Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans

262

Biochimica et Biophysica Acta, 1172 (1993) 262-266 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 92476

Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans Yoshitaka Ueda, Seiichi Taguchi 1, Ken-ichi Nishiyama, Izumi Kumagai and Kin-ichiro Miura 2 Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, Tokyo (Japan) (Received 22 September 1992)

Key words: Subtilisin inhibitor; Minor codon; bldA; tRNA; Translational efficiency; (Streptomyces)

Streptomyces are bacteria with a very high chromosomal G + C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu8°: CTG-CTC to TTA-TTA, Lys89: AAG to AAA, Serl°S-Serl°9: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in thc case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA Leu (UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.

Introduction In Escherichia coli, and yeast, Saccharomyces cerevisiae, major codon bias has been found in genes encoding highly expressed proteins [1,2]. In these two organisms, a positive correlation between the abundance of t R N A s and the occurrence of the respective codons in their protein-coding regions has been revealed [3-5]. And recently, extreme codon bias has also been revealed for highly expressed genes of Bacillus

subtilis, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens [6]. Recent studies have suggested that the major codon preference is responsible for the efficient and faithful gene expression at the translation level, and that non-optimal codons remain at the amino terminal region as a result of 'negative selection' and are used for preventing the synthesis of

Correspondence to: I. Kumagai, Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Present address: Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba 278, Japan. 2 Present address: Institute for Biomolecular Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171, Japan. Abbrebiations: SSI, Streptomyces subtilisin inhibitor; TCA, trichloroacetic acid; TSB, tryptone soya broth; PAGE, polyacrylamide gel electrophoresis.

incorrect proteins when organisms are exposed to stringent situations such as amino acid depletion [7-11]. Because translation rates differ between minor codons and common ones [12], a cluster of minor codons gives rise to a drastic reduction of gene expression [13,11] or a 50% frameshift [14]. This frameshift is suppressed by an increased dosage of cloned t R N A gene [15]. Streptomyces are Gram-positive, mycelial, soil bacteria which show a complex cycle of morphological differentiation, and produce many kinds of secondary metabolites and secretory proteins. Therefore, the secretion system of this genus has been explored for the expression of foreign genes (Refs. 16-21 and 22 for review). A notable feature of Streptomyces is a high G + C composition ( > 70 mol%) in its genomic D N A [23,24]. In particular, since the G + C content at the third positions of the codons reaches about 90%, codon usage is extremely biased [25,26]. Hence, Streptomyces seems to be a good organism for studying the relationship between codon bias and protein productivity. In the present study, we investigated the effect of minor codons on gene expression in Streptomyces. In order to monitor gene expression, we used the expression system of a secretory protein, Streptomyces subtilisin inhibitor (SSI), in Streptomyces lit'idans [27,28]. This protein was originally isolated from a culture of S. albogriseolus S-3253, and inhibits strongly the activity of serine proteinases such as subtilisin [29].

263 Materials and Methods

formed by SDS-PAGE [35] and followed by densitometry. Details are shown in the legends to Figs. 2 and 3.

Bacterial strain, plasmid and growth conditions S. licidans 66 was grown on TSB (Oxoid) agar plates or in TSB liquid medium containing 1 mg/1 thiostrepton (Sigma) at 30°C. Protoplast formation and transformation of S. licidans 66 were carried out as described by Hunter [30]. Sporulation of S. lividans was obtained on medium comprising 2 g of Bacto yeast extract, 10 g of starch, 15 g of agar and 1 mg of thiostrepton per liter. Plasmid pSV177 is a bifunctional vector between E. coli and Streptomyces. It was constructed by inserting the larger KpnI-SacI fragment of pSI177 [31], which is a derivative of pUC18 [32] containing ssi, between the same restriction sites of the Streptomyces multiple copy plasmid plJ702 [33]. Site-directed mutagenesis Site-directed mutagenesis was performed by the gapped duplex method [34]. Oligodeoxyribonucleotides used for mutation primers were synthesized on an Applied Biosystems model 381A DNA synthesizer using phosphoramidite chemistry. The mutations were confirmed by DNA sequencing analysis. Estimation of the expression level of SSI Qualitatively, the expression ldvel of SSI was evaluated by the intensity of the band of immunoprecipitation with anti-SSI antiserum on a TSB agar plate [28]. Quantitative evaluation of the expressed SSI was per-

