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

Abstract

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...

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

“…Then, the overexpression cassette of P. putida fis was cloned into pJB785TT (Santos et al, 2001). The luc gene in pJB785TT was replaced with the 2 kb BamHI-XbaI fragment from pBR-lacItac (Ojangu et al, 2000) carrying a lacI q -P tac cassette to obtain pJB-lacI q -P tac . Then, the fis gene (PP4821) was amplified from the chromosomal DNA of P. putida PaW85 by PCR using oligonucleotides fisRBSPstI (59-CACTGCAGAAGGGGTGGCCGCA-TGAC-39) and fisBamHI (59-ATGGATCCTTACAACAAGTCGTAC-TGC-39).…”

Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida

“…The pheBA promoter resembles the catBCA promoter and is also activated by CatR (Kasak et al, 1993 ;Parsek et al, 1995). Comparative studies of the interaction of CatR with the promoters of the pheBA and catBCA operons have revealed that the CatRmediated activation mechanism is well conserved, despite the different origins of these operons (Parsek et al, 1995 ;Tover et al, 2000).…”

“…Subsequently, the catBCA promoter was inserted using BamHI-and XhoI-generated ends into the promoter-probe vector pKTlacZ to obtain plasmid pZ-catBCA. For the construction of P. putida CatR overexpression strain PaWCatR + , the catR gene was cloned from plasmid pKR∆HF (Rothmel et al, 1991) by using HindIII-and EcoRI-generated ends to the vector pBRlacItac (Ojangu et al, 2000) cleaved with same enzymes (pBRlacItaccatR in Table 1). The CatR expression cassette lacI q -Ptac-catR was inserted into pUC18Not using EcoRI-and BamHI-generated ends.…”

“…Construction of the pheBA promoter-lacZ transcriptional fusion (plasmid pZ-pheBA) was described previously (Tover et al, 2000). For the cloning of the catBCA promoter into the promoter-probe plasmid pKTlacZ (Ho4 rak & Kivisaar, 1998), the 207 bp catBCA promoter region from the P. putida chromosome was amplified by using oligonucleotides cat35 (5h-GGGCTGC-CAGCCGCGGGCCC-3h) and AFC62 [5h-AGCGCGGCGG-CTCGACGACGCTG(PstI)CAGAGC-3h], complementary to the upstream and downstream regions of the catBCA promoter, respectively.…”

Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida

“…In order to construct a control strain expressing the luxAB genes under the control of the P tac promoter, the BamHI-generated DNA fragment containing the lacI q -P tac regulatory region was subcloned from pBRlacItac (Ojangu et al, 2000) into plasmid pUCNotluxAB to obtain plasmid pUCNottacluxAB. Then, the lacI q -P tac -luxAB expression cassette was inserted into the NotI site of plasmid mTn5SSgusA40.…”