materials and methods - genes &...

Suzuki et al. Supplement

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Supplemental Materials

Materials and Methods

Tables (S1-5)

Figure Legends

Figures (S1-5)

References


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Materials and methods

Production and purification of His6-CsrD.

Plasmid pYAT1 encodes the His6-tagged csrD gene lacking the membrane-spanning

regions and containing an N-terminal His6-tag was constructed using the plasmid pQDY3

and pET-16b. DNA fragments including T7 promoter region and the csrD gene lacking

the membrane-spanning regions were amplified by PCR using pET-16b and pQDY3 as

the templates (Table S2, S3), respectively. The two resulting PCR fragments were

annealed together and amplified using the primer pair for the ends of the gene. The final

PCR product was digested with XbaI and BamHI, cloned into pET-16b (Appendix Table

3), and confirmed by nucleotide sequencing. His6-CsrD was overproduced in E. coli

strain Rosetta(DE3)pLysS (Novagen). Cells were grown at 37˚C with shaking (250 rpm)

until the OD600 reached 0.2, then 1 mM IPTG was added to the culture. Cells were

collected 3 h after IPTG induction, then resuspended in lysis buffer (50 mM Tris-HCl

pH7.5, 300 mM NaCl, and 5 mM imidazole) at 1 ml per gram wet weight, and lysed by

sonication (12X10 sec bursts at 100 W). The supernatant solution was recovered by

centrifugation (10,000Xg at 4˚C for 30 min) and mixed with Ni-NTA slurry (Qiagen)

gently by shaking at 4˚C for 60 min. The lysate-Ni-NTA mixture was loaded into a

column and the purified His6-CsrD protein was recovered according to the

manufacturer’s instructions. The column was washed stepwise with lysis buffer

containing 10, 20, and 30 mM imidazole. His6-CsrD was eluted with lysis buffer

containing 250 mM imidazole and dialyzed against 10 mM Tris-HCl pH 8.0, 50 mM

KCl, and 25 % glycerol. The protein was obtained as a single homogenous product based


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on analysis by SDS PAGE with Comassie blue staining (data not shown), and was stored

in aliquots at -80˚C until needed.

Filter binding assays.

RNAs (CsrB, CsrC, rpsO, and rpsT) were synthesized in vitro using the

MEGAshortscript kit (Ambion). PCR products containing a T7 promoter sequence and

csrB (+1 to +366), csrC (5’-GG-+1 to +245), rpsO (+1 to +420), or rpsT (5’-G +90 to

+447), were used as the templates to generate CsrB, CsrC, rpsO, or rpsT RNAs. The

primer pair, T7CsrB-Bter47, TcsCF-csCter, TrpOF-rpOter, and TrpTF-rpTter were used

for synthesis of the templates for CsrB, CsrC, rpsO, and rpsT RNAs, respectively (Table

S2). Synthesized RNAs were purified from the 5%/8 M urea PAGE gel. CsrB RNA was

dephosphorylated, then 5’-labeled by T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci

mmole-1, NEN Life Science Products). Labeled CsrB RNA was recovered through a G25

spin column (Amersham Bioscience). Filter binding assay were carried out using

Minifold I 96-Place Spot-Blot System (Schleicher & Schuell BioScience) by modifying a

double-filter method, which permits experimental determination of both bound and

unbound RNA (Wong and Lohman 1993). Membranes, Hybond-N+ (Amersham

Bioscience) and Portran-BA83 (Schleicher & Schuell BioScience), were equilibrated in

10 mM Tris-HCl pH7.5, 125 mM KCl, and 1 mM MgCl2 prior to use. Binding reaction

mixtures (40 µl) contained 10 mM Tris-HCl pH7.5, 125 mM KCl, 1 mM MgCl2, 32.5 ng

yeast RNA, 1 mM DTT, 4U RNasin (Roche), 2.5 pM 5’-end-labeled CsrB RNA and

purified His6-CsrD. For competition studies, unlabeled CsrB, CsrC, rpsO, or rpsT RNAs

were added to the reaction mixture. RNAs were heated to 85˚C for 3 min then cooled for


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10 min at room temperature before adding to the reaction mixture. Binding reactions

were incubated at 37˚C for 30 min. Samples were then filtered and subsequently rinsed

twice with 40 µl of 10 mM Tris-HCl pH7.5, 125 mM KCl, and 1 mM MgCl2. Radioactive

spots were visualized and quantified using a phosphorimager (Storm® Gel and Blot

Imaging system, Amersham Bioscience) and ImageQuant software (Molecular

Dynamics). The apparent equilibrium binding constant (Kd) of His6-CsrD-CsrB RNA

complex was calculated by fitting to the cooperative binding equation as described

previously (Yakhnin et al. 2000).


