Permanent Inhibition of Angiotensinogen Synthesis by Antisense RNA Expression

ABSTRACT Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (tet) system for Staphylococcus aureus. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of tetR or a variant allele, rev-tetR r2. The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev-TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)tetR constructs, expression from a chromosomal Pxyl/tet-gfpmut2 fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native S. aureus gene (zwf) was put under tet control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev-TetR control of P xyl/tet -gfpmut2. Additionally, TetR was used in trans to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the fabI gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new tet systems and their flexible use for different purposes.

Download Full-text

Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.73.6.3568-3576.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3568-3576 ◽

Cited By ~ 29

Author(s):

Bing Wang ◽

Howard K. Kuramitsu

Keyword(s):

Streptococcus Mutans ◽

Gene Function ◽

Antisense Rna ◽

Coenzyme A ◽

Essential Gene ◽

Essential Genes ◽

Expression Vectors ◽

Rna Expression ◽

Gene Encoding ◽

Antisense Dna

ABSTRACT In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.

Download Full-text