scholarly journals New Architectures for Tet-On and Tet-Off Regulation in Staphylococcus aureus

2009 ◽  
Vol 76 (3) ◽  
pp. 680-687 ◽  
Author(s):  
Elena Stary ◽  
Rosmarie Gaupp ◽  
Sabrina Lechner ◽  
Martina Leibig ◽  
Evelyn Tichy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Antisense Rna ◽  
Target Gene ◽  
Chromosomal Location ◽  
Constitutive Expression ◽  
Rna Expression ◽  
Posttranscriptional Gene Silencing ◽  
Gene Functions ◽  
Dose Dependent

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.

scholarly journals Chimeric antisense RNA derived from chromosomal translocation modulates target gene expression

Haematologica ◽  
2012 ◽  
Vol 97 (8) ◽  
pp. 1278-1280
Author(s):  
T. Sugimoto ◽  
A. Tomita ◽  
A. Abe ◽  
C. Iriyama ◽  
H. Kiyoi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antisense Rna ◽  
Target Gene ◽  
Chromosomal Translocation ◽  
Target Gene Expression

scholarly journals Differential Target Gene Activation by the Staphylococcus aureus Two-Component System saeRS

2009 ◽  
Vol 192 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Markus Mainiero ◽  
Christiane Goerke ◽  
Tobias Geiger ◽  
Christoph Gonser ◽  
Silvia Herbert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Target Gene ◽  
Target Genes ◽  
Gene Activation ◽  
Gene Expression Pattern ◽  
Class Ii ◽  
Class I ◽  
Single Amino Acid ◽  
Single Amino Acid Change

ABSTRACT The saePQRS system of Staphylococcus aureus controls the expression of major virulence factors and encodes a histidine kinase (SaeS), a response regulator (SaeR), a membrane protein (SaeQ), and a lipoprotein (SaeP). The widely used strain Newman is characterized by a single amino acid change in the sensory domain of SaeS (Pro18 in strain Newman [SaeSP], compared with Leu18 in other strains [SaeSL]). SaeSP determines activation of the class I sae target genes (coa, fnbA, eap, sib, efb, fib, sae), which are highly expressed in strain Newman. In contrast, class II target genes (hla, hlb, cap) are not sensitive to the SaeS polymorphism. The SaeSL allele (saeSL ) is dominant over the SaeSP allele, as shown by single-copy integration of saePQRSL in strain Newman, which results in severe repression of class I target genes. The differential effect on target gene expression is explained by different requirements for SaeR phosphorylation. From an analysis of saeS deletion strains and strains with mutated SaeR phosphorylation sites, we concluded that a high level of SaeR phosphorylation is required for activation of class I target genes. However, a low level of SaeR phosphorylation, which can occur independent of SaeS, is sufficient to activate class II target genes. Using inducible saeRS constructs, we showed that the expression of both types of target genes is independent of the saeRS dosage and that the typical growth phase-dependent gene expression pattern is not driven by SaeRS.

scholarly journals Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection

1999 ◽  
Vol 181 (21) ◽  
pp. 6585-6590 ◽  
Author(s):  
Yinduo Ji ◽  
Andrea Marra ◽  
Martin Rosenberg ◽  
Gary Woodnutt
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Antisense Rna ◽  
Expression System ◽  
Toxin Gene ◽  
Critical Element ◽  
Bacterial Cells ◽  
Alpha Toxin

ABSTRACT The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted atet regulatory expression system for use inStaphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of theS. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisensehla RNA downregulated chromosomally derived hlagene expression in vitro approximately 14-fold. Similarly, induction ofhla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureusinfection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that thetet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.

scholarly journals Role of the Distal sarA Promoters in SarA Expression in Staphylococcus aureus

2005 ◽  
Vol 73 (7) ◽  
pp. 4391-4394 ◽  
Author(s):  
Ambrose L. Cheung ◽  
Adhar C. Manna
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Protein Level ◽  
Target Gene ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Regulatory Locus ◽  
Target Gene Expression

ABSTRACT The global regulatory locus sarA comprises a 375-bp open reading frame that is driven by three promoters, the proximal P1 and distal P3 and P2 promoters. We mutated the weaker P3 and P2 promoters to ascertain the effect of the change on SarA protein and target gene expression. Our results indicated that the solely active P1 promoter led to a lower SarA protein level, which has an effect on agr transcription and subsequently had corresponding effects on hla, sspA, and spa transcription, probably in both agr-independent and agr-dependent manners.

scholarly journals New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli

2015 ◽  
Vol 81 (9) ◽  
pp. 3243-3254 ◽  
Author(s):  
Sybille Schwendener ◽  
Vincent Perreten
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Fusion Proteins ◽  
Target Gene ◽  
Shuttle Vector ◽  
Expression System ◽  
Multiple Cloning Site ◽  
Content Type ◽  
E Coli

ABSTRACTFourStaphylococcus aureus-Escherichia colishuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcapcontain the strong constitutive promoter ofS. aureustype 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including theE. coliorigin ColE1, five copies of terminatorrrnBT1, and tetracycline resistance markertet(L) forS. aureusandE. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand originoriLfrom pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability inS. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcapoffer the potential to generate C-terminal RGS-His6translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links thergs-his6codons to the 3′ end of the target gene. The generation of thergs-his6codon-fusion, gene expression, and protein purification were demonstrated in bothS. aureusandE. coliusing the macrolide-lincosamide-streptogramin B resistance geneerm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Dose-Dependent Increase of Nrf2 Target Gene Expression in Mice Exposed to Ionizing Radiation

2019 ◽  
Vol 191 (2) ◽  
pp. 176 ◽  
Author(s):  
Shuta Miura ◽  
Masaru Yamaguchi ◽  
Hironori Yoshino ◽  
Yuji Nakai ◽  
Ikuo Kashiwakura
Keyword(s):  
Gene Expression ◽  
Ionizing Radiation ◽  
Target Gene ◽  
Target Gene Expression ◽  
Nrf2 Target Gene ◽  
Dose Dependent Increase ◽  
Dose Dependent
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