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Table S1. Bacterial strains, plasmids, and phages used in supplemental studies.

Strain, plasmid, Description Source or reference or phage E. coli B strain

Rosetta(DE3)pLysS For protein expression using pET vectors Novagen

Plasmids

pQE-1 T5-based expression vector Qiagen

pET-16b T7-based expression vector Novagen

pQDY3 csrD N-terminal His6-tag, lacking the membrane-spanning regions in pQE-1 This study

pYAT1 csrD N-terminal His6-tag, lacking the membrane-spanning regions in pET-16b This study


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Table S2. Oligonucleotide primers used in supplemental studies (shown 5’ to 3’).

Name DNA sequence yhdAR ATCCGCTTACTTTACCAACCACAC yhdAF CGACTTTCGTCCAGAGTCTGTACTG yhdF1 TTATCGTCCAGTTGGCTCC yhdF3 CAGTTTGGCTTATCGGAAG yhdR1 TCCGTTCGCTTCTCAATG yhdR2 TCGGTATCCTGACCACTACG yhP1 AGCGCGCATTATTCTACGTGAAAACGGATTAAACGGCAGGTGTAGGCTGGAGCTGCTTC yhP4 AGTATGCCCGCTTCCTCACTATCGGAGTTAACACAAGGATTCCGGGGATCCGTCGACC csrBR GCGTTAAAGGACACCTCCAGG csrBT7 GTAATACGACTCACTATAGGTTCGTTTCGCAGCATTCCAG csrCR GAGGACGCTAACAGGAACAATG csrCT7 GTAATACGACTCACTATAGGTCTTACAATCCTTGCAGGC rpsOR GGTTTCTGAGTTTGGTCGTGACG rpsOT7 GTAATACGACTCACTATAGTCGATGAGCTGGGTGTAACGTG rpsTR TCGTAAGCACAACGCAAGCC rpsTT7 GTAATACGACTCACTATAGTCAGACCTTTAGCAGCCTGACG ryhBR GCGATCAGGAAGACCCTCG ryhBT7 GTAATACGACTCACTATAGAAAAGCCAGCACCCGGCTG PEX1 CTGATGTTCACTTCGTTGTCTGAC adrA-F TAACTTCTGCCTTTAGCTCCGTCTC adrA-R CTCAGCAAATCCTGATGACTTTCG yhjH-F CGCCGATAATCTTTGTCGAGTC yhjH-R TGCTCATCGTTCGCTTCCTC YHDhisR CCAATGCATCTAATGATGATGATGATGATGAACCGAGTATCTTTGTG CChisR CCAATGCATCTAATGATGATGATGATGATGCAGCGTATCAAGACGGCTG DUF1R CCAATGCATCTAATGATGATGATGATGATGGTAAATAGCCCAGCTATTGC EALF GGAGTTAACACAAGGATGGGTAATGTTCGCTGGCGTAC EALR GTACGCCAGCGAACATTACCCATCCTTGTGTTAACTCC dGGF GATACGCTGATCCGCTCTTATGGACGCGGTAATGTTCGCTG dGGR CAGCGAACATTACCGCGTCCATAAGAGCGGATCAGCGTATC dPE1-3 TTCGACCTGACCATGCAGCACCATGGTTACGTTGTAGAAACTTAG dPE2-2 GGTCAGGTCGAAAGCATTGATGTTATTCGTTATTTCCATTCGTTG dTMCCF CAACGGCAACTTGCCGGGCCCAGAACCAGCAGTGCG dTMCCR CGCACTGCTGGTTCTGGGCCCGGCAAGTTGCCGTTG AatF AGTGCCACCTGACGTCTAAG dyhdArg CGCTGGTTACAACGGCAACTTGC HRAF CTGCTGGCGCGTTACGCCGCCAGTGATTTTGCTGCG HRAR CGCAGCAAAATCACTGGCGGCGTAACGCGCCAGCAG S307AF GTTACCACCGCGCTGATTTTGCTGC S307AR GCAGCAAAATCAGCGCGGTGGTAAC D308AF CCACCGCAGTGCTTTTGCTGCGCTG D308AR CAGCGCAGCAAAAGCACTGCGGTGG R235AF CGGCCTCAATAACGCACTCTTTTTCGATAATC R235AR GATTATCGAAAAAGAGTGCGTTATTGAGGCCG E430AF GTTCATCATCGCGCACTCATGTGC E430AR CACATGAGTGCGCGATGATGAACC E519AF AACTTGCAGCGGCCGATGTAGGTC E519AR ACCTACATCGGCCGCTGCAAGTTC Restriction enzyme sites (NsiI) are underlined. T7 promoter sequences and transcription initiation sites are indicated by italic and bold, respectively.

