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Promoter addresses: revelations from oligonucleotide profiling applied to the Escherichia coli genome
Karthikeyan Sivaraman, Aswin Sai Narain Seshasayee, Krishnakumar Swaminathan, Geetha Muthukumaran, Gautam Pennathur
Theoretical Biology and Medical Modelling , 2005, DOI: 10.1186/1742-4682-2-20
Abstract: We have used oligonucleotide profiling to predict regulatory regions in a bacterial genome. The method has been applied to the Escherichia coli K12 genome and the results analyzed. The information content of the putative regulatory oligonucleotides so predicted is validated through intra-genomic analyses, correlations with experimental data and inter-genome comparisons. Based on the results we have proposed a model for the bacterial promoter. The results show that the method is capable of identifying, in the E.coli genome, cis-acting elements such as TATAAT (sigma70 binding site), CCCTAT (1 base relative of sigma32 binding site), CTATNN (LexA binding site), AGGA-containing hexanucleotides (Shine Dalgarno consensus) and CTAG-containing hexanucleotides (core binding sites for Trp and Met repressors).The method adopted is simple yet effective in predicting upstream regulatory elements in bacteria. It does not need any prior experimental data except the sequence itself. This method should be applicable to most known genomes. Profiling, as applied to the E.coli genome, picks up known cis-acting and regulatory elements. Based on the profile results, we propose a model for the bacterial promoter that is extensible even to eukaryotes. The model is that the core promoter lies within a plateau of bent AT-rich DNA. This bent DNA acts as a homing segment for the sigma factor to recognize the promoter. The model thus suggests an important role for local landscapes in prokaryotic and eukaryotic gene regulation.Transcription, the first step of information flow from DNA, is regulated by sequence specific DNA-protein interactions. The regulation depends on the presence of cis-acting elements. The best examples of cis-acting elements are promoters. Other well-known examples in bacteria include the Shine Dalgarno (SD) sequence, sigma 32 binding site, LexA binding site, etc.In bacteria, promoters recognized by sigma factors initiate transcription. The responses of an organism to variou
Plasmid profiling of multidrug resistant Escherichia coli strains isolated from urinary tract infection patients  [PDF]
Sabin Khadgi,Uddhav Timilsina,Basudha Shrestha
International Journal of Applied Sciences and Biotechnology , 2013, DOI: 10.3126/ijasbt.v1i1.7918
Abstract: Introduction- Urinary tract infection is a common community-acquired bacterial disease. Escherichia coli is reported to be the major cause of urinary tract infection. Aim & Objective- The study was conducted with the aim of determining the antibiotic resistance pattern and plasmid profile of multidrug resistant Escherichia coli isolated from Urinary Tract Infection patients. Materials and Method- Antibiotic susceptibility tests were performed against E. coli following the protocol for the Kirby-Bauer disc diffusion method. Plasmid DNA was isolated following the protocol of Kado and Liu. Results- Multidrug resistant isolates exhibited high resistance to drugs like Amoxicillin, Cefixime, Ciprofloxacin, Cotrimethoxazole, Norfloxacin and Ofloxacin. The plasmid profiling showed that all, except one, isolate contained at least one plasmid. A band of approximately 23 kb was seen in most of the isolates.
The correlation between architecture and mRNA abundance in the genetic regulatory network of Escherichia coli
Yohann Grondin, Derek J Raine, Vic Norris
BMC Systems Biology , 2007, DOI: 10.1186/1752-0509-1-30
Abstract: For a cell population of E. coli, we find that there is a strong and statistically significant linear dependence between the abundance of mRNA encoding a regulatory protein and the number of genes regulated by this protein. We use this result, together with the ratio of regulatory repressors to promoters, to simulate numerically a genetic regulatory network of a single cell. The resulting model exhibits similar correlations to that of E. coli.This analysis clarifies the relationship between the static architecture of a regulatory network and the consequences for the dynamics of its pattern of mRNA abundances. It also provides the constraints on the architecture required to construct a model network to simulate mRNA production.The interactions of the many molecular constituents of a cell can be expressed in term of various networks, such as protein-protein interaction networks [1-3], metabolic networks [4,5] or genetic networks [6], in which the cell would be represented as a network of networks. Genetic regulatory networks are complex systems in which the agents or genes, that are the nodes of the network, each carry out the combined processes of transcription, translation and post-translational modifications, and the links represent the causal influences amongst these agents [7]. There are two aspects important for understanding these networks. The first is the static architecture. This comprises the overall connectivity or architecture, namely, which nodes are connected to which others, and the designation of links as either promoters or repressors. The second is the dynamics, namely, how it is determined which nodes are active at any one time, that is, the genes that are expressed, and what determines the level of activity at an active node. The architecture and dynamics gives rise to a pattern of activity over time and the corresponding time-dependent activities. In the case of cell biology, a major goal is to explain how the genetic regulatory network functions
