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Supporting InformationSonnleitner et al. 10.1073/pnas.pnas.0910308106SI Materials and MethodsConstruction of Plasmids and Gene Replacement Mutants. DNAcloning and plasmid preparations were performed according tostandard methods (1). Strains, plasmids, and oligonucleotidesused in this study are listed in Table S2.
To construct a lacZ transcriptional promoter fusion to amiE,a 127-bp fragment containing the ami promoter region wasamplified using the primer pair A1 and B1 and chromosomalDNA of PAO1 as a template. The PCR fragment was digestedwith EcoRI and PstI and ligated into the corresponding sites ofpME6016, resulting in pME9656.
A translational amiE�-�lacZ fusion was constructed by ampli-fying a 262-bp PCR fragment with primers A1 and C1. Thisfragment was digested with EcoRI and PstI and cloned into thecorresponding sites of pME6013. In the resulting plasmidpME9655 the promoter region of the ami operon, amiL, and thesequence encoding the first seven amino acids of amiE are fusedto the �lacZ reporter.
The CA motif mutations were introduced into pME9655according to the QuikChange site-directed mutagenesis protocolwith the mutagenesis primers Y1 and Z1. The parental DNAtemplate was digested with DpnI, and the mutated plasmid wastransformed into E. coli XL1-Blue, generating pME9657.
For Crc purification, the crc gene was cloned under the controlof the T7 promoter and fused to a C-terminal histidine tag. A777-bp crc fragment was amplified by PCR with primers Y4 andZ4 and cloned into the NdeI-XbaI restriction sites of pET22b,resulting in pME9659. This plasmid was used as a template forthe amplification of the histidine-tagged crc gene, which carriesthe artificial Shine-Dalgarno sequence derived from pET22b, byusing the primers B5 and C5. The resulting PCR fragment wasdigested with PstI and BamHI and cloned into the correspondingsites of pMMB67HE under the control of an inducible tacpromoter, generating pME9670.
The translational crc�-�lacZ fusion plasmid pME9668 wasconstructed by amplifying a 774-bp PCR fragment with theprimer pair F6 and H6 and chromosomal DNA of PAO1 as atemplate. The fragment was then digested with EcoRI and PstIand cloned into the corresponding sites of pME6013. Theresulting plasmid pME9668 carries the crc promoter and the first5 crc codons, which were fused to a �lacZ reporter.
The crcZ� overexpression plasmid pME9669 was constructedby amplifying a 427-bp crcZ fragment with primers B6 and C6.This fragment was digested with SpeI and BamHI and clonedinto the corresponding sites of the vector pJT19, which fused thecrcZ gene to the inducible Pm promoter.
To generate the translational �lacZ fusion to xylS, a 668-bpfragment was amplified by PCR using the primers M6 and N6and chromosomal DNA of PAO1 as template. The PCR frag-ment was digested with EcoRI and PstI and inserted into thecorresponding sites of pME6013, resulting in pME9671.
To construct a crc deletion mutant, PAO6673, we used thefollowing procedure. Two PCR products flanking the crc genewere obtained from PAO1 chromosomal DNA with primer pairsO2/P2 and Q2/R2, respectively. The combined 620-bp upstreamand 608-bp downstream fragments were used as a template fora second overlapping PCR with primers O2 and R2, which waspossible because the primers P2 and Q2 carry a complementarysequence. The resulting fragment with a 779-bp deletion, whichspans the entire crc coding region except for the A of the TGAstop codon, was digested with BamHI and EcoRI and ligatedinto the corresponding sites of the suicide vector pME3087. The
resulting plasmid pME9672 was mobilized into strain PAO1 withthe help of E. coli HB101/pRK2013 and chromosomally inte-grated with selection for tetracycline resistance. Excision of thevector by a second crossover was obtained by enrichment fortetracycline-sensitive cells (2). The chromosomal crc deletionwas confirmed by PCR using the primers T3 and U3. Ananalogous procedure was used to generate the chromosomaldeletion mutants of crcZ (PAO6679), the putative P30 gene(PAO6677), and cbrB (PAO6711) using, respectively, the suicidevectors pME9673 (deleting the crcZ promoter and 5� region ofcrcZ), pME9674 (deleting P30), and pME9675 (generating a1430-bp deletion of the entire cbrB gene, starting 10 bp upstreamof the cbrB ATG start codon; the remaining cbrB stop codon isin frame with the coding sequence of cbrA, followed by the�-independent terminator of the cbrAB operon). The followingprimer pairs were used: U1/V1 and G2/X1 (crcZ), U1/E2 andF2/X1 (P30), and D3/E3 and F3/G3 (cbrB).
