosol and is not membrane-dependent (2). Tanaka et al. (2004) isolated an Mlc 463
super repressor, which carries a H86R amino acid substitution. According to the domain 464
model, Mlc*H86R cannot be inactivated by EIICBGlc. In this paper we could demonstrate that 465
MtfA is still able to bind and inactivate Mlc*H86R both in vivo and in vitro. This clearly 466
supports the notion that MtfA-Mlc interaction de facto occurs at the C-domain of Mlc, which 467
may in contrast to the EIIBGlc-Mlc interaction, result in either severe changes in the 468
conformation of the D-domain or prevention of repressor oligomerization. Both mechanisms 469
would affect the DNA-binding activity of Mlc. Both the presence of a ternary complex in vivo 470
and that of cooperative binding effects remain uncertain and thus are areas in need of further 471
elucidation. 472
Using systematic site directed mutagenesis of conserved amino acid residues in MtfA and 473
subsequent in vivo activity assays, we identified a Mlc interaction domain within the carboxy-474
terminal part of MtfA. The results of in vivo phenotyping of the interaction between MtfA and 475
its derivatives with Mlc led to the conclusion that amino acids from residue 132 onwards 476
appear to be necessary for this interaction. Further experiments employing a bacterial two-477
hybrid assay have also supported this idea. However, two amino acid substitutions (L40E and 478
A70D) in the amino-terminus also caused a significant reduction in Mlc titration, whereas 479
conserved exchanges at these two positions had no effect. This might indicate that there are 480
two classes of amino acid substitution that affect Mlc binding: Substitutions of the first class 481
disturb the overall conformation of MtfA, whereas substitutions of the second class directly 482
affect the MtfA-Mlc interaction. 483
Recently, the crystal structure of MtfA from Klebsiella pneumonia (MtfAKpn ) was 484
characterized at the Joint Center for Structural Genomics (DOI:10.2210/pdb3khi/pdb). A 485
projection of the MtfAEc sequence onto the MtfAKpn structure revealed that at least eight of the 486
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identified amino acid residues in the carboxy-terminus are exposed on the surface of MtfA. 487
Furthermore, these residues are clearly clustered at one side of the protein (Fig. 10). Thus, it is 488
highly likely that these residues are directly involved in Mlc binding. In contrast, other 489
residues like L40 or A70 are almost or completely buried inside the protein. 490
The MtfAKpn structure revealed overall similarities to the zinc-dependent metalloprotease 491
Lethal Factor (LF) from Bacillus anthracis as well as other metalloproteases in the gluzincin 492
clan. Furthermore, we previously confirmed the presence of proteolytic activity of the 493
MtfAKpn protein and the existence of the artificial substrate L-alanine 4-nitroanilide (Qingping 494
Xu, Anna Göhler et al., manuscript in preparation). In this study, we were able to 495
demonstrate a similar activity for the E.coli protein. With an assortment of MtfA derivatives, 496
our results support the notion of MtfA as a metal dependent protease. Single substitutions of 497
amino acids involved in zinc-coordination or catalysis of the substrate show, as deduced from 498
the structure, significantly reduced in vitro activities. An MtfA mutant with a triple 499
substitution at positions 149, 150 and 153 (zinc-coordination-residues) for alanine, could not 500
be investigated in vitro, suggesting a structural instability of the residual protein without the 501
metal ion. Further analysis to identify a metal-bound and metal-unbound state by electrospray 502
ionization mass spectrometry failed due to irreversible unfolding of MtfA at low pH 503
conditions. However, once again this result points to a metal-dependent peptidase activity of 504
MtfA. 505
These findings now raise questions about the biological relevance of the MtfA-Mlc 506
interaction. Mlc is clearly not a target protein. On the contrary, addition of Mlc stimulates 507
MtfA amino-peptidase activity. Preliminary analysis of the MtfKpn structure revealed the 508
existence of a self-inhibitory complex (Qingping Xu, Anna Göhler et al., in preparation). It is 509
tempting to speculate that Mlc binding causes a conformational change in MtfA, which may 510
override self-inhibition. Transcriptome analysis by Lemuth (14) under glucose limitation 511
revealed a significant up-regulation of mtfA expression under growth conditions during the 512