Continued on following page


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Table S2. - Continued

Name DNA sequence R579AF CAGAAACATTGAGAAGGCAACGGAGAACCAGC R579AR GCTGGTTCTCCGTTGCCTTCTCAATGTTTCTG E581AF GAGAAGCGAACGGCGAACCAGCTGCTG E581AR CAGCAGCTGGTTCGCCGTTCGCTTCTC N582AF GAAGCGAACGGAGGCCCAGCTGCTGGTTC N582AR GAACCAGCAGCTGGGCCTCCGTTCGCTTC Q583AF CGAACGGAGAACGCGCTGCTGGTTCAAAGC Q583AR GCTTTGAACCAGCAGCGCGTTCTCCGTTCG L584AF GAACGGAGAACCAGGCGCTGGTTCAAAGCC L584AR GGCTTTGAACCAGCGCCTGGTTCTCCGTTC S588AF CTGCTGGTTCAAGCCCTGGTGGAAGCCTG S588AR CAGGCTTCCACCAGGGCTTGAACCAGCAG 16b CATTAGGAAGCAGCCCAGTAGTAGG HYpET GGTGATGGTGATGTTTCATGGTATATCTCCTTC GC-HYpET GAAGGAGATATACCATGAAACATCACCATCACC yhdARBam GCAGCCGGATCCTTAAACCGAGTATCTTTGTG T7CsrB GTAATACGACTCACTATAGTCGACAGGGAGTCAGACAAC Bter47 AAAAAAAGGGAGCACTGTATTCACAGCGCTCCCGGTTCGTTTCGCAG TcsCF GTAATACGACTCACTATAGGATAGAGCGAGGACGCTAACAGGAAC csCter AAGAAAAAAGGCGACAGATTACTCTGTCGCCTTTTTTCCTGACTC TrpOF GTAATACGACTCACTATAGCCGCTTAACGTCGCGTAAATTG rpOter GAAAAAAGGGGCCACTCAGGCCCCCTTTTCTGAAACTCG TrpTF GTAATACGACTCACTATAGGCCTTTGAATTGTCCATATAGAACAC rpTter AAAAAAACCCGCTTGCGCGGGCTTTTTCACAAAGCTTCAGC Restriction enzyme sites (NsiI) are underlined. T7 promoter sequences and transcription initiation sites are indicated by italic and bold, respectively.


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Table S3. Primer pairs and restriction sites for construction of plasmids encoding C-terminal His6-tag, N-terminal His6-tag, domain deletions, and site directed mutations of csrD. Name Mutation, Primer pair Parent plasmid Restriction site description pNC-His CsrD-His6 yhdF3-YHDhisR pBYH4 BstEII, NsiI

pQDY3 His6-CsrD dyhdArg-yhdAR pQE-1 PvuII, PstI (Vector) (2-135 aa deletion) NsiI (PCR product) pNCChis GGDEF and EAL yhdF1-CChisR pBYH4 XmaI, NsiI domains deletion

pDUF1 EAL domain yhdF1-DUF1R pBYH4 KpnI, NsiI deletion

pEAL EAL domain only AatF-EALR pBYH4 AatII, NsiI EALF-YHDhisR pDGGDEF GGDEF domain yhdF1-dGGR pNC-His SmaI, BstEII deletion dGGF-yhdR1

pDPERI Periplasmic region AatF-dPE1-3 pNC-His AatII, SmaI substitution dPE2-2-CChisR

pDTMCC Part of HAMP-like AatF-dTMCCR pNC-His NcoI, KpnI domain deletion dTMCCF-yhdR2

