scholarly journals Analysis of Promoter Elements Involved in the Transcriptional Initiation of RpoS-Dependent Borrelia burgdorferi Genes

2004 ◽  
Vol 186 (21) ◽  
pp. 7390-7402 ◽  
Author(s):  
Christian H. Eggers ◽  
Melissa J. Caimano ◽  
Justin D. Radolf
Keyword(s):  
Borrelia Burgdorferi ◽  
Sigma Factor ◽  
Fluorescent Protein ◽  
Core Promoter ◽  
Expression Patterns ◽  
Inducible Promoter ◽  
Transcriptional Initiation ◽  
E Coli ◽  
Promoter Recognition ◽  
Maximal Expression

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoSBb) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoSBb recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoSBb dependent, while the ospE and flaB reporters were RpoSBb independent. To compare promoter recognition by RpoSBb with that of the prototype RpoS (RpoSEc), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoSEc the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoSBb under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoSEc and RpoSBb each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(−14) in the presumptive −10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoSBb as it is for RpoSEc; (ii) the nucleotide at the −15 position determines RpoS or σ70 selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoSEc- or RpoSBb-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.

scholarly journals Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation byBorrelia burgdorferi, the Lyme Disease Spirochete

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Arianna P. Grove ◽  
Dionysios Liveris ◽  
Radha Iyer ◽  
Mary Petzke ◽  
Joseph Rudman ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Sigma Factor ◽  
Fluorescent Protein ◽  
Core Promoter ◽  
Host Adaptation ◽  
Mammalian Host ◽  
Alternative Sigma Factor ◽  
The Core ◽  
Rpos Mutant ◽  
Green Fluorescent

ABSTRACTThe alternative sigma factor RpoS plays a key role modulating gene expression inBorrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identifycisregulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genesospAB, theglpoperon, andbba74. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboringospAB- andglp-gfpconstructs containing only the minimal (−35/−10) σ70promoter elements had significantly lower expression in DMCs relative to growthin vitroat 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression ofbba74required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivatedB. burgdorferiproduced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g.,ospABand theglpoperon) involves primarily sequence elements near the core promoter, while the other (e.g.,bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout theB. burgdorferienzootic cycle.IMPORTANCEBorrelia burgdorferi, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a “gatekeeper” due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with anin vivomodel to definecisregulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression ofospABand theglpoperon requires only sequences near their core promoters, whereas modulation ofbba74expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.

scholarly journals Molecular Characterization of Borrelia burgdorferi erp Promoter/Operator Elements

2004 ◽  
Vol 186 (9) ◽  
pp. 2745-2756 ◽  
Author(s):  
Kelly Babb ◽  
Jason D. McAlister ◽  
Jennifer C. Miller ◽  
Brian Stevenson
Keyword(s):  
Borrelia Burgdorferi ◽  
Dna Sequences ◽  
Fluorescent Protein ◽  
Factor H ◽  
Surface Proteins ◽  
Virulence Determinants ◽  
Transcriptional Initiation ◽  
Bacterial Proteins ◽  
Complement Regulator ◽  
Green Fluorescent

ABSTRACT Many Borrelia burgdorferi Erp outer surface proteins have been demonstrated to bind the host complement regulator factor H, which likely contributes to the ability of these organisms to evade the host innate immune system. B. burgdorferi controls Erp protein synthesis throughout the bacterial infectious cycle, producing the proteins during mammalian infections but repressing their synthesis during tick infections. Defining the mechanism by which B. burgdorferi regulates the expression of these virulence determinants will provide important insight into the biological and pathogenic properties of the Lyme disease spirochete. The present study demonstrates that two highly conserved DNA sequences located 5′ of erp operons specifically bind bacterial proteins. Analyses with B. burgdorferi of transcriptional fusions between erp promoter/operator DNAs and the gene for green fluorescent protein indicated that the expression of these operons is regulated at the level of transcriptional initiation. These analyses also indicated significant differences in the promoter strengths of various erp operons, which likely accounts for reported variations in expression levels of different Erp proteins. Mutagenesis of promoter-gfp fusions demonstrated that at least one of the proteins which bind erp operator DNA functions as a repressor of transcription.

Transformation of Escherichia coli K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiology†

2006 ◽  
Vol 69 (2) ◽  
pp. 276-281 ◽  
Author(s):  
T. P. OSCAR ◽  
K. DULAL ◽  
D. BOUCAUD
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Predictive Models ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
E Coli ◽  
Contaminated Food ◽  
Green Fluorescent ◽  
K 12 ◽  
High Copy Plasmid

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (μmax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40°C, μmax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

scholarly journals Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Virtu Solano-Collado ◽  
Sofía Ruiz-Cruz ◽  
Fabián Lorenzo-Díaz ◽  
Radoslaw Pluta ◽  
Manuel Espinosa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulation Of Gene Expression ◽  
E Coli ◽  
Core Enzyme ◽  
Protein Architecture ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

scholarly journals Molecular Cloning and Functional Characterization of Bisabolene Synthetase (SaBS) Promoter from Santalum album

Forests ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 85
Author(s):  
Haifeng Yan ◽  
Yuping Xiong ◽  
Jaime A. Teixeira da Silva ◽  
Jinhui Pang ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Transgenic Arabidopsis ◽  
Core Promoter ◽  
Functional Characterization ◽  
Promoter Sequence ◽  
Inducible Promoter ◽  
Gus Activity ◽  
Regulation Mechanism ◽  
Santalum Album ◽  
Transcriptional Initiation ◽  
Key Enzyme