IS3 profiling identifies the enterohaemorrhagic Escherichia coli O-island 62 in a distinct enteroaggregative E. coli lineage
Iruka N Okeke, Louissa R Macfarlane-Smith, Jonathan N Fletcher, Anna M Snelling
Gut Pathogens , 2011, DOI: 10.1186/1757-4749-3-4
Abstract: To identify genetic loci conserved within significant EAEC lineages, but absent from non-EAEC, IS3-based PCR profiles were generated for 22 well-characterised EAEC strains. Six bands that were conserved among, or missing from, specific EAEC lineages were cloned and sequenced. One band corresponded to the aggR gene, a plasmid-encoded regulator that has been used as a diagnostic target but predominantly detects EAEC bearing the plasmid already marked by CVD432. The sequence from a second band was homologous to an open-reading frame within the cryptic enterohaemorrhagic E. coli (EHEC) O157 genomic island, designated O-island 62. Screening of an additional 46 EAEC strains revealed that the EHEC O-island 62 was only present in those EAEC strains belonging to the ECOR phylogenetic group D, largely comprised of sequence type (ST) complexes 31, 38 and 394.The EAEC 042 gene orf1600, which lies within the EAEC equivalent of O-island 62 island, can be used as a marker for EAEC strains belonging to the ECOR phylogenetic group D. The discovery of EHEC O-island 62 in EAEC validates the genetic profiling approach for identifying conserved loci among phylogenetically related strains.Enteroaggregative Escherichia coli (EAEC) were originally associated with persistent diarrhoea in developing countries but are now known to cause both acute and persistent diarrhoea worldwide [1]. EAEC strains all demonstrate a characteristic aggregative adherence to human epithelial cells in vivo or in culture. There are no other phenotypic or genotypic properties known to be shared by all EAEC strains, and the contribution of potential EAEC virulence factors to human disease is yet to be assessed. Volunteer studies and outbreaks have unequivocally demonstrated that at least some EAEC strains are pathogens [2-5]. However, epidemiological studies have always recovered EAEC from healthy people as well as individuals with diarrhoea. Although host factors are one reason for this observation [6,7], it is al
Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042  [PDF]
Roy R. Chaudhuri,Mohammed Sebaihia,Jon L. Hobman,Mark A. Webber,Denisse L. Leyton,Martin D. Goldberg,Adam F. Cunningham,Anthony Scott-Tucker,Paul R. Ferguson,Christopher M. Thomas,Gad Frankel,Christoph M. Tang,Edward G. Dudley,Ian S. Roberts,David A. Rasko,Mark J. Pallen,Julian Parkhill,James P. Nataro,Nicholas R. Thomson,Ian R. Henderson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008801
Abstract: Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation.
Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling  [PDF]
Donghyuk Kim equal contributor,Jay Sung-Joong Hong equal contributor,Yu Qiu,Harish Nagarajan,Joo-Hyun Seo,Byung-Kwan Cho,Shih-Feng Tsai,Bernhard ?. Palsson
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002867
Abstract: Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5′ RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5′ UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5′ UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5′ UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization.
Molecular, Serological And Microbiological Profiling Evidence Of The Formit Transmission Of Escherichia Coli O157: Nm To A 1-Year Old Nigerian Child
I I Luga
Nigerian Veterinary Journal , 2007,
Abstract: Transmission of Escherichia Coli O157 by the formit route is under reported since interest in E. Coli O157 is usually arisen during outbreak situations which are mostly linked to consumption of food of bovine origin (Armstrong et al., 1996; Fukushima et al., 1999 and Allison et al., 2000). In December 2005 while working on the isolation of E. Coli O157 from cattle herds in Zaria, Kaduna State, a 1– year old child of a member of the team came down with a mild watery diarrhea. He was not presented to any Physician, since he was observed to be alert, active and with a normal appetite. However, stool specimens were collected from the child on days 2 and 3 of the diarrhea, (which was self limiting on day 3). Stool specimens were also collected from all the five members of his family. All items that the boy had contact with including a laboratory coat, bunch of keys and shoes were swabbed. Finally samples of all the boy's food and drinks were taken. Microbiological, Serological and Polymerase Chain Reaction (PCR) Profiling Assays. l the samples were cultured on Sorbitol - MacConkey (SMAC) agar, after enrichment using Modified Tryptone Soya Broth (MTSB) supplemented with Novobiocin (Oxoid Ltd, Basingstoke, England). All colourless colonies on SMAC agar from the samples were biochemically confirmed as E. coli by the methods described by (Cowan, 1981) and