For the construction of a chromosomally inserted crcZ-lacZfusion in strains PAO1, PAO6358 (�rpoN), and PAO6711(�cbrB), the crcZ promoter region was amplified by PCR withthe primers crcZfw and crcZrev. The resulting 1.58-kb fragmentwas digested by EcoRI and PstI and cloned into the correspond-ing sites of pME6016, generating pME9806, which was thendigested either with EcoRI and EcoRV or with EcoRV andXhoI. The resulting 2.0-kb and 2.8-kb fragments containingcrcZ-lacZ were ligated together, blunted with T4 DNA poly-merase (Promega), and inserted into the SmaI restriction site ofthe miniTn7 vector pME3280a, generating pME9812. For chro-mosomal insertion of miniTn7 carrying crcZ-lacZ into the Tn7attachment site of PAO1, PAO6358, and PAO6711, these re-cipient strains were grown overnight at 43 °C; E. coli S17–1/pME9812 was used as donor and E. coli SM10�pir/pUXBF-13 ashelper for transposition (3). P. aeruginosa strains carrying acrcZ-lacZ insertion were selected with gentamicin (10 �g mL�1)and chloramphenicol (10 �g mL�1) and confirmed by PCR.
RNA Techniques. RNA purification was performed by the hotphenol method as described elsewhere (4). Samples containing10 �g of total RNA were analyzed by Northern blotting asdescribed previously (5) with DIG-labeled probes. For thesynthesis of DIG-labeled, double-stranded probes of crcZ and 5SrRNA, PCR fragments were synthesized using PAO1 genomicDNA as template and the primer pairs B6 and C6 (for crcZ) and5S-rRNA-1 and 5S-rRNA-2 (for 5S rRNA), respectively. ThesePCR fragments and the same set of primers were used for theDIG-labeling procedure, as described by the manufacturer (DIGDNA Labeling Mix; Roche). To generate the crcZ single-stranded probe, a PCR fragment was amplified with primers L3and M3, where L3 carries the T7 promoter sequence. This PCRfragment was used as template for the DIG RNA Labeling Kit(Roche) with T7 RNA polymerase (Roche). The resultingDIG-labeled RNA is complementary to crcZ and was purified ona 6% polyacrylamide/8 M urea gel by extraction with a 0.5 MNa-acetate (pH 5.5) and 1 mM EDTA solution and subsequentphenol-chloroform extraction and ethanol precipitation.
For gel mobility shift assays, the amiE�, amiE�-�CA-motif andCrcZ� RNAs were transcribed in vitro using T7 RNA polymerase(Roche) and PCR fragments as templates. For the truncatedamiE� and amiE�-�CA-motif mRNAs (for details see legends ofFig. 6 and Fig. S6), primers A5 and C1 were used, with pME9655and pME9657, respectively, as templates. For the amplificationof the truncated crcZ� PCR fragment the primer pair E6-E2 was
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used. Primers A5 and E6 carry the sequence for the T7 promoter.RNAs were dephosphorylated with calf intestinal alkaline phos-phatase (Roche) and subsequently 5� end-labeled using [�-32P]-ATP (Hartmann Analytic) and polynucleotide kinase (Fermen-tas). Labeled RNAs were gel-purified and dissolved indiethylpyrocarbonate-treated water.