1 ~ G C C

CCG

TCC

GCG

CTC

TAC

GCC

CCC

ASP ALA

PRO

SER

ALA

LEU

TYR ALA

PRO

GCG

ACG

ACC

Gee IGCAICCG ~ C G C

GCG

GGC ACC

CAC

CCG

GCG

GCC

GGC

TCG

GCC

GLY

HIS

PRO ALA

ALA

GLY

SER

ALA

Distribution of minor codons in ssi The amino acid and nucleotide sequences of mature SSI are shown in Fig. 1. Five minor codons (codons that have A or T in the third positions) are found among 113 codons. The first minor codon (GAT for Asp-l) is located at the amino terminus of SSI mature protein. The second and third (GCA for Ala-26 and GAA for Glu-28) and fourth and fifth (TGT for Cys-35 and GCT for Ala-36) are located on both sides of the region encoding the /32-strand in the secondary structure of SSI. No minor codons are found in the carboxyl terminal region. This good correspondence between the positions of minor codons and the boundaries of the protein domains might suggest that the presence of a minor codon acts not only to prevent mistranslation under stringent conditions but also affects the mode of protein folding. This possibility has also been discussed in Refs. 36-38 from the viewpoint of translation rates. Effect of minor codons on gene expression To investigate the effect of minor codons on gene expression in S. lividans 66, we substituted the codons of tandemly arranged Leu (at position 79-80), single Lys (at 89) and tandem Ser (at 108-109) in ssi for the respective minor codons by site-directed mutagenesis

i0 TCC GCC SER ALA

GTG CTG ACC

GTC

GGC

AAG

GGC

LEU

VAL

LEU

VAL

GLY

LYS

GLY VAL

GTC

ACC

CTG

ACC [TGTIIGCT~CCG

GGC

CCG

TGC

GCG

GAC

CTG

GCC

Gee

GTC

GGC

GGC

GAC

CTG

CYS

ALA

ASP

LEU ALA

ALA

VAL

GL¥

GLY

ASP

LEU

THR

30

41

40 TCG

50 THR

I TTA TTAI A 8o

1 61 AAC ASN

2O GTC

CTG

21 AGe

Results

70 GCG ALA

CTG LEU

ACG THR

CGG GGC ARG GLY

GAG GAC GTC GLU ASP VAL

81 ACC GTG

GAC

GGC

GTC

TGG

CAG GGC

THR

ASP

GLY

VAL

TRP

GLN GLY

VAL

~G

4 101 TGC GAG ATG CYS GLU MET

LYS

ATG MET

TGC CYS

90 CGG GTC ARG VAL

C C G A T G G T G TAC G A C PRO MET VAL TYR ASP

CCG PRO

GTG VAL

CTG LEU

CTC LEU

TCC

TAC

GAG

CGC

GTC

TTC

TCC

AAC

GAG

SER

TYR GLU

ARG

VAL

PHE

SER

ASN

GLU

GCC ALA

TTC PHE

i00

ITeT TCT I pl~ AAC ASN

GCG ALA

CAC HIS

110

GGC TCG AGC GTC GLY SER SER VAL

113 TTC PHE

TAG ***

o~2 Fig. 1. Amino acid and nucleotide sequence of SSI. Boxed codons are minor codons, a and /3 indicate the a-helix and /3-sheet in the secondary structure of SSI, respectively [52]. Arrows show the codon replacements carried out for Leu-79-Leu-80 (CTG-CTC to TTA-TTA), Lys-89 (AAG to AAA) and Ser-108-Ser-109 (TCG-AGC to TCT-TCT).

264 W

Wild type

Leu79- Leu8° ('n-A-'IrA)

Lys 89 (AAA)

L

K

S

Ser1°8" S erl°9 (TCT-TCT)

Fig. 2. Effect of minor codons on SSI production. Qualitative analysis of the SSI secretion level was performed as described by Obata et al. [28]. Transformed 3-day-old cells of S. liuidans 66 carrying pSV177 with wild type ssi and its minor codon mutants were placed on TSB agar plates. After 3 h, anti-SSI antiserum was spotted at the positions indicated by dots beside transformants. In the course of diffusion of both SSI secreted from the transformants and anti-SSI antibodies, lines of immunoprecipitation appeared where the two substances met. The intensity of these lines was taken as the level of expression of SSI.

