海洋科学技術センター試験研究報告　第40号　JAMSTECR, 40（February 2000）

15

Cloning and characterization of the rpoA gene encoding the ααααα sub-

unit of RNA polymerase from deep-sea barophilic

Shewanella violacea strain DSS12

Kaoru NAKASONE＊１

Chiaki KATO＊１　Koki HORIKOSHI＊１

The rpoA gene encoding the α subunit of RNA polymerase from deep-sea barophilic Shewanella violacea strain DSS12 was

cloned and sequenced using a PCR-based approach. The rpoA gene was found to encode a polypeptide consisting of 329 aa with a

molecular mass of 36, 238 Da. The 6xHis-tagged wild-type α protein was overexpressed in an E. coli strain with the mutation

rpoA112, a ts mutant, to determine whether the cloned rpoA gene was functional or not. This gene complemented the rpoA112

mutation, indicating that chimeric RNA polymerase is assembled and functional in E. coli, allowing the ts mutant to survive at a non-

permissive temperature. These findings will facilitate further studies on the structure and function of RNA polymerase from this deep-

sea barophilic strain.

Key Words : Deep-sea barophilic bacterium, RNA polymerase α subunit, Complementation analysis

深海由来好圧性細菌 Shewanella violacea DSS12株のRNAポリメ

ラーゼαサブユニット遺伝子 rpoAのクローニングとその解析

仲宗根　薫＊２

加藤 千明＊２　掘越 弘毅＊２

　深海由来好圧性細菌Shewanella violacea DSS12株より、RNAポリメラーゼαサブユニットをコードする遺伝子rpoAを

クローン化し塩基配列決定を行った。単離された rpoA遺伝子は、329個のアミノ酸を有する、分子量 36,238 Daの蛋白

をコードしていた。本菌株由来 rpoA遺伝子の機能を確かめるため、高温感受性 rpoA変異株・大腸菌 rpoA112株菌体内

にHis6-a蛋白を発現させたところ、DSS12株 rpoA遺伝子はこの変異株を相補した。この結果は、この変異株菌体内に

DSS12株由来αサブユニットを含むキメラRNAポリメラーゼが再構成され機能したことで、高温下においても生育可能

になったことを示しており、今後の、深海由来好圧性細菌RNAポリメラーゼの構造と機能の研究に貢献すると考えられる。

キーワードキーワードキーワードキーワードキーワード：：：：：深海由来好圧性細菌、RNAポリメラーゼαサブユニット、相補性試験

＊１　The DEEP STAR Group

＊２　海洋科学技術センター　深海微生物研究グループ

JAMSTECR, 40 (2000)16

1　Introduction

The moderately barophilic deep-sea bacterium Shewanella

violacea strain DSS12, isolated from the Ryukyu trench (depth

: 5, 110 m), grows optimally at 30 MPa and 8°C, but also at

atmospheric pressure (0.1 MPa) and 8°C1), 2). It is useful as a

model for comparison of various features of bacterial physiol-

ogy under high and low pressure conditions. Recently an op-

eron identified as a pressure-regulated operon, with a promoter

activated under high pressure conditions, was cloned and char-

acterized from this strain3). We have reported that gene expres-

sion from this operon, which has five transcription initiation

sites, is controlled at the transcriptional level by elevated pres-

sure3), 4). However, the molecular basis of transcription of this

operon remains to be elucidated. Knowledge of the regulation

of gene expression and the transcriptional machinery of this

bacterium is necessary for a critical understanding of how these

organisms adapt, survive and grow in high pressure environ-

ments. Thus, cloning of the genes for RNA polymerase sub-

units from alkaliphilic bacteria and the establishment of an in

vitro system for reconstitution of the enzyme are essential.

Transcription in eubacteria is mediated by an RNA poly-

merase holoenzyme which governs the selectivity of the pro-

moter sequence of a gene. RNA polymerase is a multisubunit

complex composed of α (rpoA), β (rpoB), β´ (rpoC) and

one of several s subunits5). Among these subunits, the α sub-

unit is an important component involved in not only the assem-

bly of the core enzyme but also the activation of transcription6).

Here, we report the cloning of the rpoA gene encoding the

RNA polymerase α subunit of barophilic Shewanella violacea

strain DSS12 and characterization of the gene by means of a

complementation experiment using an E. coli mutant7).

2　Materials and Methods

2.1　Bacterial strains and plasmids

Barophilic Shewanella violacea strain DSS12 was the source

of chromosomal DNA encoding the rpoA gene. E. coli strain

JM109 was used as the recipient for cloning. The following

strains of E. coli K12 described previously were used for

complementation analysis : HN198, a malA derivative of

AB2834 F- aroE thi Su-; HN317ts112, a rpoA112 derivative of

HN1987). For the cloning of PCR products, the T-overhanging

vector pCR2.1 (Invitrogen) was used.