Purification of Crc Protein. Histidine-tagged Crc was overproducedin E. coli BL21(DE3)/pME9659 by adding 1 mM isopropyl-�-D-thiogalactoside (IPTG) to cells grown to mid-exponentialphase. Cells were harvested 3 h later, and Crc was purified byNi-NTA agarose (Qiagen) according to the manufacturer’s
protocol for batch purification of 6xHis-tagged proteins from E.coli under native conditions (Qiagen). The protein was dialyzedagainst 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 5 mM�-mercaptoethanol. Labeled RNA (20 nM) was incubated withincreasing amounts of purified Crc protein in 10 mM Tris-HCl(pH 8.0), 10 mM MgCl2, 25 mM NaCl, 10 mM DTT, and 25 ngtRNA in 10 �L. Competition assays also contained unlabeledRNA competitor (see legend of Fig. 6 for details). Reactionmixtures were incubated at 37 °C for 30 min to allow Crc–RNAcomplex formation. Samples were then fractionated through 4%polyacrylamide gels in Tris-borate buffer (1).
1. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY).
2. Ye RW, et al. (1995) Anaerobic activation of the entire denitrification pathway inPseudomonas aeruginosa requires Anr, an analog of Fnr. J Bacteriol 177:3606–3609.
3. Bao Y, Lies DP, Fu H, Roberts GP (1991) An improved Tn7-based system for thesingle-copy insertion of cloned genes into chromosomes of gram-negative bacteria.Gene 109:167–168.
4. Leoni L, Ciervo A, Orsi N, Visca P (1996) Iron regulated transcription of the pvdA genein Pseudomonas aeruginosa: Effect of Fur and PvdS on promoter activity. J Bacteriol178:2299–2313.
5. Valverde C, Heeb S, Keel C, Haas D (2003) RsmY, a small regulatory RNA, is required inconcert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonasfluorescens CHA0. Mol Microbiol 50:1361–1379.
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Time after induction with IPTG (h)
amiE’-’lacZ
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Fig. S1. The crc� plasmid pME9670 complements the crc deletion mutant PAO6673 for loss of catabolite repression. �-Galactosidase activity of an amiE�-�lacZfusion (in pME9655) was measured in PAO1/pMMB67HE (filled squares), PAO1/pME9670 (open squares), PAO6673/pMMB67HE (filled triangles), and PAO6673/pME9670 (open triangles), after growth in succinate minimal medium containing 1 mM IPTG for induction of crc expression.
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TTTTTTCGTCCCGAAAAAATTCAGTAGCGAGGTGATATCCATGCGTCACGGCGATATTTC
TTTTTTCGTCCCGAAAAAATAACAACAAGAGGTGATATCCATGCGTCACGGCGATATTTCTCACTCAATATTTGGTTGGTAACAACAAATGCCTGATGTCCGCGTTCTGCCTTGCACGTTACCGATGATTATGTTGTAAAAACAACAATTGATAGTCTGTAGGAGAATCTGCCATGCACCCCTGGCGCGGGGCATTGCGGTTCAACGACGATGAATAACGACAACAAGAGAAGCGCCGGCGCTTGGTGCCGCGCGGCGATAACAACAACGATAGCAGCCTGAGTGTCCTGCCCATGAATGCGGTAGGGCGCGGCGACCAGAACAATAACGATACCGAGTGCCTTGCCTATGGAAAGCCGC
CAmotif
amiEzwfeddglkxylSbenR P.p.
Fig. S2. Nucleotide sequence comparison of translation initiation regions of genes known to be regulated by catabolite repression. The following Crc-regulatedgenes of P. aeruginosa (1, 2) were chosen: amiE, aliphatic amidase; zwf, glucose-6-phosphate dehydrogenase; edd, phosphogluconate dehydratase; glk,glucokinase; and xylS, transcriptional regulator involved in toluate degradation and homologue of benR in P. putida (benR P.p.), which is also known to beregulated by Crc (3). The conserved AACAACAA sequence, named CA motif, is highlighted in gray. Start codons are in boldface and ribosomal binding sites areunderlined. The random sequence of the CA motif mutation of amiE (black box) is given in the first line (�CA motif).