pR235A R235A yhdF1-R235AR pNC-His KpnI, MluI R235AF-DUF1R

pHRA HR305AA yhdF1-HRAR pNC-His KpnI, MluI HRAF-DUF1R

pS307A S307A yhdF1-S307AR pNC-His KpnI, MluI S307AF-DUF1R pD308A D308A yhdF1-D308AR pNC-His KpnI, MluI D308AF-DUF1R

pE430A E430A yhdF2-E430AR pBYH4 BstEII, MluI E430AF-yhdR1

pE519Ahis E519A yhdF3-E519AR pBYH4 BstEII, NsiI E519AF-YHDhisR


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Continued on following page

Table S3. – Continued Name Mutation, Primer pair Parent plasmid Restriction site description pR579A R579A yhdF3-R579AR pBYH4 BstEII, NsiI R579AF-YHDhisR

pE581A E581A yhdF3-E581AR pBYH4 BstEII, NsiI E581AF-YHDhisR pN582A N582A yhdF3-N582AR pBYH4 BstEII, NsiI N582AF-YHDhisR

pQ583A Q583A yhdF3-Q583AR pBYH4 BstEII, NsiI Q583AF-YHDhisR pL584A L584A yhdF3-L584AR pBYH4 BstEII, NsiI L584AF-YHDhisR pS588A S588A yhdF3-S588AR pBYH4 BstEII, NsiI S588AF-YHDhisR pYAT1 His6-CsrD 16b-HYpETa pET-16b XbaI, BamHI (2-135 aa deletion) GC-HYpET-yhdARBamb

Plasmid construction is described in Materials and Methods. Primer sequences are listed in Table S2. Primer pairs for the first PCR reaction are listed internally, while primer pairs for the second PCR reactions are underlined. The plasmid vector pQE-1 (QIAGEN) was used for construction of clones encoding N-terminal His6-tagged CsrD proteins. The plasmid vector pET-16b (Novagen) was used for construction of clones encoding N-terminal His6-tagged CsrD proteins. a and b, Templates used for the PCR reactions were pET-16b (a) and pQDY3 (b).


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Table S4. Site directed mutagenesis of CsrD and c-di-GMP-metabolizing GGDEF and EAL domain proteins.

Positiona Protein WTb Mutationb Activityc Quantitative assaysd Reference Seq. csrB-lacZ Biofilm

1 CsrD R235A Active 98 ± 14 96 ± 1 This study

2 CsrD HRSDF AASDF Active 90 ± 2 98 ± 1 This study

HRADF Active 81 ± 8 90 ± 1

HRSAF Active 98 ± 8 95 ± 0

PleD GGEEF DEDEF Inactive (Paul et al. 2004)

AdrA GGDEF AADEF Inactive (Simm et al. 2004)

STM1987 GGEEF GGGSF Inactive (García et al. 2004)

HmsT GGEEF AGEEF Inactive (Kirillina et al. 2004)

GAEEF Inactive

GGAEF Inactive

GGEAF Inactive

3 CsrD ELM ALM Active 94 ±12 94 ± 0 This study

VieA EAL AAL Inactive (Tamayo et al. 2005)

HmsP EAL AAL Inactive (Kirillina et al. 2004)

Arr EAL AAL Inactive (Hoffman et al. 2005)

CC3396 EAL QAL Active (Christen et al. 2005)

4 CsrD ELAE ELAA Partial 24 ± 3 71 ± 1 This study

YhjH ELVE ELVA Inactive (Simm et al. 2004)

5 VieA DDFGTG ADFGTG Inactive (Tischler & Camilli 2004)

a, Numbers indicate the positions of the amino acid changes, as shown in Fig. 5. b, Sequences in the wild type (WT) and site-directed mutant of the indicated proteins are shown. c, Results of published findings for other GGDEF and EAL proteins are summarized for comparison to those of CsrD. d, Plasmids encoding the site-directed mutations of csrD were transferred into KDKSB837 (ΔcsrD::kan). Quantitative assays for csrB-lacZ and biofilm formation were conducted as described in Materials and methods and the relative activity (% ± standard deviation) with respect to that of a strain harboring the isogenic wild type csrD plasmid is shown.


