scholarly journals m-Xylene-Responsive Pu-PnifH Hybrid σ54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

2005 ◽  
Vol 187 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Manuel Carmona ◽  
Silvia Fernández ◽  
María J. Rodríguez ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Promoter Sequence ◽  
Growth Conditions ◽  
Integration Host Factor ◽  
Vector System ◽  
Wild Type ◽  
Physiological Control ◽  
Accurate Comparison ◽  
Rnap Binding

ABSTRACT The sequences surrounding the −12/−24 motif of the m-xylene-responsive σ54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH σ54 promoter variants known to have various degrees of efficacy and affinity for σ54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the −12/−24 region of the wild-type PnifH and its higher σ54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the −12/−24 region of Pu dictates the coupling of promoter output to growth conditions.

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

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-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 ςS 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 ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting

2011 ◽  
Vol 82 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Marc Valls ◽  
Rafael Silva-Rocha ◽  
Ildefonso Cases ◽  
Amalia Muñoz ◽  
Víctor de Lorenzo
Keyword(s):  
Functional Analysis ◽  
Pseudomonas Putida ◽  
Integration Host Factor ◽  
Host Factor

scholarly journals TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

2007 ◽  
Vol 189 (24) ◽  
pp. 9037-9043 ◽  
Author(s):  
Carolina Elvira César ◽  
Matxalen Llosa
Keyword(s):  
Promoter Sequence ◽  
Dna Topology ◽  
Integration Host Factor ◽  
Host Factors ◽  
Elongation Complex ◽  
Site Specific ◽  
Double Stranded Dna ◽  
Transcript Elongation ◽  
A Site

ABSTRACT R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a “closed” conformation. In an IHF-deficient (IHF−) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF− background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an “open” form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

scholarly journals Involvement of H-NS in Transpositional Recombination Mediated by IS1

2001 ◽  
Vol 183 (8) ◽  
pp. 2476-2484 ◽  
Author(s):  
Yasuyuki Shiga ◽  
Yasuhiko Sekine ◽  
Yasunobu Kano ◽  
Eiichi Ohtsubo
Keyword(s):  
Dna Binding ◽  
Wild Type Strain ◽  
Integration Host Factor ◽  
Deficient Mutant ◽  
Wild Type ◽  
Transposase Gene ◽  
In The Wild ◽  
Transposition Reaction ◽  
Target Plasmid

ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

scholarly journals Evidence of In Vivo Cross Talk between the Nitrogen-Related and Fructose-Related Branches of the Carbohydrate Phosphotransferase System of Pseudomonas putida

2008 ◽  
Vol 190 (9) ◽  
pp. 3374-3380 ◽  
Author(s):  
Katharina Pflüger ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Cross Talk ◽  
Complete System ◽  
Growth Conditions ◽  
High Energy ◽  
Phosphotransferase System ◽  
Primary Flow ◽  
Fructose Intake ◽  
C Metabolism

ABSTRACT The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded by ptsP (EINtr), ptsO (NPr), and ptsN (EIIANtr), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the EIIANtr enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to EINtr to NPr to EIIANtr. We also found that in the presence of fructose, unlike in the presence of succinate, EIIANtr can be phosphorylated in a ptsP strain but not in a ptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.

scholarly journals Transcription from Fusion Promoters Generated during Transposition of Transposon Tn4652 Is Positively Affected by Integration Host Factor in Pseudomonas putida

2000 ◽  
Vol 182 (3) ◽  
pp. 589-598 ◽  
Author(s):  
Riho Teras ◽  
Rita Hõrak ◽  
Maia Kivisaar
Keyword(s):  
Pseudomonas Putida ◽  
Specific Binding ◽  
Stationary Growth Phase ◽  
Integration Host Factor ◽  
Host Factor ◽  
Mobile Dna ◽  
Mobility Shift ◽  
Transposase Gene ◽  
Gel Mobility Shift

ABSTRACT We have previously shown that both ends of the Tn3family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA inPseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822–2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the −35 hexamer provided by the terminal inverted repeats of Tn4652 and the −10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts fromP. putida cells allowed us to detect specific binding ofP. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.