1. Collier DN, Hager PW, Phibbs PV (1996) Catabolite repression control in the pseudomonads. Res Microbiol 147:551–561.2. Wolff JA, MacGregor CH, Eisenberg RC, Phibbs PV (1991) Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J Bacteriol
173:4700–4706.3. Moreno R, Rojo F (2008) The target for the Pseudomonas putida Crc global regulator in the benzoate degradation pathway is the BenR transcriptional regulator. J Bacteriol
190:1539–1545.
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BSM + lactamide+ succinate
BSM + lactamide+ glucose
BSM + lactamide+ mannitol
crc’-’lacZ
Fig. S3. Crc levels do not change with different C sources at constant OD values. A translational crc�-�lacZ fusion carried by pME9668 was measured in thewild-type PAO1 cultivated to OD600 � 1.5 in BSM amended with succinate (gray bar), glucose (white bar), or mannitol (black bar). Lactamide (40 mM) was addedto all media to induce amiE expression.
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Fig. S4. Sequence and secondary structure of the full-length CrcZ sRNA, as predicted by mfold (1, 2). CA motifs are highlighted by contour lines.
1. Mathews DH, Sabina J, Zuker M, Turner DH (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol288:911–940.
2. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415.
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A
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Time after induction (h)
L broth BSM+acetamide+toluate BSM+acetamide
C
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Fig. S5. (A) Positive effect of CrcZ sRNA on amiE expression and complementation of a crcZ mutation with the crcZ� overexpression plasmid pME9669. Strainswere grown in BSM amended with succinate to an OD600 of 0.3 and induced with 2 mM toluate. Thereafter, samples were taken every hour. �-Galactosidaseactivities of an amiE�-�lacZ fusion carried by pME9655 were measured in the wild-type strain PAO1/pJT19 (open squares), in the crcZ mutant PAO6679/pJT19 (opentriangles), in PAO1/pME9669 (filled squares), and in PAO6679/pME9669 (filled triangles). (B) Growth on acetamide as the sole carbon source: strains PAO1/pJT19(bottom), PAO6679/pME9669 (Left) and PAO6679/pJT19 (Right) were streaked out on an L broth plate (Left), a plate containing BSM with 40 mM acetamide and2 mM toluate (Center), and a plate containing BSM with 40 mM acetamide (Right) and incubated at 37 °C for 24 h.
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Crc
mRNA:Crc 1:0 1:10 1:25 1:50 1:100M Crc 0 0.2 0.5 1 2
amiE’
Crc
amiE’- CAmotif
1:0 1:10 1:25 1:50 1:100 0 0.2 0.5 1 2
Crc
sRNA:Crc 1:0 1:5 1:10 1:15 1:20 1:25M Crc 0 0.1 0.2 0.3 0.4 0.5
CrcZ’
A
B
Fig. S6. RNA-binding specificity of the Crc protein. (A) Binding of Crc to wild-type amiE� mRNA (Left) and to amiE� mRNA with the mutated CA-motif (Right).The mRNAs where 5�-end-labeled with �-[32P]ATP and increasing amounts of Crc protein were added as indicated. The amiE� and amiE�-�CAmotif mRNAs aretruncated and consist of the first 154 nt of the transcript, which corresponds to the fragment used for constructing the translational �lacZ fusion in pME9655 andpME9657. (B) Binding of Crc to CrcZ sRNA. A 151-nt CrcZ� fragment truncated at the 3� end was labeled with �-[32P]ATP and incubated with increasing amountsof purified Crc protein.
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Fig. S7. CbrB and RpoN are required for crcZ expression. �-Galactosidase activities of a chromosomally encoded crcZ-lacZ fusion were measured in the wild-typePAO1 (filled squares), in the �cbrB mutant PAO6711 (open squares), and in the �rpoN mutant PAO6358 (filled triangles) in L broth [Sambrook J, Russell DW (2001)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY)].
