Transcription from Fusion Promoters Generated during Transposition of Transposon Tn
            <i>4652</i>
            Is Positively Affected by Integration Host Factor in
            <i>Pseudomonas putida</i>

Teras, Riho; Hõrak, Rita; Kivisaar, Maia

doi:10.1128/jb.182.3.589-598.2000

Cited by 21 publications

(30 citation statements)

References 43 publications

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“…Strain Generation-Introduction of the lacI Q /P tac -ihfAB mini-Tc transposon carried on the suicide plasmid pUTtetPF (24) into the chromosome of P. putida KT2440::dmpR-Tel was by conjugation from the E. coli donor host S17pir. Tellurite and tetracycline were used as counter selection for the donor and recipient parental strains, respectively.…”

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confidence: 99%

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

Bernardo¹,

Johansson²,

Skärfstad

et al. 2009

Journal of Biological Chemistry

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The 54 -factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of 54 -dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of 54 -dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of 54 -dependent transcription in combination with a series of 54 -promoters with graded affinities for 54 -RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted 54 -transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous 54 -RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive 54 -transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity 54 -promoters that is conferred by the strong DNA binding properties of the P. putida 54 -RNA polymerase holoenzyme.To ensure promoter specificity, the bacterial multisubunit catalytic core RNA polymerase (RNAP) 4 (␣ 2 ␤␤Ј) is guided to the distinct promoter classes within the genome by a dissociable -factor. In addition to the household 70 -factor, most bacteria encode a variable number of alternative -factors that fall into two families based on primary amino acid sequences and regulatory properties. The largest of these is the 70 -family, which encompasses most alternative -factors within four subgroups that all form RNAP holoenzymes that can spontaneously initiate transcription (1). Orthologs of the structurally distinct 54 -subunit are the only members of the second family. 54 imposes kinetic constrains that lock the 54 -RNAP holoenzyme in a non-productive closed complex. Thus, 54 -dependent transcription always requires activators, called bacterial enhancer-binding proteins or bEBPs, which use ATP catalysis to remodel 54

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confidence: 99%

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

Bernardo¹,

Johansson²,

Skärfstad

et al. 2009

Journal of Biological Chemistry

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“…This indicated that the fusion promoters are certainly stationary phase specific. Previously we have shown that transcription from the fusion promoters PRA1 and PLA1, containing sequences of either the right or left end of Tn4652, respectively, is modulated by IHF and that the positive effect of IHF becomes apparent in stationary-phase cells of P. putida (43). However, the fusion promoters cloned into the pKTLacZ reporter plasmid lacked functional IHF binding sites but were still expressed at an increased level in stationary-phase cells.…”

Section: Resultsmentioning

confidence: 98%

“…The S -independent increase in transcription became evident with all of the fusion promoters. Gel mobility shift experiments with Tn4652 ends and crude lysate of P. putida have revealed that in addition to IHF, some other proteins form specific complexes with the ends of Tn4652 (18,43). The amount of the protein-DNA complexes detected depends on the growth phase of the bacteria used for the preparation of cell extracts (43).…”

Section: Resultsmentioning

confidence: 99%

“…Gel mobility shift experiments with Tn4652 ends and crude lysate of P. putida have revealed that in addition to IHF, some other proteins form specific complexes with the ends of Tn4652 (18,43). The amount of the protein-DNA complexes detected depends on the growth phase of the bacteria used for the preparation of cell extracts (43). It is therefore possible that a protein(s) which binds termini of Tn4652 could sequester transcription from the fusion promoters in a growth phase-dependent manner.…”

Section: Resultsmentioning

confidence: 99%

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Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς ^S -Dependent Transcription in Pseudomonas putida

et al. 2000

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The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is S . In contrast to other minor sigma factors, RNA polymerase holoenzyme containing S (E S ) recognizes a number of promoters which are also recognized by that containing 70 (E 70 ). We have previously shown that transposon Tn4652 can activate silent genes in starving Pseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the 70 -specific promoter consensus. The ؊10 hexameric sequence and the sequence downstream from the ؊10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of S on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the ؊10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the ؊10 element can affect In their natural environment, most bacteria are challenged by widely changing nutrient availability and by exposure to various forms of physical stress (temperature shock, oxidative stress, etc.). When starvation or various other stress factors cause a reduction or cessation of growth, many genes are shut down while others are induced to help the cells to survive. One way to modulate gene expression is replacement of the main sigma factor with an alternative sigma factor that recognizes a specific promoter of the stimulus response gene (reviewed in reference 19). In Escherichia coli, S regulates the expression of more than 100 genes involved in cell survival in the stationary phase and in response to different stresses (reviewed in references 14 and 15). The rpoS gene encoding S has been described also for nonenteric bacteria, e.g., fluorescent pseudomonads (21,36,37,40). Despite clearly different physiological roles, S is similar to the major sigma factor 70 in terms of structure and molecular function (7,26,42). No clear differences between 70 -and S -dependent promoters are apparent. A compilation of S -dependent promoters deduced a Ϫ10 consensus sequence, CTATACT, that is slightly different from the typical TATAAT sequence recognized by 70 (11). However, the binding patterns of E S and E 70 revealed by DNase I protection experiments are not completely identical (29,41). E S appears to be less dependent on contacts in the Ϫ35 region (7,17,41). The activity of E S and E 70 is differentially influenced by salt concentrations and by the degree of negative supercoiling of the DNA template (2, 10, 23). Additionally, a number of global regulators and histone-like proteins, such as H-NS, Lrp, CRP, IHF, and Fis, are involved in determination of sigma factor specificity (29; see also references 14 and 15 for a review and references cited therein).We have previously shown the generation of constitutively ex...

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“…Furthermore, the efficient site-specific resolution of the Tn4651-mediated cointegrate required the two gene products, TnpS and TnpT, and the 2.4-kb region carrying tnpSres-tnpT was 48 kb apart from the tnpA gene (34). Although the cointegration step of Tn4651 has been extensively analyzed by Kivisaar's group (13,14,15,32), no detailed analysis of the resolution system has been carried out in more than a decade.…”

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Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651

2002

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The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323-and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.

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Transcription from Fusion Promoters Generated during Transposition of Transposon Tn 4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

Cited by 21 publications

References 43 publications

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς ^S -Dependent Transcription in Pseudomonas putida

Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651

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Transcription from Fusion Promoters Generated during Transposition of Transposon Tn 4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

Cited by 21 publications

References 43 publications

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς S -Dependent Transcription in Pseudomonas putida

Site-Specific Recombination System Encoded by Toluene Catabolic Transposon Tn4651

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Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς ^S -Dependent Transcription in Pseudomonas putida