Bisabolene-type sesquiterpenoids, which have multiple bioactivities, including anticancer activity, are one of the main groups of compounds in the essential oil extracted from Santalum album L. and other Santalum species. Bisabolene synthetase (SaBS) is a key enzyme for the synthesis of bisabolene in S. album, but the regulation of the SaBS gene’s expression is poorly understood. In this study, a 1390-bp promoter sequence of the SaBS gene was isolated from the leaves of six-year-old S. album. A bioinformatics analysis showed that certain environment stresses and phytohormone-activated cis-acting elements were distributed in different regions of the SaBS promoter (PSaBS). Transgenic Arabidopsis carrying full-length PSaBS had significantly higher β-glucuronidase (GUS) activity than the untreated control after treatment with salicylic acid (SA), suggesting that PSaBS is a SA-inducible promoter. Histochemical GUS staining and GUS fluorometric assays of transgenic Arabidopsis showed that the GUS activity directed by PSaBS was mainly expressed in stem tissue, followed by leaves and flowers. Moreover, different regions of PSaBS showed significantly different GUS activity. A 171-bp fragment upstream of the transcriptional initiation codon (ATG) is the core promoter region of PSaBS. Our results provide insight into and a greater understanding of the transcriptional regulation mechanism of the SaBS gene, which could serve as an alternative inducible promoter for transgenic plant breeding.

scholarly journals Structure of a Core Promoter in Bifidobacterium longum NCC2705

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Tomoya Kozakai ◽  
Ayako Izumi ◽  
Ayako Horigome ◽  
Toshitaka Odamaki ◽  
Jin-zhong Xiao ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Core Promoter ◽  
Bifidobacterium Longum ◽  
Sequence Motifs ◽  
Reporter Assay ◽  
Spacer Length ◽  
Content Type ◽  
Promoter Recognition ◽  
N Terminus ◽  
Consensus Motifs

ABSTRACT Bacterial promoters consist of core sequence motifs termed –35 and –10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on Escherichia coli. However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of Bifidobacterium longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSSs) of the B. longum NCC2705 strain were identified using transcriptome sequencing (RNA-Seq) analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the –35 and –10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed 37 plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the –35 and –10 boxes, namely, TTGNNN and TANNNT, respectively. We further analyzed spacer lengths between the –35 and –10 boxes via a bioinformatics approach. The spacer lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer lengths indicated that the 11-bp spacer length produced unusually high activity. IMPORTANCE The structures of sigma factors vary among bacterial strains, indicating that recognition rules may also vary. Therefore, we investigated the promoter structure of Bifidobacterium longum NCC2705 using a bioinformatics approach and wet analyses. The most frequent and optimal motifs were similar to other bacterial consensus motifs. The optimal spacer length between the two boxes was reported to be 17 bp. It is widely applied to a bioinformatics approach for other bacteria. Unexpectedly, conserved spacer lengths were 11 bp as well as 17 bp in the genus Bifidobacterium. Moreover, the sigma factor of the genus Bifidobacterium has a characteristic domain in the N terminus which may contribute to the additional functions. Hence, it would be valuable to reevaluate the promoter in other organisms.

scholarly journals Selective Promoter Recognition by Chlamydial σ28 Holoenzyme

2006 ◽  
Vol 188 (21) ◽  
pp. 7364-7377 ◽  
Author(s):  
Li Shen ◽  
Xiaogeng Feng ◽  
Yuan Yuan ◽  
Xudong Luo ◽  
Thomas P. Hatch ◽  
...  
Keyword(s):  
Core Promoter ◽  
Sigma Factors ◽  
Promoter Sequences ◽  
E Coli ◽  
The Core ◽  
Recognition Specificity ◽  
Promoter Recognition ◽  
Transcription In Vitro

ABSTRACT The σ transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma factors, σ66, σ54, and σ28. We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis σ28 (σ28 Ct). One involved the arabinose-induced expression of plasmid-encoded σ28 Ct in a strain of Escherichia coli defective in the σ28 structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant σ28 Ct. These approaches were used to investigate the interactions of σ28 Ct with the σ28 Ct-dependent hctB promoter and selected E. coli σ28 (σ28 Ec)-dependent promoters, in parallel, compared with the promoter recognition properties of σ28 EC. Our results indicate that RNAP containing σ28 Ct has at least three characteristics: (i) it is capable of recognizing some but not all σ28 EC-dependent promoters; (ii) it can distinguish different promoter structures, preferentially activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than σ28 EC in recognizing variants with different spacing lengths separating the −35 and −10 elements of the core promoter.

scholarly journals Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

1998 ◽  
Vol 180 (20) ◽  
pp. 5375-5383 ◽  
Author(s):  
Wilma Ross ◽  
Sarah E. Aiyar ◽  
Julia Salomon ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Core Promoter ◽  
Consensus Sequence ◽  
Α Subunit ◽  
Core Promoters ◽  
E Coli ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Up Elements

ABSTRACT The α subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, λp R, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to thelac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

Gene array analysis of Yersinia enterocolitica FlhD and FlhC: regulation of enzymes affecting synthesis and degradation of carbamoylphosphate

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Vinayak Kapatral ◽  
John W. Campbell ◽  
Scott A. Minnich ◽  
Nicholas R. Thomson ◽  
Philip Matsumura ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Yersinia Enterocolitica ◽  
Sigma Factor ◽  
Expression Patterns ◽  
Gene Array ◽  
Enteric Bacteria ◽  
Wild Type ◽  
E Coli ◽  
Aspartate Carbamoyltransferase ◽  
Genes Encoding

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 °C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of l-histidine to l-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 °C was compared to the same strain grown at 37 °C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 °C and reduced at 4 and 37 °C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC.

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