2.2　Cloning of the rpoA gene, nucleotide sequence

and computer analysis

In order to clone a portion of the rpoA gene from strain DSS12,

two synthetic degenerate oligonucleotide primers, A1(5’-ATY

CTD CTB TCD TCD ATG-3’) and A2(5’-YTC KGC YTT HAR

RCA GTT-3’), were synthesized. A target DNA fragment con-

taining a part of the rpoA gene was amplified by PCR using

these degenerate primers. PCR was performed in a 50 μ1 vol-

ume with 2.5 U of ExTaq (Takara Shuzo) for 30 cycles of 30 s

at 94°C, 1 min at 45°C and 30 s at 72°C. The PCR product

(approx. 300 bp) was cloned into the vector pCR2.1 and se-

quenced. This fragment was labeled with digoxygenin

(Boehringer Mannheim) to prepare a hybridization probe.

Plaque hybridization was carried out with the probe and a posi-

tive clone was isolated from a DSS12λlibrary constructed in

λEMBL3. The nucleotide sequence of the DNA insert of this

clone was determined for both strands by the dye terminator

method using an ABI-Prism 377 automatic DNA sequencer (Ap-

plied Biosystems). The nucleotide and amino acid sequences

were analyzed using the computer program GENETYX-MAC

ver. 10.

2.3　Complementation analysis

An expression plasmid containing the rpoA gene was con-

structed for complementation analysis using an E. coli rpoA

mutant. PCR was performed to amplify the rpoA gene and the

resulting fragment, digested with both BamHI and HindIII, was

cloned into expression plasmid pQE30 (pQSVA). For comple-

mentation experiments, the plasmids pQE30 and pQSVA were

introduced into HN198 and HN317 cells by the standard trans-

formation procedure7). Ampicillin-resistant colonies were picked

up and suspended in LB medium. The cell suspensions were

streaked onto LB agar plates containing 50 μ g/ml ampicillin

and incubated overnight at 30°C and 42°C.

3　Results and discussion

3.1　Cloning of the rpoA gene from S. violacea

strain DSS12

First, based on a comparison of the amino acid sequences of α

proteins from several Gram-negative bacteria, degenerate oli-

gonucleotide primers, A1 and A2(Fig. 1B) were designed and

synthesized for use in the PCR reaction. To clone a portion of

the rpoA gene, the corresponding region of S. violacea chro-

mosomal DNA was amplified by PCR. A 300 bp fragment of

the expected size was obtained as the only PCR product. It was

cloned into the vector pCR2. 1 and its nucleotide sequence was

determined. The nucleotide sequence of this fragment and the

deduced amino acid sequence showed strong similarity to those

of the rpoA genes of E. coli and P. putida. Next, this product

labeled with digoxygenin, was used as a probe for plaque hy-

bridization of clones in a S. violacea λ library constructed in

the λ EMBL3 vector. As a result of this screening, a single

positive clone containing a DNA insert approximately 18.5 kb

in size, designated λ PA, was isolated and the nucleotide se-

quence was determined. Sequence analysis revealed that the

λPA fragment contained the complete rpoA gene and four other

genes for ribosomal proteins. The structure of a 3.8 kb DNA

fragment containing the α operon, part of λ PA, is shown in

Fig. 1A. In E. coli, the αoperon contains the genes for riboso-

mal proteins S13 (rpsM), S11 (rpsK), S4 (rpsD), a (rpoA) and

L17 (rplQ), arranged in this order8). As shown in Fig. 1A, the

genetic organization of these genes of the α operon in strain

DSS12 is exactly the same as that in E. coli. This result sug-

JAMSTECR, 40 (2000) 17

gest that the mechanisms of cotranscription of these genes and

assembly of the ribosomes and RNA polymerase have been

conserved during evolution.

3.2　Structural analysis of the amino acid sequence

of RpoA

The nucleotide sequence encoding the α subunit is shown in

Fig. 1B together with the predicted amino acid sequence. The

open reading frame of the α gene consists of 990 nucleotides.

It codes for a protein consisting of 329 amino acid residues

(Fig. 1B) with a molecular mass of 36,238 Da and a pI value of

4.6, whereas the α subunit in E. coli consists of 329 amino

acid residues (Fig. 2) with a molecular mass of 36,500 Da and

a pI value of 4.8.