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transition from the exponential to the stationary phase. According to the Mlc regulation 513
model, running out of glucose would lead to a rephosphorylation of EIICBGlc and thus to a 514
release of Mlc into the cytoplasm. Both effects would boost the overall MtfA protease activity 515
at this growth transition point. This leads to a model according to which the glucose sensor 516
Mlc/EIICBGlc not only controls gene expression by Mlc, but that at least in E. coli it also 517
regulates the proteolytic activity of MtfA in a glucose-dependent manner. This reflects a 518
novel regulatory output signal by the glucose-PTS. 519
Questions still remain regarding which proteins are natural targets for MtfA. For example, the 520
structurally related endopeptidase LF of B. subtilis cleaves off the amino-terminal peptides of 521
mitogen-activated protein kinase kinases (MAPKKs) during host infections (9, 31). It is 522
tempting to speculate that MtfA also may cleave off short peptides from its natural target 523
proteins rather than working just as an amino-peptidase. In a series of unspecific crosslinking 524
experiments to identify further interaction partners of MtfA, several putative target proteins 525
were identified (our unpublished results). Among others, the γ-subunit of the ATP synthase 526
was identified 4 times by 5 screening approaches under different conditions. Variations in the 527
number of ATP synthase complexes might be necessary for growth adaptation under changing 528
environmental conditions, making the γ-subunit a promising target candidate of MtfA. 529
In conclusion, by elucidating that MtfA is a glucose-regulated peptidase, we have identified a 530
novel regulatory output mechanism of the glucose-PTS, which may be involved in the 531
adaptation of E. coli cells during the transition from the glucose-phase to the next consecutive 532
growth phase. Since the physiologically relevant target is not known yet, further investigation 533
is necessary. 534
535
Acknowledgements 536
We gratefully acknowledge Kurt Schmid, Hans-Peter Schmitz, Katja Bettenbrock, 537
Gabriele Deckers-Hebestreit, Karin Lemuth and Jürgen Heinisch for helpful discussions, 538
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Katrin Fänger for excellent technical support and Lucille Schmieding and Jennifer Manne for 539
help with the manuscript. 540
This work was financially supported by the German Research Foundation (DFG) through the 541
“Sonderforschungsbereich” 431 (Teilprojekt P14 to K. Jahreis) and by the German Ministry 542
of Education and Research through the FORSYS-program (grant FKZ 0315285C to K. 543
Jahreis). 544
References 545
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33. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning 641 vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. 642 Gene 33:103-119. 643
34. Zeppenfeld, T., C. Larisch, J. W. Lengeler, and K. Jahreis. 2000. Glucose 644 transporter mutants of Escherichia coli K-12 with changes in substrate recognition of 645 IICB(Glc) and induction behavior of the ptsG gene. J Bacteriol 182:4443-4452. 646
647 648
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Tables 649 650
TABLE 1. Escherichia coli strains used in this study 651
Strains Relevant Genotype or Phenotype Source or Reference
JM109 thi-1 Δ(lac-proA,B) recA1 hsdR1 / F´traD36 proA+ B+ lacIqZ M15
(33)
LZ110 LJ110 ΔlacU169 zah-735::Tn10tet (34)
LZ150 LZ110 Δ(ptsG::cat+) (34)
JEK5 LZ110 Δ(dgsA::Tn10kan) this study
JSW1 JEK5 pheU::dgsA+ cat+ this study
JSW2 JEK5 pheU:: dgsA*(Mlc*H86R) cat+ this study
XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac /F´proAB+ lacIq ZΔM15 Tn10TcR
Stratagene
SU202 lexA71::Tn5 su1A211 sulA::lacZ ΔlacU169/ F´lacIq lacZΔM15::Tn9
(8)
652
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Legends 653
Figure 1. Multiple Alignment of MtfAEco and ortholog proteins from other bacteria. 654
The amino acid sequence of MtfAEco was aligned with the presented homologues using the 655
ClustalW program. Abbreviations are as follows: Eco: Eschericha coli gi|90111364; 656
Escherichia fergusonii gi|22034302; Kpn: Klebsiella pneumoniae gi|152970975; Sty: 657
Salmonella enterica serovar Typhimurium gi|16420534; Ype: Yersinia pestis gi|22126457; 658
Cvi: Chromobacterium violaceum ATCC gi|34104211; Psy: Pseudomonas syringae pv tomato 659
DC3000 gi|28851187; Xca: Xanthomonas campestris pv. Campestris strain ATCC 33913 660
gi|21115196; Rso: Ralstonia solanacearum GMI 1000 gi|17428198; Npu: Nostoc punctiforme 661
PCC 73102 gi|23129869. Identical residues are marked by a *, highly conserved residues by 662