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Table S5. Alanine replacement of CsrD amino acids that are conserved in proposed orthologs, but not in c-di-GMP metabolizing proteins.a

Plasmid Mutation csrB-lacZ Biofilm Westernb pR579A R579A 118 ± 4 89 ± 4 +

pE581A E581A 110 ± 9 94 ± 3 + pN582A N582A 105 ± 11 89 ± 3 +

pQ583A Q583A 73 ± 12 86 ± 3 + pL584A L584A 29 ± 5 52 ± 2 +

pS588A S588A 89 ± 7 84 ± 1 +

a, Plasmids encoding mutant proteins (Fig. 5B, Region II) were transferred into KDKSB837 (ΔcsrD::kan). Assays for csrB-lacZ and biofilm formation were conducted as described in Materials and methods. Values for csrB-lacZ expression and biofilm formation depict activity of each alanine replacement mutant (% ± standard deviation), and were normalized with respect to the same strain harboring the isogenic plasmid with wild-type csrD gene (pNC-His; Fig. S3; 100% activity) and the cloning vector pBR322 (0% activity). b, Western blotting of His6-tagged proteins from 6 h, 37˚C cultures used HisProbe-HRP under the recommended conditions (Pierce Biotechnology, Inc.); +, recombinant CsrD protein detected at essentially the same concentration as that of the pNC-His control.


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Supplemental Figure legends

Figure S1. Comparison of single and dual RNase E and CsrD deficiency on CsrB

decay. Northern blot of CsrB RNA following shift to nonpermissive temperature and

rifampicin addition to MG1693 and isogenic mutants of this strain, as indicated, at the

transition from exponential to stationary phase of growth. Curves were plotted as in Fig.

2.

Figure S2. Effect of CsrD on CsrB/C decay in a csrA mutant strain. Northern blot of

CsrB and CsrC RNAs, following rifampicin addition to cultures of MG1655 (WT),

DCMG (csrD), TRMG 1655 (csrA) and DCTRMG1655 (csrD csrA) at the transition to

stationary phase of growth. Half-lives were determined as in Fig. 2.

Figure S3. Decay of rpsO, rpsT, and RyhB RNAs in csrD wild type and mutant

strains. (A) Northern blot analysis of rpsO and rpsT mRNAs following rifampicin

addition, in strains MG1655 (WT) and DCMG (csrD). Total RNA was separated by

electrophoresis on 6% polyacrylamide gels containing 7M urea. (B) MG1655 (WT),

DCMG (csrD), and DCMG containing pBR322 or pBYH4 (csrD++) were grown and

RyhB stability was determined as described previously (Massé et al. 2003). Arrows

indicate the addition of 250 µM of 2,2’-dipyridyl (dip) and 100 µM of FeSO4 (5 min.

after addition of dip). Total RNA was prepared from cultures harvested at the times

indicated and separated by electrophoresis on 1.5% agarose gels containing

formaldehyde.


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Figure S4. Effects of domain deletions and sequence substitutions on CsrD activity

in vivo. KDKSB837 (ΔcsrD::kan csrB-lacZ ) was transformed with plasmids encoding

wild type and various mutant CsrD proteins, or KDMG (ΔcsrD::kan) was transformed

with pQDY2 and 3. IPTG (1 mM) was added to the culture for strains harboring plasmid

pQDY2 and 3. Plasmid pQDY3 expresses a protein with the N-terminal sequence

MKHHHHHHQ fused to Arg156 of CsrD. Plasmid pQDY2 is identical, except that it has

a frame shift mutation at Arg156. In pDPERI, the putative periplasmic region (residue

33-129) was replaced by the periplasmic sequence (16 aa) of ArcB (Kwon et al. 2000).

Activity of the clones was determined by their ability to complement the ΔcsrD::kan

deletion in glycogen synthesis, biofilm formation and (with the exception of pQDY2, 3)

csrB-lacZ expression assays, as described in Materials and Methods. Western blotting of

His6-tagged proteins from cultures at 6 h of 37˚C growth used HisProbe-HRP under the

recommended conditions (Pierce Biotechnology, Inc.); +, protein detected; NT, not

tested.

Fig. S5. Binding of His6-CsrD to radiolabeled CsrB RNA. Panels A and B depict the

equilibrium binding reactions of CsrD with labeled CsrB RNA and competition assays,

respectively. The concentration of labeled CsrB was 2.5 pM in these experiments. CsrD

protein concentration in panel B and was 31 nM. Other experimental conditions for these

filter-binding assays are given in the Supplemental Materials and Methods.


