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A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein  [PDF]
Kervin Rex, Sanjay Kumar Bharti, Shivjee Sah, Umesh Varshney
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094669
Abstract: Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β451-β452-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L45 loop and the β6 strand of MtuSSB.
Cloning and Expression of Mycobacterium tuberculosis Major Secreted ProteinAntigen 85B (Ag85B) in Escherichia coli
Reza Zarif,Mojtaba Sankian,Aida Gholubi,Zahra Farshadzadeh
Jundishapur Journal of Microbiology , 2013,
Abstract: Background: The 30 kDa major secretory protein of Mycobacterium tuberculosis (antigen 85B) is a primary vaccine candidate. This secreted antigen induces a protective immune response and stimulates the production of IFN-? in animal models. Objectives: The aim of this study was cloning and expression of Ag 85B of M. tuberculosis in Escherichia coli. Materials and Methods: To produce recombinant Ag85B, the fbpB gene was amplified by PCR method. Then inserted into the pET101/D vector and transported into E. coli strain TOPO10. Plasmid containing pET101/D: Ag85B was transformed into competence E.coli BL21 (DE3). The transformed E.coli strain BL21 was effectively expressed recombinant Ag85B. Results: The expressed fusion protein was found almost entirely in the insoluble form. Followed by sonication to disrupt the cells, Solution of the cell debris was centrifuged and after use of Ni–NTA column and 6 molar urea and 6 M guanidine-HCl solutions recombinant protein was purified. Conclusions: These results could serve as a basis for further studies in endemic regions of tuberculosis on the usefulness of this gene and its expression product in the development of subunit vaccine and DNA vaccine against tuberculosis.
Growth of Mycobacterium avium subsp. paratuberculosis, Escherichia coli, and Salmonella Enteritidis during Preparation and Storage of Yogurt  [PDF]
K. Cirone,Y. Huberman,C. Morsella,L. Méndez,M. Jorge,F. Paolicchi
ISRN Microbiology , 2013, DOI: 10.1155/2013/247018
Abstract: The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180?min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. 1. Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease. MAP affects domestic and wild animals and, in cows, causes chronic enteritis, diarrhea, weight loss, and progressive emaciation that can eventually lead to death [1]. MAP has also been linked to human Crohn’s disease, a systemic disorder that causes mainly a chronic inflammation of the intestine [2]. It is suggested that humans might be infected through contaminated milk, although relatively little is known about MAP survival during industrial milk manipulation. Some authors have suggested that pasteurization is capable of destroying mycobacteria. Thus, laboratory assays were performed to evaluate MAP heat resistance according to differential distribution of heat treatment during pasteurization [3–6]. In contrast, other authors support the theory that MAP is able to resist pasteurization when it is present in raw milk [7–13]. Viable MAP was detected in commercial pasteurized milk in the UK, the USA, the Czech Republic, and India [10, 11, 14–16]. Furthermore, in the Czech Republic, using F-57 or IS900 real-time PCR, MAP was detected in 49% of samples of powdered infant milk, with one study yielding viable MAP [17]. Nevertheless, little was published regarding MAP in dairy products other than
Min-protein oscillations in Escherichia coli with spontaneous formation of two-stranded filaments in a three-dimensional stochastic reaction-diffusion model  [PDF]
Nenad Pavin,Hana Cipcic Paljetak,Vladimir Krstic
Physics , 2005, DOI: 10.1103/PhysRevE.73.021904
Abstract: We introduce a three-dimensional stochastic reaction-diffusion model to describe MinD/MinE dynamical structures in Escherichia coli. This model spontaneously generates pole-to-pole oscillations of the membrane-associated MinD proteins, MinE ring, as well as filaments of the membrane-associated MinD proteins. Experimental data suggest MinD filaments are two-stranded. In order to model them we assume that each membrane-associated MinD protein can form up to three bonds with adjacent membrane associated MinD molecules and that MinE induced hydrolysis strongly depends on the number of bonds MinD has established.
Accurate Prediction of Protein Functional Class from Sequence in the Mycobacterium tuberculosis and Escherichia coli Genomes Using Data Mining
Ross D. King,Andreas Karwath,Amanda Clare,Luc Dehaspe
Comparative and Functional Genomics , 2000, DOI: 10.1002/1097-0061(200012)17:4<283::aid-yea52>3.0.co;2-f
Abstract: The analysis of genomics data needs to become as automated as its generation. Here we present a novel data-mining approach to predicting protein functional class from sequence. This method is based on a combination of inductive logic programming clustering and rule learning. We demonstrate the effectiveness of this approach on the M. tuberculosis and E. coli genomes, and identify biologically interpretable rules which predict protein functional class from information only available from the sequence. These rules predict 65% of the ORFs with no assigned function in M. tuberculosis and 24% of those in E. coli, with an estimated accuracy of 60–80% (depending on the level of functional assignment). The rules are founded on a combination of detection of remote homology, convergent evolution and horizontal gene transfer. We identify rules that predict protein functional class even in the absence of detectable sequence or structural homology. These rules give insight into the evolutionary history of M. tuberculosis and E. coli.
Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing

Jianping Wang,Bingjie Zou,Zhiyao Chen,Yinjiao M,Shu Xu,Guohua Zhou,

生物工程学报 , 2011,
Abstract: We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (Tm) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the Tm of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the Tm difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
Escherichia coli enterohemorrágica
Margall,Núria; Domínguez,àngela; Prats,Guillem; Salleras,Lluís;
Revista Espa?ola de Salud Pública , 1997, DOI: 10.1590/S1135-57271997000500002
Abstract: groups of escherichia coli enteropathogen are described, with special attention to escherichia coli enterohaemorragic. some serotypes of escherichia coli vero citotoxin-producing are able to produce hemorrhagic enteritis, which can develop a complication with hemolityc uraemic syndrome. this complication is most frequent in children and has a high mortality rate. the transmission takes place via food and its capacity to cause epidemic outbreaks together with the seriousness of the complications caused by enteritys make this microorganism of great importance to public health. the epidemiology of this microorganism in spain is reviewed.
Response of Escherichia coli containing mycobacterial carotene genes to UV radiation  [cached]
Houssaini-Iraqui Mohamed,Khamlichi Naima,El Yamani Jamal,Rastogi Nalin
Journal of Biomedicine and Biotechnology , 2001,
Abstract: The plasmid pC5, which encodes biogenesis of lycopene in Mycobacterium aurum A+, was partially digested by restriction endonucleases and generated fragments were cloned. After transformation of Escherichia coli (colorless bacteria) with the plasmids so constructed, seven orange clones were detected and found to carry the same recombinant plasmid (pC51). E. coli cells containing this plasmid synthesize neurosporene and lycopene, and were more resistant to ultraviolet irradiation than non pigmented strain.
Enterotoxigenic Escherichia coli - an overview
Guth, Beatriz Ernestina Cabilio;
Memórias do Instituto Oswaldo Cruz , 2000, DOI: 10.1590/S0074-02762000000700017
Abstract: enterotoxigenic escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world. in this presentation we will focus on the main virulence attributes of this pathogenic category of e. coli, and discuss the evolution of studies conducted in our laboratory.
Enterotoxigenic Escherichia coli - an overview  [cached]
Guth Beatriz Ernestina Cabilio
Memórias do Instituto Oswaldo Cruz , 2000,
Abstract: Enterotoxigenic Escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world. In this presentation we will focus on the main virulence attributes of this pathogenic category of E. coli, and discuss the evolution of studies conducted in our laboratory.
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