Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector

Ismo Ulmanen (Corresponding Author), Kenneth Lundström, Päivi Lehtovaara, Matti Sarvas, Marja Ruohonen, Palva Ilkka

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

Original languageEnglish
Pages (from-to)176 - 182
Number of pages7
JournalJournal of Bacteriology
Volume162
Issue number1
Publication statusPublished - 1985
MoE publication typeNot Eligible

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beta-Lactamases
Bacillus subtilis
alpha-Amylases
Genes
Exopeptidases
Protein Sorting Signals
Northern Blotting
Methionine
Culture Media
Plasmids
Escherichia coli
Gene Expression

Cite this

Ulmanen, I., Lundström, K., Lehtovaara, P., Sarvas, M., Ruohonen, M., & Ilkka, P. (1985). Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. Journal of Bacteriology, 162(1), 176 - 182.
Ulmanen, Ismo ; Lundström, Kenneth ; Lehtovaara, Päivi ; Sarvas, Matti ; Ruohonen, Marja ; Ilkka, Palva. / Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. In: Journal of Bacteriology. 1985 ; Vol. 162, No. 1. pp. 176 - 182.
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abstract = "Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.K{\"a}{\"a}ri{\"a}inen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10{\%} and that of E1 was ca. 0.01{\%} of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.",
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language = "English",
volume = "162",
pages = "176 -- 182",
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Ulmanen, I, Lundström, K, Lehtovaara, P, Sarvas, M, Ruohonen, M & Ilkka, P 1985, 'Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector', Journal of Bacteriology, vol. 162, no. 1, pp. 176 - 182.

Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. / Ulmanen, Ismo (Corresponding Author); Lundström, Kenneth; Lehtovaara, Päivi; Sarvas, Matti; Ruohonen, Marja; Ilkka, Palva.

In: Journal of Bacteriology, Vol. 162, No. 1, 1985, p. 176 - 182.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector

AU - Ulmanen, Ismo

AU - Lundström, Kenneth

AU - Lehtovaara, Päivi

AU - Sarvas, Matti

AU - Ruohonen, Marja

AU - Ilkka, Palva

PY - 1985

Y1 - 1985

N2 - Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

AB - Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

UR - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC218971/pdf/jbacter00221-0186.pdf

M3 - Article

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JO - Journal of Bacteriology

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Ulmanen I, Lundström K, Lehtovaara P, Sarvas M, Ruohonen M, Ilkka P. Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. Journal of Bacteriology. 1985;162(1):176 - 182.