a :, conserved residues by a . Numbers of identical residues in binary comparisons between E. 663
coli MtfA and the respective homologues of all other bacteria are given in the box. 664
Substituted amino acids are marked in grey. 665
666 Figure 2. Test system to quantify the MtfA interaction with Mlc in vivo. 667
This figure illustrates the test system for phenotyping MtfA mutants. The strain background 668
LZ150F´::Ф(ptsGop-lacZ) provides a chromosomally encoded dgsA+ gene (mlc+), 669
chromosomal deletions of the ptsG and the lacZ genes, and a single copy ptsGop-lacZ fusion 670
on an F´-plasmid. MtfA or its derivatives were introduced via the IPTG inducible expression 671
plasmid pTMByeeI-S or derivatives thereof, respectively. Since Mlc cannot bind to the 672
EIIBGlc domain in this strain, it can only be inactivated by MtfA. Thus, inactivation of Mlc by 673
MtfA is reflected by high β-galactosidase activities. 674
675 Figure 3. β-galactosidase activities for the quantification of the interactions between MtfA 676
wild type or derivatives thereof with Mlc in vivo. 677
The results of phenotyping MtfA and defined mutants with regard to Mlc titration are shown. 678
The test system presented in figure 2 was used. Cells harboring the pTMByeeI-S vector or 679
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derivatives thereof were grown in LBo, mtfA expression was induced with IPTG, and cells 680
were harvested for β-galactosidase activity assays. The activity of the wild type control 681
(pTMByeeI-S), which was ususally around 850 nmol per mg protein and min, was set to 682
100% in each set of experiments. The empty expression vector pTM30 served as a negative 683
control (represented by grey bars). Mutants, which exhibited β-galactosidase activities of 684
more than 80% of the wild type level, were drawn in white columns. In contrast, MtfA 685
mutants with less than 80% activity, which reflected a severe reduction of Mlc binding, are 686
presented by black columns. MtfA mutants K/Q/K/Q and A/A/A denote substitutions 687
H149K/E150Q/H153K/E212Q and H149A/E150A/H153A, respectively. Averages of at least 688
three independent tests with standard deviations are given. 689
690 Figure 4. Bacterial-two-hybrid assays to test in vivo interaction of MtfA with Mlc. 691
Cells were grown in LBo without (-) and with (+) IPTG, harvested and analyzed for their β-692
galactosidase activities. Control samples (SU202: test strain without any plasmid as a negative 693
control; pMS (LexA-Fos-hybrid protein)/pDL (LexA*-Jun-hybrid protein) as a positive 694
control) are shown in grey. The results for the analyzed test samples (pMM3 and pDM3: Mlc 695
full-length constructs; pMM2: MtfA full-length construct; pMM4: MtfAΔ1-83 construct) are 696
illustrated by the white bars. All test combinations showed reduced β-galactosidase activities 697
(given in nmol per min and mg total cell protein) under inducing conditions, which indicates 698
an interaction of the fusion proteins. Averages of at least three independent measurements and 699
standard deviations are given. 700
701 Figure 5. Electrophoretic mobility shift assays (EMSA) with purified Mlc and MtfA proteins. 702
a. A fluorescence-labeled DNA fragment containing the Mlc-binding sites in front of the ptsG 703
gene (ptsGop) was incubated with different combinations of purified Mlc and MtfA and 704
subsequently run on a native gel. A control sample without any protein (-) led to an unshifted 705
signal of the DNA. After incubation of the operator fragment with purified Mlc (+), a retarded 706
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Mlc-DNA complex could be detected. Addition of purified MtfA (150 nM in the assay) 707
caused not band shift. Incubation of Mlc (usually 25nM in the assay) with rising amounts of 708
MtfA (usually 25 to 150nM in the assay) as indicated prior to the incubation with ptsGop led 709
to an increasing release of operator fragments. 710
b. Incubation of the ptsG-operator fragment with purified super repressor Mlc*H86R led to 711
the generation of an Mlc-DNA complex. Similar to the results obtained for the wild type 712
repressor, addition of MtfA to the band shift assay prior to the incubation with ptsGop led to 713
released operator fragments. 714
c. Incubation of the ptsG-operator fragment with purified wild type Mlc and increasing 715
amounts of MtfA E150D. 716
d. Incubation of the ptsG-operator fragment with purified wild type Mlc and increasing 717
amounts of MtfA E150Q, further explanations are given in the text. 718