(Fig. 1). These codons were chosen because (i) each one is located in the carboxyl-terminal region where no minor codons are present, (ii) Leu and Set exist as consecutive amino acids, and T I ' A and T C T are extremely rare codons [25,26], hence their influence on gene expression is presumed to be significant. Fig. 2 shows the results of qualitative immuno diffusion analysis of the effect of various codon substitutions on SSI secretory expression. As compared with the wild type, the double substitution of Leu codons caused a decrease of SSI productivity. In contrast, the expression level of ssi with a single A A A codon seemed to be increased slightly and that with double T C T codons was found to be almost the same. For exact measurement of the extent of each secretion level, we attempted to minimize the distibution of SSI expression level among transformants carrying the same gene by allowing all the transformants to sporulate and suspending the resultant spores in 10% glycerol because each spore was supposed to grow synchronously after germination. Then, the spore suspensions were spread onto TSB plates and incubated at 30°C. After 2 days, each colony was scraped off the plate into 3 ml of TSB liquid medium and incubated for 3 days. Then, values of SSI production relative to total extracellular proteins were evaluated by S D S - P A G E and subsequent densitometry. One typical example of SDSP A G E pattern is shown in Fig. 3 and the statistical results of SSI production are represented in Fig. 4. Though we managed to allow each transformant grow syncronously, the distribution of SSI productivity found in Fig. 4 appears to reflect subtle differences of growth phases. A negative effect of minor codons on gene expression was found only for the gene harboring the double leucine T T A codons, as had also been seen in the qualitative analysis (Fig. 2), and the level of SSI secretion was reduced to 60% of the wild-type level on average.

Fig. 3. SDS-PAGE analysis of extracellular proteins from S. lit,idans 66 carrying SSI genes. All secreted proteins including SSI in TSB culture supernatant were precipitated with trichloroacetic acid (TCA) at a final concentration of 6%. Next, these proteins were electrophoresed on 0.1% SDS-15% polyacrylamide gel [35] and stained with Coomassie brilliant blue. The position of SSI is indicated by an arrow. W, L, K and S denote wild type, Leu-79-Leu-80 (TTA-TTA), Lys-89 ( A A A ) and Ser-108-Ser-109 (TCT-TCT), respectively.

1~~ ' ~ i "

WildType

6

°i E ~

o

"6 8 E z

4 2 o 8 6 4

2

$' Level of SSI relative to total e x t r a c e l l u l a r p r o t e i n s (%)

Fig. 4. The level of SSI relative to total extracellular proteins. About 30 transformants, which were derived from individual primary transformants, harboring the wild and mutated ssi with minor codons, were cultured. The culture supernatant of each transformant was subjected to analysis of extracellular proteins as described in the legend to Fig. 3. The density of the bands of total proteins including SSI on SDS-polyacrylamide gel was evaluated by densitometer (Atto, ACD-25DX, Japan).

265 2

3

4

5

6

days

W L W L W L W L W L iii!

Fig. 5. Temporal effect of TTA codon on SSI productivity. As the experiment shown in Fig. 3, accumulated SSI in the supernatant cultivated for 2 to 6 days was precipitated with TCA and subjected to SDS-PAGE. The position of SSI is indicated by an arrow. W and L represent the wild type and Leu-79-Leu-80 (TTA-TTA) mutant, respectively. Numerals above capital letters indicate the days of cultivation.