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Table S1. Substrate utilization by P. aeruginosa cbrB and crcZ mutants
Respiratory activity
Substrate PAO1 (wild type) PAO6711 (�cbrB) PAO6679 (�crcZ)
Amino acidsD-alanine �� � �
L-asparagine �� �� �
L-histidine �� � �
L-leucine � � �
L-ornithine �� � �
L-proline �� � �
Other C sources�-Aminobutyric acid �� �� �
L-aspartic acid �� �� �
D-fructose �� � �
D-gluconic acid �� � �
Glycerol �� � �
�-Hydroxybutyric acid �� � �
p-Hydroxyphenylacetic acid �� � �
D-mannitol �� � �
Propionic acid �� � �
Succinic acid �� � �
The GN2 microplate (Biolog) was used to measure the respiratory activity on different carbon sources. ��, respiratory activity within 24 h; �, respiratoryactivity within 48 h; �, no respiratory activity within 48 h. An overnight culture grown in BSM amended with 40 mM succinate was diluted in 0.9% (wt/vol) NaClto an OD600 of 0.045; 150 �L of this dilution was used to inoculate each well of the plate. The microplate was incubated at 35 °C and rotated at 500 rpm in aTHERMOstar� apparatus (BMG Labtech).
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Table S2. Strains, plasmids, and oligonucleotides used in this study
Strains/plasmid Genotype/relevant features Source
P. aeruginosaPAO1 Wild type 1PAO6673 �crc This studyPAO6677 �P30 (crcZ-internal deletion) This studyPAO6679 �crcZ promoter This studyPAO6711 �cbrB This studyPAO6358 �rpoN 2
E. coliBL21(DE3) F– ompT hsdSB(rB-, mB-) dcm gal �(DE3) NovagenDH5� recA1 endA1 hsdR17 thi-1 supE44 gyrA96 relA1 deoR �(lacZYA-argF) U169 (�80lacZ�M15) 3HB101 thi-1 hsdS20(rB-, mB-) supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 3S17–1 thi pro hsdR recA chromosomal RP4–2 �Tcr::Mu, Kmr::Tn7� 4SM10/�pir thi-1 thr-1 leu-6 tonA21 lacY1 supE44 recA chromosomal RP4–2 �Tcr::Mu Kmr::Tn7� �pir 5XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17(rK-, mK�) supE44 relA1 lac �F� proAB lacIqZ�M15::Tn10(Tcr)� Stratagene
PlasmidspET22b T7 expression vector, C-terminal histidine tag; Apr NovagenpJT19 IncP expression vector carrying an inducible Pm promoter; Kmr 6pMMB67HE IncQ expression vector carrying an inducible tac promoter; Ap/Cbr 7pUC18 Cloning vector, ColE1 replicon; Apr 8pUX-BF13 Helper plasmid containing Tn7 transposition functions, R6K replicon; Apr 9pRK2013 Helper plasmid, ColE1 replicon, Tra; Kmr 10pME3087 Suicide vector, ColE1 replicon, Mob; Tcr 11pME3280a Chromosomal integration vector, mini-Tn7; Gmr Apr 12pME6013 Cloning vector for translational lacZ fusions; Tcr 13pME6016 Cloning vector for transcriptional lacZ fusions; Tcr 13pME9655 