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A

CsrB RNA

1

10

100

1000

0 15 30 45 60 75 90Time (min)

RN

A re

mai

ning

(%)

csrDcsrD rne-1rne-1

B

CsrB

0 2 15 30 45 60 90(min)

csrD

csrD rne-1

rne-1

Half-life(min)

>90

>90

>90

Fig. S1


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Fig. S2

WT

csrD

csrD csrA

(min) CsrB

Half-life (min)

1.8

>8

>8

0 2 4 8

WT

csrD

csrD csrA

(min) CsrC

Half-life (min)

4.1

>8

3.2

0 2 4 8

csrA 3.0 csrA 2.5

csrC 1.5 csrB 3.2


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16

0 2 4 8 12 18

rpsO

(min)

csrD

WT

csrD

WT

rpsT

WT

csrD

csrD[pBR322]

csrD[pBYH4]

(min) 0 2 4 6 10 15 20

dip FeSO4

RyhB

A

B

Fig. S3


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Fig. S4

Plasmid Domains of CsrD encoded Western

pNC-His

pQDY3

pQDY2

pNCC

pDUF1

pEAL

pDGGDEF

pDPERI

pDTMCC

Activity

+

+

-

-

-

-

-

+

-

+

NT

NT

+

+

+

+

+

+

His6

219

His6

385

646 aa10 32 130 152 199 219

220 381 395 630

His6

csrD

PromoterGGDEF EAL

His6

393223

His6

32 130

ArcB

16 aa

His6

164 192

His6

395M

156MHis6T5 promoter

156His6T5 promoter

Frame shift at Arg156M

Coiled CoilHAMP-likeTM


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Fig. S5

Frac

tion

boun

d

CsrD (nM)

Kd = 24.52n = 0.7919

A

B

0

20

40

60

80250 pM2500 pM

Rel

ativ

e bin

ding

(%)

CsrB CsrC rpsO rpsT

Kd = 25 nM n = 0.8


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References for Supplemental Material

Christen, M., Christen, B., Folcher, M., Schauerte, A., and Jenal, U. 2005. Identification

and characterization of a cyclic di-GMP-specific phosphodiesterase and its

allosteric control by GTP. J. Biol. Chem. 280: 30829-30837.

García, B., Latasa, C., Solano, C., García-del Portillo, F., Gamazo, C., and Lasa, I. 2004.

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and

biofilm formation. Mol. Microbiol. 54: 264-277.

Hoffman, L.R., D'Argenio, D.A., MacCoss, M.J., Zhang, Z., Jones, R.A., and Miller, S.I.

2005. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436:

1171-1175.

Kirillina, O., Fetherston, J.D., Bobrov, A.G., Abney, J., and Perry, R.D. 2004. HmsP, a

putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control

Hms-dependent biofilm formation in Yersinia pestis. Mol. Microbiol. 54: 75-88.

Kwon, O., Georgellis, D., Lynch, A.S., Boyd, D., and Lin, E.C. 2000. The ArcB sensor

kinase of Escherichia coli: genetic exploration of the transmembrane region. J.

Bacteriol. 182: 2960-2966.

Massé, E., Escorcia, F.E., and Gottesman, S. 2003. Coupled degradation of a small

regulatory RNA and its mRNA targets in Escherichia coli. Genes & Dev. 17:

2374-2383.


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Paul, R., Weiser, S., Amiot, N.C., Chan, C., Schirmer, T., Giese, B., and Jenal, U. 2004.

Cell cycle-dependent dynamic localization of a bacterial response regulator with a

novel di-guanylate cyclase output domain. Genes & Dev. 18: 715-727.

Simm, R., Morr, M., Kader, A., Nimtz, M., and Römling, U. 2004. GGDEF and EAL

domains inversely regulate cyclic di-GMP levels and transition from sessility to

motility. Mol. Microbiol. 53: 1123-1134.

Tamayo, R., Tischler, A.D., and Camilli, A. 2005. The EAL domain protein VieA is a

cyclic diguanylate phosphodiesterase. J. Biol. Chem. 280: 33324-33330.

Tischler, A.D. and Camilli, A. 2004. Cyclic diguanylate (c-di-GMP) regulates Vibrio

cholerae biofilm formation. Mol. Microbiol. 53: 857-869.

Wong, I. and Lohman, T.M. 1993. A double-filter method for nitrocellulose-filter

binding: application to protein-nucleic acid interactions. Proc. Natl. Acad. Sci. 90:

5428-5432.

Yakhnin, A.V., Trimble, J.J., Chiaro, C.R., and Babitzke, P. 2000. Effects of mutations in

the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of

Bacillus subtilis. J. Biol. Chem. 275: 4519-4524.

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