Organization and Expression of a
            <i>Thermus thermophilus</i>
            Arginine Cluster: Presence of Unidentified Open Reading Frames and Absence of a Shine-Dalgarno Sequence

Aims: To obtain data concerning gene expression in Thermus thermophilus and demonstrate the use of the b-galactosidase gene from Thermus sp. A4 as a convenient reporter gene. Methods and Results: Thermus thermophilus PPKU was constructed, in which the b-gal gene was deleted from the chromosome. Two inducible promoters PdnaK (regulating the DnaK heat shock-inducible protein) and Parg (regulating expression of an arginine-inducible protein) and a carbon-regulated promoter, Pscs-mdh (regulating expression of succinyl-coA and malate dehydrogenase) were cloned upstream of the b-gal reporter gene derived from Thermus sp. A4 to construct vectors pTEX7, pTEX8 and pTEX9, respectively. The amount of b-galactosidase activity produced by the PdnaK promoter in pTEX7 was substantially above the background level of 0AE3 U mg )1 , and increased from 5AE2 to 10AE4 U mg )1 after heat-shock induction indicating that significant amounts of DnaK are produced even when T. thermophilus is grown at its optimum temperature. The Parg promoter was found to be maximally induced by 10-30 mM M arginine, but was inhibited by higher concentrations. The Pscs-mdh promoter was maximally active in the presence of malate while lower levels of activity were observed in the presence of succinate, pyruvate, glutamate, glucose and the presence of yeast extract or peptone.Conclusions: These results demonstrate that several inducible and regulated promoters are available for genetic studies in Thermus and that b-galactosidase can be used as a convenient reporter gene for studies of transcriptional regulation in Thermus. Significance and Impact of the Study: The availability of characterized inducible and regulated promoters will facilitate the development of improved gene expression vectors for Thermus. The demonstration that b-galactosidase activity in T. thermophilus PPKU can be used to allow reliable screening for b-gal-positive transformant colonies on agar plates will add to the convenience of performing genetic manipulations in T. thermophilus. Future studies of transcriptional regulation in Thermus will benefit from the b-gal host-vector system reported here.

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“…The P arg promoter of T. thermophilus was previously shown to be inducible by arginine at the transcriptional level (Sanchez et al. 2000).…”

Section: Discussionmentioning

confidence: 99%

“…1978). Arginine and uracil‐free liquid medium with 20 mmol l −1 pyruvate as the carbon source and 10 mmol l −1 ammonium sulphate as the nitrogen source (Sanchez et al. 2000) was used for arginine induction experiments.…”

Section: Growth Conditionsmentioning

confidence: 99%

Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh

Park

Kilbane

2004

Lett Appl Microbiol

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“…This region has been referred to as the downstream box (DB) (Sprengart et al ., 1996) or the downstream region (DR) (Stenström et al ., 2001a). Several suggestions have been presented as explanations to effects by this region, such as codon usage (Robinson et al ., 1984; Bulmer, 1988; Sorensen et al ., 1989), involvement in secondary structures (de Smit and van Duin, 1994), mRNA stability (Kuroda and Maliga, 2001), peptidyl‐tRNA drop‐off (Karimi and Ehrenberg, 1994; Dincbas et al ., 1999) or complementary binding to the 16S rRNA (Sprengart et al ., 1990; Faxen et al ., 1991; Hartz et al ., 1991; Etchegaray and Inouye, 1999; Sanchez et al ., 2000). However, the last suggestion appears to be experimentally refuted (Resch et al ., 1996; Blasi et al ., 1999; O’Connor et al ., 1999; La Teana et al ., 2000).…”

Section: Introductionmentioning

confidence: 99%

Influences on gene expression in vivo by a Shine–Dalgarno sequence

Jin

Zhao

Valdivia

et al. 2006

Molecular Microbiology

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SummaryThe Shine-Dalgarno (SD + : 5 ¢ -AAGGAGG-3 ¢ ) sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD + sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD + is confirmed. A downstream SD + gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD + is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD + is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD + and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD + located between them. A minor positive contribution to upstream initiation resulting from 3 ¢ to 5 ¢ ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD + or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.

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“…For T. thermophilus, Sanchez et al (2000) previously suggested that the argF promoter is regulated by ArgR. For the initial investigation, we aligned the sequences between the argF promoter region containing the ARG box and the hcs promoter region (Fig.…”

Section: Identification Of Ttc1194 As the Argr Gene Of T Thermophilusmentioning

confidence: 99%

Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus

et al. 2006

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Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an α-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.

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Organization and Expression of a Thermus thermophilus Arginine Cluster: Presence of Unidentified Open Reading Frames and Absence of a Shine-Dalgarno Sequence

Cited by 16 publications

References 37 publications

Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh

Gene expression studies of Thermus thermophilus promoters PdnaK, Parg and Pscs-mdh

Influences on gene expression in vivo by a Shine–Dalgarno sequence

Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus

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