scholarly journals Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor

1999 ◽  
Vol 181 (14) ◽  
pp. 4245-4249 ◽  
Author(s):  
Eun Hee Cho ◽  
Chan-Eun Nam ◽  
Renato Alcaraz ◽  
Jeffrey F. Gardner
Keyword(s):  
Integration Host Factor ◽  
Host Factor ◽  
Maximum Efficiency ◽  
Wild Type ◽  
Bacteriophage P22 ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Mutant Cells ◽  
Formation Of Complexes

ABSTRACT Site-specific recombination by phages λ and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates λ site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting λ site-specific recombination than wild-type cells. Although the attPregion of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.

scholarly journals Activation and Repression of Transcription at the Double Tandem Divergent Promoters for the xylR and xylS Genes of the TOL Plasmid of Pseudomonas putida

1998 ◽  
Vol 180 (11) ◽  
pp. 2889-2894 ◽  
Author(s):  
Silvia Marqués ◽  
María-Trinidad Gallegos ◽  
Maximino Manzanera ◽  
Andreas Holtel ◽  
Kenneth N. Timmis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Pseudomonas Putida ◽  
Integration Host Factor ◽  
Tol Plasmid ◽  
Divergent Promoters ◽  
Upstream Promoter ◽  
And Control ◽  
Background Expression ◽  
Very High

ABSTRACT The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive ς70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive ς70-dependent promoter, and by the regulatable ς54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions −2 to −30 (−2/−30 region) and the −137/−156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a ς54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing ς54 modulates expression from the downstream Ps2 ς70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the ς54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing ς54 at the upstream promoter may modulate expression from the Ps2 promoter.

NGHIÊN CỨU SỬ DỤNG MÀNG BAO SINH HỌC TỪ DỊCH CHIẾT VI KHUẨN Pseudomonas putida 199B ĐẾN KHÁNG NẤM Aspergilus flavus T1 TRONG QUÁ TRÌNH BẢO QUẢN HẠT GIỐNG NGÔ

2017 ◽  
Vol 126 (3C) ◽  
Author(s):  
Nguyễn Thỵ Đan Huyền ◽  
Lê Thanh Long ◽  
Trần Thị Thu Hà ◽  
Nguyễn Cao Cường ◽  
Nguyễn Hiền Trang
Keyword(s):  
Pseudomonas Putida ◽  
28S Rrna

Chủng T1 phân lập từ các mẫu ngô nếp NK66 nhiễm nấm mốc tự nhiên được sử dụng để nghiên cứu khả năng kháng nấm của dịch chiết vi khuẩn Pseudomonas putida 199B. Đặc điểm hình thái của chủng T1 đã được quan sát đại thể (màu sắc, hình dáng, kích thước khuẩn lạc) trên môi trường PDA và vi thể (hình dáng bào tử) trên kính hiển vi kết hợp so sánh với loài Aspergilus flavus đối chứng. Kết quả phân tích trình tự gen mã hóa 28S rRNA của chủng T1 cho thấy sự tương đồng trình tự cao với các trình tự tương ứng của loài Aspergilus flavus trên ngân hàng gen. Kết quả khảo sát ảnh hưởng của dịch chiết vi khuẩn P. putida lên sự phát triển của nấm A.  flavus gây bệnh trên hạt ngô sau thu hoạch và bảo quản ở điều kiện in vitro cho thấy, ở nồng độ P. putida 24% đã ức chế 74,50% sự phát triển đường kính tản nấm sau 10 ngày nuôi cấy, ức chế 79,63% sự hình thành sinh khối sợi nấm sau 7 ngày nuôi cấy. Ở điều kiện in vivo, sự nảy mầm của hạt giống ngô sau 30 ngày được tạo màng bao sinh học bằng dịch chiết vi khuẩn P. putida nồng độ 18% đạt 97,91%, tỉ lệ hạt nhiễm nấm mốc giảm còn 20% so với 72% ở mẫu đối chứng.

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