A broad-host-range expression vector series including a Ptac test plasmid and its application in the expression of the dod gene of Serratia marcescens (coding for ribulose-5-phosphate 3-epimerase) in Pseudomonas stutzeri

Graupner, Stefan; Wackernagel, Wilfried

doi:10.1016/s1389-0344(00)00061-7

Cited by 18 publications

(12 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When ␤-galactosidase activities were measured, tadp was at least as strong as unrepressed tacp, if not slightly stronger (data not shown). Because tacp is known to be a strong synthetic promoter (14), these results indicate that tadp is also a strong promoter. We also compared tadp and tacp in E. coli, where the synthetic tacp is known to be strong.…”

Section: Resultsmentioning

confidence: 74%

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

et al. 2008

View full text Add to dashboard Cite

The tad (tight adherence) locus of Aggregatibacter actinomycetemcomitans includes genes for the biogenesis of Flp pili, which are necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis. Although studies have elucidated the functions of some of the Tad proteins, little is known about the regulation of the tad locus in A. actinomycetemcomitans. A promoter upstream of the tad locus was previously identified and shown to function in Escherichia coli. Using a specially constructed reporter plasmid, we show here that this promoter (tadp) functions in A. actinomycetemcomitans. To study expression of the pilin gene (flp-1) relative to that of tad secretion complex genes, we used Northern hybridization analysis and a lacZ reporter assay. We identified three terminators, two of which (T1 and T2) can explain flp-1 mRNA abundance, while the third (T3) is at the end of the locus. T1 and T3 have the appearance and behavior of intrinsic terminators, while T2 has a different structure and is inhibited by bicyclomycin, indicating that T2 is probably Rho dependent. To help achieve the appropriate stoichiometry of the Tad proteins, we show that a transcriptional-termination cascade is important to the proper expression of the tad genes. These data indicate a previously unreported mechanism of regulation in A. actinomycetemcomitans and lead to a more complete understanding of its Flp pilus biogenesis.

show abstract

Section: Resultsmentioning

confidence: 74%

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

et al. 2008

View full text Add to dashboard Cite

show abstract

“…EF435043) was derived from pRK415 (Keen et al, 1988) by insertion of a 1.2 kbp PCR product covering the lacI q gene. The plasmid pQLICE consists of a derivative of RSF1010 (Scholz et al 1989) containing the 1.9 kbp BamHI-AgeI fragment covering the lacI q gene and the p tac promoter (de Boer et al, 1983) of plasmid pYanni1 (Graupner & Wackernagel, 2000), and also has the strAB genes (streptomycin resistance). The GenBank accession number of that plasmid is EF189157.…”

Section: Methodsmentioning

confidence: 99%

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi

2007

View full text Add to dashboard Cite

In prokaryotes, homologous recombination is essential for the repair of genomic DNA damage and for the integration of DNA taken up during horizontal gene transfer. In Escherichia coli, the exonucleases RecJ (specific for 59 single-stranded DNA) and RecBCD (degrades duplex DNA) play important roles in recombination and recombinational double-strand break (DSB) repair by the RecF and RecBCD pathways, respectively. The cloned recJ of Acinetobacter baylyi partially complemented an E. coli recJ mutant, suggesting functional similarity of the enzymes. A DrecJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a DrecBCD mutant was UV-sensitive, and had a low viability and altered transformation. Compared to wild-type, transformation with large chromosomal DNA fragments was decreased about 5-fold, while transformation with 1.5 kbp DNA fragments was increased 3.3-to 7-fold. A DrecD mutation did not affect transformation, viability or UV resistance. However, double mutants recJ recBCD and recJ recD were non-viable, suggesting that the RecJ DNase or the RecBCD DNase (presumably absent in recD) becomes essential for the recombinational repair of spontaneously inactivated replication forks if the other DNase is absent. A model of recombination during genetic transformation is discussed in which the two ends of the single-stranded donor DNA present in the cytoplasm frequently integrate separately and often with a time difference. If replication runs through that genomic region before both ends of the donor DNA are ligated to recipient DNA, a double-strand break (DSB) is formed. In these cases, transformation becomes dependent on DSB repair.

show abstract

“…Although there are a number of broad-host-range vectors with expression systems based on lacI q -Plac (2,7,16,18,30), cloned genes often are expressed at unacceptably high levels in the absence of induction. Full repressional control of the lac operon by LacI requires cooperative interaction of the tetrameric repressor at two of the three operators, O 1 , O 2 , and O 3 , that are located in the operon (38).…”

Section: Construction and Properties Of The Psrk Family Of Vectorsmentioning

confidence: 99%

Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing

Khan

Gaines

Roop

et al. 2008

Appl Environ Microbiol

306

267

View full text Add to dashboard Cite

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI q -lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alphaand gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ␣. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10-to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

show abstract

A broad-host-range expression vector series including a Ptac test plasmid and its application in the expression of the dod gene of Serratia marcescens (coding for ribulose-5-phosphate 3-epimerase) in Pseudomonas stutzeri

Cited by 18 publications

References 31 publications

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

Transcriptional Regulation of the tad Locus in Aggregatibacter actinomycetemcomitans : a Termination Cascade

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi

Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing

Contact Info

Product

Resources

About