pME6013 with amiE�-�lacZ This studypME9656 pME6016 with amiE-lacZ This studypME9657 pME9655 with �CA motif mutation in amiE leader This studypME9659 pET22b with crc fused to C-terminal histidine tag This studypME9668 pME6013 with crc�-�lacZ This studypME9669 pJT19 with crcZ This studypME9670 pMMB67HE with crc-his6 This studypME9671 pME6013 with xylS�-�lacZ This studypME9672 pME3087 with a 779-bp deletion of crc This studypME9673 pME3087 with a 160-bp deletion in crcZ This studypME9674 pME3087 with a 193-bp deletion of P30 This studypME9675 pME3087 with a 1430-bp deletion of cbrB This studypME9806 pME6016 with crcZ-lacZ This studypME9812 pME3280a with crcZ-lacZ in mini-Tn7 This study
DNA oligonucleotides Sequence (5�33�) Restrictionsite
A1 TTTTTTGAATTCGGCTGCATGCTATCTCAGGCGC EcoRIB1 TTTTTTCTGCAGCTGATGCGCACGACCTGAT PstIC1 TTTTTTCTGCAGGAAATATCGCCGTGACGCAT PstIU1 ACGTGGATCCACCGCGACCTGAAAACCC BamHIV1 CGGTGGGTCGGCGGAGGGCAC
G2 GTGCCCTCCGCCGACCCACCGATTCTTTTGGAGAGGAGTTG
X1 ACGTGAATTCGGCGCGGACCTGC EcoRIY1 CGTCCCGAAAAAATTCAGTAGCGAGGTGATATCCATGCGTCACG
Z1 CGTGACGCATGGATATCACCTCGCTACTGAATTTTTTCGGGACG
E2 CTTCTTCCGACTGGCTGCGGG
F2 CCCGCAGCCAGTCGGAAGAAGACTTGGGGGGGAGCTTCGG
O2 ACGTGGATCCGCTCACCACCGGCATGCC BamHIP2 AAATGGCCCCCAAAATC
Q2 GATTTTGGGGGCCATTTAGCCCTGTCAGACACGAAAAAG
R2 ACGTGAATTCGCTTCATCGAGGTGCAGGG EcoRID3 ACGTGGATCCCTTCCATCGGCCGCCTAG BamHIE3 CTCTCGACGTGCTCGTCTAC
F3 GTAGACGAGCACGTCGAGAGCCTCGGCCGACTCGTAACAC
G3 ACGTGAATTCGGATTCTAGCATTGGATGGGG EcoRIL3 TCTAGACGTAATACGACTCACTATAGGCCGAAGCTCCCCCCCAAG XbaIM3 CCACCGAAAACCTCAACCC
T3 GCAGAACCCCGCGCTCG
U3 GCTGGTGGTGATCGGCTTC
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Strains/plasmid Genotype/relevant features Source
Y4 ATGCCATATGCGGATCATCAGTGTGA NdeIZ4 ATGCCTCGAGGATGCTCAACTGCCAGTCG XhoIA5 AATCTAGACGTAATACGACTCACTATAGATCAGGTCGTGCGCATCAG XbaIB5 TTTTCTGCAGGGATAACAATTCCCCTCTAGAAAT PstIC5 TTTTGGATCCCTTCCTTTCGGGCTTTGTTAG BamHIB6 ATGCACTAGTGCACAACAACAATAACAAGC SpeIC6 ATGCGGATCCGAAATGGTGTAAGGCGAAGG BamHIE6 TCTAGACGTAATACGACTCACTATAGGCACAACAACAATAACAAGC XbaIF6 TTTTAAGCTTGAATTCATCCTGTCCGACCATGC EcoRIH6 TTTTTTCTGCAGACACTGATGATCCGCAT PstIM6 TTTTTTGAATTCCATCCCCTTCGCCCTCACC EcoRIN6 TTTTTTCTGCAGTCGAACACCCGGCTGCGC PstI5S-rRNA-1 GCCTAAGCTTTTGGACAGGATGGGGTTGGA HindIII5S-rRNA-2 CAAAGAATTCGACGATTGTGTGTTGTAAGG EcoRIcrcZfw TTTTGAATTCCCGATCTGCATTGCGACG EcoRIcrcZrev TTTTTTCTGCAGCCAATACATAAGCAGATGCCGTGC PstI
For the sequences listed for oligonucleotides, restriction sites are highlighted in boldface, T7 promoter sequences are underlined, and the CA motif mutationis italic.
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