serologically as E. Coli O157:NM using the Remel Wellcolex E. coli O157:H7 kit (Remel Europe Ltd, Kent UK). Shigatoxin profiling was done using the VTEC-RPLA kit (Oxoid Ltd, Basingstoke England). Antimicrobial resistance profiling was carried out by the method of Bauer et al. (1966) using 16 antimicrobial agents: Ampicillin (AMP10), Ceftazidime (CAZ30), Cefuroxime (CXM30), Cephazolin (KZ30), ciprofloxacin (CIP5), compound Sulphonamides (S3300), gentamicin (CN10), Kanamycin (K30), Chloramphenicol (C30), Nalidixic acid (NA30), Neomycin (N10), Norfloxacin (NOR2), streptomycin (S10), Sulphamethoxazole (RL25), Co-trimoxazole (SXT25) and tetracycline (TE30) (Oxoid Ltd, Basingstoke, England). The assays were controlled using a control strain of E. Coli O157:H7 (ATCC 43895). Total DNA was isolated from 1 ml of brain heart infusion (BHI) broth culture grown overnight based on the method of Silhavy et al (1984). The Eae primer (Operon Ltd, Dusseldorf, Germany) was used (Germani et al., 1997). The PCR mix consisted of 2.50μl 10 x buffer, 1.50μl MgCl2, 0.50 dNTPs, eae-f 0.40μl, Taq polymerase (Ampli Taq Gold, Applied Biosystems, California, U.S.A.) 0.50μl, and 4.0μl of template DNA. The volume of the mix was adjusted to 25μl by addition of 15.2μl of nuclease free sterile water. DNA amplification was carried out in a Gene Amp PCR System 9700 Thermocycler (Applied Biosystems, California, U.S.A.) using an initial denaturation step at 94oC for 5 minutes, followed by 30 cycles of amplification with denaturation step at 94oC for 1 minute, annealing at 53oC for 1 minute, and extension at 72oC for 10 minutes and cooled to 4
Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins
Chung-Hsien Cheng, Wen-Chien Lee
Microbial Cell Factories , 2010, DOI: 10.1186/1475-2859-9-63
Abstract: Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction.Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.Under the regulation of strong promoters, as in numerous commercial plasmid-based vectors, heterologous proteins are typically expressed at high levels in Escherichia coli. The overexpression of plasmid-encoded genes can trigger transcription of heat-shock genes and other stress responses and often result in the aggregation of the encoded proteins as inclusion bodies
Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains
Zhou Chuanli,Liu Zhengzhu,Jiang Jicai,Yu Ying
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-330
Abstract: Background Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains. Results A total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (CF4ab), with F4ac ETEC (CF4ac) and with F18ac ETEC (CF18ac) compared to the cells without infection (control), respectively. The number of differentially expressed genes between CF4ab and CF4ac, CF4ab and CF18ac, and CF4ac and CF18ac were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in CF4abvs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in CF4acvs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between CF18acvs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc. Conclusions The discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects.
Virulence profiling of Shiga toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico  [PDF]
Bianca A. Amézquita-López,Beatriz Qui?ones,Bertram G. Lee,Cristóbal Chaidez
Frontiers in Cellular and Infection Microbiology , 2014, DOI: 10.3389/fcimb.2014.00007
Abstract: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen that causes human gastrointestinal illnesses. The present study characterized the virulence profiles of O157 and non-O157 STEC strains, recovered from domestic animals in small rural farms within the agricultural Culiacan Valley in Mexico. Virulence genes coding for adhesins, cytotoxins, proteases, subtypes of Shiga toxin (Stx), and other effectors were identified in the STEC strains by PCR. The genotyping analysis revealed the presence of the effectors nleA, nleB, nleE, and nleH1-2, espK, and espN in the O157:H7 and O111:H8 STEC strains. Furthermore, the genes encoding the autoagglutinating adhesin (Saa) and subtilase (SubA) were exclusively identified in the O8:H19 eae-negative strains. The adhesin (iha) and the silent hemolysin (sheA) genes were detected in 79% of the O157 and non-O157 strains. To examine the relative toxicities of the STEC strains, a fluorescent Vero cell line, Vero-d2EGFPs, was employed to measure the inhibition of protein synthesis by Stx. Analysis of culture supernatants from serotype O8:H19 strains with the stx gene profile stx1a, stx2a, and stx2c and serotypes O75:H8 and O146:H8 strains with the stx gene profile stx1a, stx1c, and stx2b, resulted in a significant reduction in the Vero-d2EGFP fluorescent signal. These observations suggest that these non-O157 strains may have an enhanced ability to inhibit protein synthesis in Vero cells. Interestingly, analysis of the stx2c-positive O157:H7 strains resulted in a high fluorescent signal, indicating a reduced toxicity in the Vero-d2EGFP cells. These findings indicate that the O157 and non-O157 STEC strains, recovered in the Culiacan Valley, display distinct virulence profiles and relative toxicities in mammalian cells and have provided information for evaluating risks associated with zoonotic STEC in this agricultural region in Mexico.
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