Figure S1. Effect of the plasmid copy number on silencing of lacZ. (A) For the LacZ assay, wild-type cells were transformed with plasmids harboring the indicated features and cultured in the presence of IPTG to mid-logarithmic phase. LacZ activities are represented as the mean ± standard deviation of three replicates. (B) lacZ mRNA was quantified by real-time quantitative RT-PCR. Used transformants and culture conditions are same as in the panel (A). Relative lacZ mRNA level is shown as fold-change compared with lacZ mRNA level of a transformant with “pBR322 ori, -” (the leftmost bar).

0102030405060708090

0

0.5

1

1.5

2

2.5

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

ori

asRNA

lacZ-

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

lacZ-

lacZ-

lacZ-

lacZ-

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

lacZ-

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

lacZ-

lacZ-

lacZ-

lacZ-

LacZ

act

ivity

(Uni

ts)

Rel

ativ

e la

cZ m

RN

A le

vel (

fold

)

(A) (B)

Figure S2. Effect of the plasmid copy number on silencing of ackA. For the AckA assay, wild-type cells were transformed with plasmids harboring the indicated features and cultured in the presence of IPTG to mid-logarithmic phase. AckA activities are represented as the mean ± standard deviation of three replicates.

0

20

4060

80

100

120140

160

180

Ack

A (U

nits

)

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

ackA-

pBR

322

pAC

YC

pSC

101

RK

2

pSC

101H

ackA-

ackA-ackA-

ackA-

ori

asRNA

Figure S3. Silencing of pepN using an arabinose-inducible vector. (A) Arrows indicate open reading frames or promoters, and a circle indicates an ori. Details of pHN649 are described in our previous report (Nucleic Acids Res., 34, e138). The pepN antisense sequence same as described in the body text was inserted into the multiple cloning site (MCS) of pHN649, yielding pHN1365. (B) For the PepN assay, wild-type cells were transformed with plasmids harboring the indicated features and cultured to mid-logarithmic phase. The names of the plasmids used are pHN649 and pHN1365. PepN activities are represented as the mean ± standard deviation of three replicates. Open and gray bars are results with the absence and presence of 0.3% L-arabinose, respectively. (C) The pepN PTasRNA was detected by northern blot (lower panel). The rRNA bands stained with ethidium bromide (upper panel) are shown as loading controls.

0

5

10

15

20

25

30

35

40

pAC

YC

pAC

YC

asRNA

pepN-

ori

rRNAs

pepN asRNA

(C)

arabinose - + - +

pAC

YC

pAC

YC

asRNA

pepN

-

ori

Pep

N (U

nits

)

pHN6493.9 kb

araC

PbadChlr

pACYC ori

PT-MCS

(A) (B)

Figure S4. The ackA-pta loci of wild-type (WT) and disruptants (ackA, pta, and ackA pta) are shown, along with their AckA and Pta activities (% relative to that of WT). N.D. indicates not detected. The disruptants were generated from the WT strain using the “marker-less gene disruption method” (Mol. Microbiol., 5, 1447-1457). Dashed lines are deleted parts.

ackA(473-1675)

pta(1761-3890)

0.5 kb

yfcC(4080->4345)

ackA pta

yfbV(<1-135)

WT

514 3834

ackA514 1339

1846 3834pta

AckA (%) Pta (%)

62 ±12 N.D.

100 100

N.D. 69 ± 15

N.D. N.D.

Figure S5. Rescue of growth retardation that is caused by ackA and/or pta PTasRNAs by overexpressing ackA and/or pta genes. (A) The plasmid pHN1032 is an IPTG-inducible vector. Note that it is not a vector for expressing PTasRNAs but is an ordinary expression vector. It was constructed by cloning a 1.5-kb fragment excised from pTrc-99a (Amersham Biosciences Corp.) by digestion with SphI and EcoRI into the SphI-EcoRI moiety of pHN540u (Nucleic Acids Res., 34, e138). The ackA and pta full-length fragments were PCR-amplified using primers (TTCCATGGCGAGTAAGTTAGTACTGGT and GTCTCGAGTCAGGCAGTCAGGCGGCTCGC, and AACTCGAGGAGATATACCATGCTGATCCCTACCGGAACCAGC and GAACTAGTTACTGCTGCTGTGCAGACTGAA, respectively) and the genomic DNA of the MG1655 strain as a template. The ackA fragment was cloned into the NcoI-XhoI moiety of pHN1032, thereby yielding pHN1371. To construct pHN1372, the pta fragment was cloned into the XhoI-SpeI moiety of pHN1371. (B and C) Growth curves of the indicated transformants are shown. The wild-type strain was co-transformed with the PTasRNA vector(s) carrying the indicated features and one of pHN1032, pHN1371, or pHN1372. Then, the co-transformants were cultured in the presence of IPTG.

0

200

400

600

800

1000

2 4 6 8Time (hr)

Turb

idity

(Arb

itrar

y un

its)

10 12 14

pBR322 -pSC101H -

pSC101H -pBR322 -

pBR322 ackApSC101H pta

pSC101H ptapBR322 ackA

(+ pHN1372)

(+ pHN1372)

0

200

400

600

800

1000

2 4 6 8Time (hr)

Turb

idity

(Arb

itrar

y un

its)

10 12 14

pBR322 - (+ pHN1032)

pBR322 - (+ pHN1371)

pBR322 ackA (+ pHN1032)

pBR322 ackA (+ pHN1371)

(B) (C)

pHN10324.1 kb

lacIq

PtrcChlr

pACYC ori

MCS

pHN1371

ptaackA

(A)

(+ pHN1032)

(+ pHN1032)

ackA

pHN1372

ori asRNA (+ rescue vector)

ori asRNA (+ rescue vector)

Figure S6. Effect of the different asRNA sequence on silencing of lacZ. (A) The partial DNA sequence of lacZ is shown. A boxed nucleotide indicates the transcription start position, and the putative ribosome-binding site and translation start codon are shown in blue and red characters, respectively. Four versions of lacZ asRNA sequences were used to construct PTasRNA expression vectors, and those sequences are underlined with red (version 1), blue (version 2), green (version 3), and magenta (version 4). (B) For the LacZ assay, wild-type cells were transformed with plasmids harboring the indicated features and cultured in the presence of IPTG to mid-logarithmic phase. LacZ activities are represented as the mean ± standard deviation of three replicates.

0

5

10

15

20

25

30

35

(A)

(B)

LacZ

act

ivity

(Uni

ts)

aggctttacactttatgcttccggctcgtatgttgtgtggaattgtg

agcggataacaatttcacacaggaaacagctatgaccatgattacgg

attcactggccgtcgttttacaacgtcgtgactgggaaaaccctggc

gttacccaacttaatcgccttgcagcacatccccctttcgccagctg

gcgtaatagcgaagaggcc

Ver. 1Ver. 2Ver. 3

Ver. 4

pAC

YC

pAC

YC

lacZ ver. 1

-

ori

asRNA

pAC

YC

lacZ ver. 2

pAC

YC

lacZ ver. 3

pAC

YC

lacZ ver. 4