Studies on deletion mutagenesis and limited proteolysis of the

E. coli αsubunit indicate that it consists of two independently

folded domains, an N-terminal domain (αNTD ; residues 8

to 235) and a C-terminal domain (α CTD ; residues 249 to

329), connected by a flexible, 14-residue linker9). To search for

conserved domains thought to be involved in subunit assembly

and domains involved in the contact between transcriptional

factors and DNA, a comparison of the amino acid sequence of

the α subunit of strain DSS12 and that of E. coli was carried

out. One stretch near the N-terminus, residues 24 to 69 in the

αmotif, designated as such by Zhang and Darst10), was found

to be identical, while the 82 C-terminal residues (αCTD) were

highly conserved (Fig. 2). The N-terminal two-thirds of the α

subunit plays an important role in the assembly of E. coli RNA

polymerase9). One of the residues in this domain, Arg45 in the

αmotif, is essential for the assembly of RNA polymerase ; it

is one of the determinants of the interaction between the α and

β subunits11). As shown in Fig. 2, the residue Arg45 is also con-

served in strain DSS12, suggesting that the mechanisms of

RNA polymerase assembly are evolutionarily conserved. There

are contact sites on αCTD for some transcription factors6) and

the promoter UP element12). Our finding that αCTD is highly

conserved suggests that common transcription factors and the

specific affinity in binding to the UP element are conserved.

3.3　Complementation analysis

An E. coli mutant with the rpoA112 mutation produces RNA

polymerase showing altered thermostability and reduced fidel-

ity of transcription in vitro13). In complementation analysis stud-

ies using this mutant as the host for an E. coli rpoA-expression

plasmid, it has been shown that the rpoA112 mutation is re-

sponsible for temperature-sensitive growth7). This mutation is

known to involve a single transition which leads to the substi-

tution of Cys for Arg at position 45, resulting in a defect in

RNA polymerase assembly7). These studies suggest that the

amino terminal region of the α subunit, including the residue

at position 45 plays an important role in subunit assembly7). A

complementation experiment using E. coli rpoA mutant HN317

(rpoA112) 7) as the host strain for plasmid pQSVA carrying the

S. violacea rpoA gene was performed to test whether this gene

that we cloned was functional in E. coli or not. Plasmid pQE30

lacking the rpoA gene was used as a control. Strains HN198

(rpoA+) and HN317 (rpoA112) were transformed with these

plasmids and the growth of transformants was examined at per-

missive and non-permissive temperatures (30°C and 42°C, re-

spectively) (Fig. 3). Growth of HN198 transformed with pQSVA

or pQE30 was the same at both temperatures (Fig. 3).

Transformants of strain HN317 harboring pQSVA could grow

at 42°C, but HN317 cells transformed with pQE30 remained

temperature-sensitive and were unable to grow at 42°C (Fig.

3). These results taken together suggest that, in the E. coli mu-

tant transformed with pQSVA, a chimeric RNA polymerase

composed of α2 from S. violacea and ββ’ from E. coli was

assembled, allowing the cells to survive at 42°C, the non-per-

missive temperature for this mutant. As the chimeric polymerase

is functional, these findings will facilitate further studies on

Fig. 1　Restriction map, nucleotide and deduced amino acid sequences of the RNA

polymerase α subunit gene of S. violacea strain DSS12.

(A) Restriction map of the DSS12 a operon containing the genes for riboso-

mal protein S13 (rpsM), S11(rpsK), S4 (rpsD), a (rpoA), and L17 (rplQ).

(B) Nucleotide and deduced amino acid sequences of the rpoA gene of strain

DSS12.

The amino acid sequence encoded by the gene is shown in single-letter nota-

tion below the codons ; an in-frame stop codon is indicated by an asterisk. The

nucleotide sequences corresponding to the two degenerate oligonucleotide PCR

primers, A1 and A2, are indicated by arrows. The numbers to the right refer to

nucleotide positions.

JAMSTECR, 40 (2000)18

Fig. 2　Comparison of the amino acid sequence of the S. violacea strain DSS12 a subunit and those of two E. coli strains. The alignment was made with E. coli wild-type

rpoA and mutant rpoA112 sequences. Dots in each sequence represent the amino acid residues identical to those of the strain DSS12 a subunit. Identical residues

in the a subunit of all of these strains are marked by an asterisk, whereas a conserved substitution is marked by a dot.

Fig. 3　Complementation of a ts mutation (rpoA112) in E. coli by an expression

plasmid encoding the S. violacea DSS12 rpoA gene. E. coli with wild-

type rpoA and an E. coli mutant with the rpoA112 mutation were trans-

formed with pQE30 (control, lacking rpoA) or pQSVA (carrying the

rpoA gene from strain DSS12). Two ampicillin-resistant colonies ob-

tained as a result of each transformation were streaked onto LB-agar

plates. Incubation was at 30°C (upper) or 42°C (lower).

the structure and function of RNA polymerase from this deep-

sea barophilic strain.

4　Acknowledgements

We would like to thank Dr. A. Ishihama (National Institute

of Genetics) for the generous gift of strain HN198 and Dr. Mary

Berlyn (E. coli Genetic Stock Center of Yale University) for

strain HN317. We also thank Dr. W. R. Bellamy for assistance

in editing the manuscript.

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（原稿受理：NVVV年U月Q日）