scholarly journals Regulation of Expression of the adhE Gene, Encoding Ethanol Oxidoreductase in Escherichia coli: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS

1999 ◽  
Vol 181 (24) ◽  
pp. 7571-7579 ◽  
Author(s):  
Jorge Membrillo-Hernández ◽  
E. C. C. Lin
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Start Site ◽  
Start Codon ◽  
Start Site ◽  
Rnase Iii ◽  
Regulation Of Expression ◽  
Experimental Conditions ◽  
Stem Loop ◽  
Translational Start ◽  
Transcriptional Start Sites

ABSTRACT The adhE gene of Escherichia coli, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of adhEis dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the lacZ gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activatesadhE transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.

scholarly journals Transcriptional Analysis and Regulation of Expression of the ScrFI Restriction-Modification System of Lactococcus lactis subsp.cremoris UC503

2001 ◽  
Vol 183 (15) ◽  
pp. 4668-4673 ◽  
Author(s):  
Derek Butler ◽  
Gerald F. Fitzgerald
Keyword(s):  
Transcriptional Start Site ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Northern Analysis ◽  
Start Site ◽  
Regulation Of Expression ◽  
Modification System ◽  
Reading Frame ◽  
Restriction Modification System ◽  
Restriction Modification

ABSTRACT ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (orfX) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, andscrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5′ extension analysis indicated that the start site for thescrFIAM promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for theorfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M.ScrFIA). In order to determine if this motif played a role in regulation of the ScrFI locus, M.ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream oforfX and the other located just upstream ofscrFIAM. M.ScrFIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of theScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in regulation of both promoters on the ScrFI locus.

scholarly journals Promoter Architecture of the Porphyromonas gingivalis Fimbrillin Gene

1999 ◽  
Vol 67 (7) ◽  
pp. 3227-3235 ◽  
Author(s):  
Hua Xie ◽  
Richard J. Lamont
Keyword(s):  
Porphyromonas Gingivalis ◽  
Promoter Activity ◽  
Transcriptional Start Site ◽  
Start Codon ◽  
Base Substitution ◽  
Transcriptional Level ◽  
Regulatory Sequences ◽  
Start Site ◽  
Environmental Signals ◽  
Translational Start

ABSTRACT Porphyromonas gingivalis fimbriae can mediate adherence to many of the available substrates in the oral cavity. Expression ofP. gingivalis fimbriae is regulated at the transcriptional level by environmental signals, such as temperature and hemin concentration. The arrangement of the upstream promoter and regulatory sequences required for transcription and control of the fimbrial structural gene (fimA) was investigated. Primer extension analysis demonstrated that the transcriptional start site of the fimA gene is located 41 bp upstream from the translational start codon. A region (upf) spanning 648 bp upstream of the start codon to 44 bp downstream of the translational start site was cloned upstream of a promoterless lacZreporter gene. A series of deletion and base substitution mutations were then generated in the upf region. The constructs were introduced into the chromosome of P. gingivalis, and promoter activity measured by assaying levels of β-galactosidase. The results showed that fimA contains sequences resembling ς70 promoter consensus sequences, consisting of a −10 region (TATGAC) located at −18 to −23 and a −35 region (TTGTTG) located at −41 to −46 from the transcriptional start point. The AT-rich upstream sequences spanning bases −48 to −85 and bases −90 to −240 were required for full expression of thefimA gene, indicating the existence of positive regulation regions. Moreover, the −48 to −64 region may constitute an UP element, contributing to promoter activity inP. gingivalis. Thus, our data suggest that the P. gingivalis fimA gene has a transcription complex consisting of −10 and −35 sequences, an UP element, and additional AT-rich upstream regulatory sequences.

scholarly journals Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

2002 ◽  
Vol 184 (12) ◽  
pp. 3242-3252 ◽  
Author(s):  
Françoise Blain ◽  
A. Lydia Tkalec ◽  
Zhongqi Shao ◽  
Catherine Poulin ◽  
Marc Pedneault ◽  
...  
Keyword(s):  
Transcriptional Start Site ◽  
Expression System ◽  
Regulatory Region ◽  
Start Codon ◽  
Upstream Region ◽  
Wild Type ◽  
Heparinase I ◽  
High Level ◽  
Flavobacterium Heparinum ◽  
Transcriptional Start Sites

ABSTRACT A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

scholarly journals The Pseudomonas aeruginosa Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS

2000 ◽  
Vol 182 (22) ◽  
pp. 6401-6411 ◽  
Author(s):  
Klaus Winzer ◽  
Colin Falconer ◽  
Nachman C. Garber ◽  
Stephen P. Diggle ◽  
Miguel Camara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Transcriptional Start Site ◽  
Immunoblot Analysis ◽  
Homoserine Lactone ◽  
Start Codon ◽  
Virulence Determinants ◽  
Putative Promoter Region ◽  
Consensus Sequences ◽  
Translational Start

ABSTRACT In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators,N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in alasR mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene, lecA, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of thelecA translational start codon. A lux box-type element together with RpoS (ςS) consensus sequences was identified upstream of the putative promoter region. InEscherichia coli, expression of alecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in P. aeruginosaPAO1, the expression of a chromosomallecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of rpoS abolished lectin synthesis inP. aeruginosa, demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the lecA::lux reporter in E. coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P. aeruginosa. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation oflecA expression.

scholarly journals Regulation of the furA and catC Operon, Encoding a Ferric Uptake Regulator Homologue and Catalase-Peroxidase, Respectively, in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (13) ◽  
pp. 3767-3774 ◽  
Author(s):  
Ji-Sook Hahn ◽  
So-Young Oh ◽  
Jung-Hye Roe
Keyword(s):  
Streptomyces Coelicolor ◽  
Transcriptional Start Site ◽  
Protein Product ◽  
Negative Regulator ◽  
Start Codon ◽  
Multicopy Plasmid ◽  
Ferric Uptake Regulator ◽  
Start Site ◽  
Reading Frame ◽  
Catalase Peroxidase

ABSTRACT We isolated the catC gene, encoding catalase-peroxidase in Streptomyces coelicolor, using sequence homology with the katG gene from Escherichia coli. Upstream of the catC gene, an open reading frame (furA) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the furA gene was cotranscribed with the catC gene. The transcriptional start site of the furA-catC mRNA was mapped to the translation start codon ATG of the furA gene. The putative promoter contains consensus −10 and −35 elements similar to those recognized by ςHrdB, the major sigma factor of S. coelicolor. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the furA gene was introduced into S. lividans on a multicopy plasmid, the increased production of catC transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the furA-catC operon. FurA protein bound to its own promoter region between −59 and −39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni2+, Mn2+, Zn2+, or Fe2+. Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.

The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc

2004 ◽  
Vol 19 (3) ◽  
pp. 303-318 ◽  
Author(s):  
D.J. Culp ◽  
L.R. Latchney ◽  
M.A. Fallon ◽  
P.A. Denny ◽  
P.C. Denny ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Genomic Organization ◽  
Splice Variants ◽  
Transcriptional Start Site ◽  
Mucous Cell ◽  
Complete Sequence ◽  
Full Length ◽  
Start Codon ◽  
Rich Domain ◽  
Translational Start

We previously demonstrated expression of full-length transcripts for sublingual mucin apoprotein, Muc19, of ∼24 kb (Fallon MA, Latchney LR, Hand AR, Johar A, Denny PA, Georgel PT, Denny PC, and Culp DJ. Physiol Genomics 14: 95–106, 2003). We now describe the complete sequence and genomic organization of the apomucin encoded by 43 exons. Southern analyses indicate a central exon of ∼18 kb containing 36 tandem repeats, each encoding 163 residues rich in serine and threonine. Full-length transcripts are an estimated 22,795 bp in length that span 106 kb of genomic DNA. The transcriptional start site is 24 bp downstream of a TATA box and 42 bp upstream of the conceptual translational start codon. The putative apoprotein has an estimated mass of 693.4 kDa and contains 7,524 amino acids (80% serine, threonine, glycine, alanine, and proline). We present a model for rat Muc19 transcripts and compare the conceptually translated Muc19 proteins for mouse, rat, pig, and the 3′ end of human Muc19. Conserved among these apoproteins are a signal peptide, a large tandem repeat region, von Willebrand factor type C and D domains, a trypsin inhibitor-like Cys-rich domain, and a COOH-terminal cystine knot-like domain. Southern blot analyses indicate transcripts for Muc19 and Smgc (submandibular gland protein C) are splice variants of a larger gene, Muc19/ Smgc. Comparative Northern analyses between the major salivary glands demonstrate highly selective Muc19 expression in neonatal and adult sublingual glands, whereas Smgc is expressed in neonatal submandibular and sublingual glands. Regulation of Muc19/ Smgc gene expression is discussed with respect to alternative splicing and mucous cell cytodifferentiation.

scholarly journals Differential Expression of the Smb Bacteriocin in Streptococcus mutans Isolates