In Fig. 5, the temporal effect of T I ' A codon on the expression of SSI was shown. The accumulated SSI in the culture supernatant for 2 to 6 days were estimated by SDS-PAGE as described in the quantitative experiment. Little amount of SSI could be detected from the culture of the transformant harboring a pair of T T A codons within 2 days. On the third day, the production of SSI was clearly observed from the Leu mutant. In old culture (4-6 days), although the expression level of accumulated SSI from the Leu mutant was still low, the production of SSI seemed to be increased slightly. And the expressed SSI was found to be a smaller size protein which was a minor component of the wild-type SSI. This might be due to accessibility of a smaller amount of SSI to amino terminal proteolysis [39]. In order to exclude the possibility that the product of the transformant harboring double T T A codons incorporated erroneous amino acids at these codons, we purified SSI from the supernatant obtained from a large scale of culture of this transformant and investigated its anti-subtilisin activity and amino acid composition. No differences were found in comparison with SSI derived from the wild-type transformant (data not shown). Discussion

In E. coli and S. cerevisiae, codon usage is closely related to the intracellular concentration of tRNAs [3-5], and the same situation might be assumed for Streptomyces. The relationship between codon usage and gene expression has been well examined in E. coli both experimentally [8] and theoretically [40,41], and it has been reported that translation rates differ among various codons [12]. In particular, consecutive unfavorable codons cause a marked reduction in the level of protein synthesis [11,13] or provoke frameshift [14].

The results obtained b y replacement of the codons of ssi for minor ones revealed that only the pair of leucine T T A codons brought about a conspicuous reduction of SSI expression. And the temporal effect was shown to be strict at the early stage (within 2 days) of growth, and not strict but still strong at the stationary phase when cells are grown for 4 or more days. This is in line with the regulatory expression of bldA (reviewed by Ref. 42). This gene regulates pleiotropically the morphological differentiation and secondary metabolism of S. coelicolor A3(2), and its product is one of, and perhaps the only, candidate for the tRNA recognizing U U A codon [43]. The promoter of bldA works late in growth, and therefore the product is thought to be available only under such conditions [44]. On the other hand, the promoter of ssi operates strongly and constitutively from an early stage (Fig. 5 and Ref. 31), hence the expression of the ssi mutant possessing double TTA codons would be delayed in S. lividans, which is very closely related to S. coelicolor (the counterpart of bldA of S. lividans is identical to that of S. coelicolor [45], thus bldA of S. lividans is presumed to express in the same fashion). Similar results have been reported by Leskiw et al. [46], who found that phenotypic expression of some genes (carB, lacZ, ampC) containing q-TA codons depended strongly on the presence of bldA and other genes (hyg and aad) containing T T A codons also partially. Besides bldA, other factors might influence expression of the ssi variants. The transcription level of ssi is assumed to be invariant because each version contains the same promoter and terminator. The secondary structure of each mRNA between nucleotide position 40 and 339 in Fig. 1 (which corresponds to amino acid position from 14 to 113) was predicted by computer analysis (program SECST of SDC-GENETYX, Japan). The mRNA of the wild type, Leu and Ser mutants were predicted to have almost the same structure. Therefore, it seemed unlikely that the secondary structure of these mRNAs affected their translational efficiency. On the other hand, the mRNA of the Lys mutant showed slight differences in its predicted local structure, although its overall structure was very similar to that of the wild type. In the case of the Lys mutant, the level of SSI secretion increased slightly. In E. coli, only one species of tRNA recognizing lysine is known and its anticodon is U * U U where U * is 5-methylaminomethyl-2thiouridine [47]. Hence, both the A A G and the AAA lysine codons are decoded by a single tRNALyL The modified base U* has been shown to have much higher affinity for A than G [48,49], and therefore the interaction of E. coli tRNA Ly~ with the AAA codon may be strong compared with its interaction with an A A G codon [50]. As a result, the translation rate of the AAA codon is presumed to be faster than that of the

266 A A G codon. However, hitherto the putative S. lividans tRNA TM genes have been shown to have the anticodon CTT, and not T T T [51]. Thus, maybe two tRNA species (anticodons CUU and UUU) for lysine exist in Streptomyces cells, and codon-anticodon interaction as seen for the case of E. coli tRNA Lys cannot be applied as a cause of slightly increased level of SSI expression from the gene harboring the AAA codon. From these results, it can be concluded that one or two minor codons seem not to have a significant negative effect on gene expression in Streptomyces, just as has been shown in E. coli [11], if rare tRNAs, which recognize minor codons, are expressed constitutively. However, the TI'A codon does impare gene expression strictly early in growth, and not strictly but still strongly even late in growth, probably due to the late expression of bldA.

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (03222105). We are thankful to Miss K. Ono for her excellent technical assistance.

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