719 Figure 6. β-galactosidase assays for the characterization of the in vivo interaction between 720
MtfA wild type and either Mlc wild type or Mlc*H86R super repressor. 721
The test system was similar to the one described in figure 2. LZ110 was used as dgsA+ wild 722
type control (as indicated by grey bars). JEK5 carries a dgsA deletion. JSW1 carries a dgsA 723
deletion and a single wild type copy of dgsA+ at a different position in the chromosome. 724
JSW2 carries also a dgsA deletion and a single copy dgsA* gene encoding the Mlc*H86R 725
super repressor. All strains harbor the mtfA wild type expression plasmid pTMByeeIs. Cells 726
were grown in the absence (-) or presence of IPTG (+) and tested for their ß-galactosidase 727
activities, which are given in nmol per min and mg total cell protein. All activities are mean 728
values of at least three independent experiments with standard deviations. 729
730 Figure 7. Peptidase assays of purified MtfA with different substrates. 731
The activity measurements were carried out as described in methods. For L-amino 4-732
nitroanilide substrates, activity was monitored by measuring the change of the optical density 733
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at 390 nm (ΔOD390) triggered by the release of 4-nitroanilide after cleavage. The specific 734
activity [U/mg*min] was calculated. The highest cleavage rate was reached with L-alanine 4-735
nitroanilide as substrate and set as 100%. The typical activity level under the test conditions 736
for this substrate was around 9 mU per mg protein and min. Lower activities were detected 737
for L-arginine 4-nitroanilide as a basic amino acid or for other none polar amino acid 738
substrates such as L-proline or L-valine fused to 4-nitroanilide. Glutamic-acid 4-nitroanilide 739
as an acidic amino acid or alanine-alanine-alanine 4-nitroanilide (data not shown) could not 740
be cleaved by MtfA. Substrates for carboxy peptidase were also investigated. Neither 741
hippuryl-arginine nor hippuryl-L-phenylalanine was cleaved sufficiently by MtfA. The 742
cleavage of L-alanine 4-nitroanilide was not strongly inhibited by the protease inhibitor 743
AEBSF. In contrast, the chelator reagents EDTA and phenantroline reduced proteolytic 744
activity severely as it is shown in the last three columns. 745
All activities are given in % relative to the cleavage of L-alanine 4-nitroanilide. Data are 746
mean values of three independent experiments with standard deviations. 747
748 Figure 8. Protease activity of MtfA wild type and defined MtfA mutants. 749
MtfA and defined mutants (as indicated) were purified and the amino-peptidase activities 750
towards the cleavage of L-alanine 4-nitroanilde were monitored. Each activity was calculated 751
and is diagrammed relatively to the activity of the MtfA wild type protein, which was 752
analyzed in parallel. Wild type activity, which was typically around 9 mU per mg protein and 753
min was set to 100% and is presented by the grey bars. Activities of mutant proteins, which 754
showed more than 80% wt-activity, are displayed by the white columns. In contrast, black 755
columns represent MtfA derivatives, which showed less than 80% wt- proteolytic activity. 756
The MtfA derivative 149/150/153/212 carries the substitutions 757
H149K/E150Q/H153K/E212Q. Standard deviations and average values of at least three 758
independent experiments are indicated. 759
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760
Figure 9. Protease activities of MtfA wild type and MtfAN145D in the absence or presence 761
of Mlc. 762
The cleavage efficiency of L-alanine 4-nitroanilide by MtfA or MtfAN145D was assayed in 763
the presence or absence of Mlc as indicated. Control samples were carried out incubating 764
MtfA either without any supplements or with accessory bovine serum album (BSA). Addition 765
of purified Mlc with an amino-terminal his-tagged or addition of an untagged Mlc version 766
stimulated MtfA enzyme activity up to 175%. In contrast, the MtfA derivative N145D with 767
reduced Mlc binding capacity was only stimulated to 123% of the wild type activity. Activity 768
values are averages of at least three independent experiments with standard deviations. 769
770
Figure 10. Projected MtfAEc protein structure. 771
We used the POLYVIEW-3D web-based tool (http://polyview.cchmc.org/polyview3d.html ; 772
(22)) for molecular structure visualization of MtfAKpn. The positions of amino acid residues 773
132, 142, 145, 149, 153, 205, 207, 216, 219 are indicated in red color. 774
775
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