2008 ◽  
Vol 52 (8) ◽  
pp. 2742-2749 ◽  
Author(s):  
Hideo Yonezawa ◽  
Howard K. Kuramitsu ◽  
Shu-ichi Nakayama ◽  
Jiro Mitobe ◽  
Mizuho Motegi ◽  
...  
Keyword(s):  
Differential Expression ◽  
Streptococcus Mutans ◽  
Transcription Initiation ◽  
Start Codon ◽  
Nucleotide Position ◽  
Start Site ◽  
Low Level ◽  
Translational Start ◽  
High Level

ABSTRACT The two-component lantibiotic Smb is produced by Streptococcus mutans GS5. In the present study, we identified seven strains of S. mutans containing the smb gene cluster. These strains could be classified into high- and low-level Smb producers relative to the levels of Smb production by indicator strains in vitro. This classification was dependent upon the transcription levels of the structural smbA and smbB genes. Sequence analysis upstream of smbA in the high- and low-level Smb-producing strains revealed differences at nucleotide position −46 relative to the smbA start codon. Interestingly, the transcription start site was present upstream of the point mutation, indicating that both groups of strains have the same promoter constructs and that the differential expression of smbA and smbB mRNA occurred subsequent to transcription initiation. In addition, smbA::lacZ fusion expression was higher when it was regulated by the sequences of strains with high-level Smb activity than when it was regulated by the comparable region from strains with low-level Smb activity. Taken together, we conclude that high- or low-level Smb expression is dependent on the presence of a G or a T nucleotide at position −46 relative to the smbA translational start site in S. mutans Smb producers.

scholarly journals Characterization of the Fructosyltransferase Gene of Actinomyces naeslundii WVU45

2000 ◽  
Vol 182 (13) ◽  
pp. 3649-3654 ◽  
Author(s):  
Lori J. Bergeron ◽  
Evangelia Morou-Bermudez ◽  
Robert A. Burne
Keyword(s):  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Initiation Site ◽  
Start Codon ◽  
Gram Positive ◽  
Regulation Of Expression ◽  
Significant Similarity ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Actinomyces Naeslundii

ABSTRACT Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of d-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated fromActinomyces naeslundii WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5′ from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving ftfexpression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimalftf expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that ftf was constitutively transcribed. Thus, the A. naeslundii FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.

scholarly journals Regulation of Expression of the Region 3 Promoter of the Escherichia coli K5 Capsule Gene Cluster Involves H-NS, SlyA, and a Large 5′ Untranslated Region

2008 ◽  
Vol 191 (6) ◽  
pp. 1838-1846 ◽  
Author(s):  
Peng Xue ◽  
David Corbett ◽  
Marie Goldrick ◽  
Clare Naylor ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Transcription Start Site ◽  
Untranslated Region ◽  
Regulatory Element ◽  
Gene Clusters ◽  
Regulation Of Transcription ◽  
Start Site ◽  
Transcription Start ◽  
Regulation Of Expression ◽  
Group 2

ABSTRACT Escherichia coli group 2 capsule gene clusters are temperature regulated, being expressed at 37°C but not at 20°C. Expression is regulated at the level of transcription by two convergent promoters, PR1 and PR3. In this paper, we show that regulation of transcription from PR3 involves a number of novel features including H-NS, SlyA, and a large 741-bp 5′ untranslated region (UTR). H-NS represses transcription from PR3 at 20°C and binds both 5′ and 3′ of the transcription start site. The 3′ downstream regulatory element (DRE) was essential for temperature-dependent H-NS repression. At 37°C, SlyA activates transcription independent of H-NS but maximal transcription requires H-NS. The UTR is present between the transcription start site and the first gene in the operon, kpsM. We demonstrate that the UTR, as well as containing the H-NS DRE, functions to moderate the extent of transcription that reaches kpsM and allows the binding of antitermination factor RfaH.

scholarly journals Positive and Negative Transcriptional Regulation of the Escherichia coli Gluconate Regulon Gene gntTby GntR and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex

1998 ◽  
Vol 180 (7) ◽  
pp. 1777-1785 ◽  
Author(s):  
Norbert Peekhaus ◽  
T. Conway
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Catabolite Repression ◽  
Transcriptional Start Site ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Start Site ◽  
Camp Receptor Protein ◽  
Camp Receptor

ABSTRACT The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor ofgntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the −10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntTexpression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntRmutants to the inducer is termed ultrarepression. Transcription ofgntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the gntT transcription start site.

Sign in / Sign up

Export Citation Format

Share Document