DOCSLIB.ORG

Differential Gene Expression of E. Coli O157:H7 in Ground Beef Extract

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Differential Gene Expression of E. Coli O157:H7 in Ground Beef Extract Differential Gene Expression of E. coli O157:H7 in Ground Beef Extract Compared to Tryptic Soy Broth Pina M. Fratamico, Siyun Wang, Xianghe Yan, Wei Zhang, and Yuesheng Li Abstract: E. coli O157:H7 is an important foodborne pathogen, and ground beef is a common vehicle of infection. DNA microarrays have been used for transcriptomic studies of E. coli O157:H7 using laboratory media; however, analysis of gene expression in complex matrices such as food are lacking. This study compared gene expression profiles of E. coli O157:H7 Sakai strain in raw ground beef extract (GBE) and tryptic soy broth (TSB). Total RNA was isolated from GBE and TSB after 2 h of incubation with E. coli O157:H7. Following reverse transcription (RT) of the RNA, labeled cDNA was hybridized to microarrays representing 9608 open reading frames (Operon; Genome Array-Ready Oligo Set) corresponding to 4 genomes of E. coli strains and 3 plasmids. There were 74 up-regulated (genes involved in protein and polysaccharide biosynthesis, transcription factors, membrane transport proteins, and acid shock proteins) and 54 down-regulated (encoding proteins for energy metabolism, biosynthesis of cofactors, transporters of small molecules, and transcription factors and enzymes responsible for protein degradation) genes in E. coli O157:H7 grown in GBE compared to TSB, respectively. Furthermore, compared to incubation in TSB, E. coli O157:H7 incubated in GBE for 2 h showed significantly increased survival when exposed to synthetic gastric fluid, pH 1.5. This study demonstrated that microarray analyses can be performed using complex food matrices, and gene expression of E. coli O157:H7 differs in TSB compared to GBE. The information will be useful for identification of genes that can be employed as potential targets for interventions to control E. coli O157:H7. Keywords: biotechnology, E. coli O157: H7, meat safety, microbial survival, molecular biology & Safety M: Food Microbiology Introduction mit the development of effective interventions to inactivate this E. coli O157:H7 is a leading cause of foodborne illness world- pathogen or control growth in beef. wide. Ruminant animals, particularly cattle, are reservoirs for this Gene expression profiling using DNA microarray technology pathogen; however, other animals, including birds and pigs can provides a measurement of the activity (expression) of every gene also be carriers (Smith and Fratamico 2005). Outbreaks have been in a genome allowing a global analysis of cellular function in re- linked to contaminated drinking water, fresh produce, and fruit sponse to specific environmental variables. Altered levels of specific juices; however, food of bovine origin, particularly, raw or un- mRNAs may indicate a change in the level of the proteins encoded dercooked ground beef, has been implicated in a large number by the mRNA required in response to specific environmental con- of the documented outbreaks (Smith and Fratamico 2005). Cat- ditions. This technology can also be utilized to identify genes that tle harboring Shiga toxin-producing E. coli (STEC) O157:H7 on are controlled by specific regulators and to study the function of hides and in feces can result in contamination of carcasses during particular genes by comparing gene expression in wild-type bacte- slaughter. There have been numerous recalls of ground beef due ria and their isogenic mutants. Generally, gene expression studies to contamination with STEC O157:H7 (http://www.fsis.usda. in foodborne bacteria have been conducted in growth medium gov/FSIS_Recalls/Open_Federal_Cases/index.asp), resulting in and not in complex matrices such as food. The potential for the major ramifications for meat processors. An understanding of how analysis of gene expression of pathogens in food environments has STEC O157:H7 survives and proliferates in ground beef will per- not yet been completely realized due to the substantial techni- cal challenges associated with accurately measuring bacterial gene expression in complex matrices. The isolation of stable mRNA from complex materials, such as food, soil, and fecal samples that contain nucleases and PCR inhibitors is a challenge. There have MS 20100705 Submitted 6/23/2010, Accepted 9/30/2010. Authors Fratamico been very few reports on gene expression analyses in food. Liu and and Yan are with U.S. Dept. of Agriculture, Agricultural Research Service, Eastern Ream (2008) examined gene expression of Listeria monocytogenes in Regional Research Center, Wyndmoor, PA 19038, U.S.A. Authors Wang and Zhang ◦ are with Natl. Center for Food Safety and Technology, Illinois Inst. of Technology, 6502 ultra-high temperature-processed skim milk at 4 C. Compared S. Archer Rd, Summit, IL 60501, U.S.A. Author Li is with Fox Chase Cancer to growth in brain heart infusion broth, there were 14 down- Center Genomic Facility, 333 Cottman Ave., Philadelphia, PA 19111, U.S.A. regulated genes and 26 up-regulated genes encoding for transport Direct inquiries to author Fratamico (E-mail: [email protected]). and binding proteins, transcriptional regulators, proteins involved Mention of trade names or commercial products is solely for the purpose in amino acid biosynthesis and energy metabolism, protein syn- of providing specific information and does not imply recommendation or thesis, and cell division. Makhzami and others (2008) developed endorsement by the U.S. Dept. of Agriculture. a mRNA extraction method suitable for analyses of Enterococcus R Journal compilation C 2010 Institute of Food Technologists No claim to original government works r doi: 10.1111/j.1750-3841.2010.01952.x Vol. 76, Nr. 1, 2011 Journal of Food Science M79 Further reproduction without permission is prohibited E. coli O157:H7 gene expression in beef . faecalis gene expression profiles in cheese samples using DNA ar- RNA was then isolated and purified using the RNeasy kit (Qi- rays. Their study revealed that there were differences in transcript agen) according to manufacturer’s instructions. The total RNA levels of an E. faecalis food isolate compared to a clinical isolate, isolated was quantified using a Nanodrop 1000 (ThermoFisher suggesting that the food isolate may have adapted to a dairy envi- Scientific, Waltham, Mass., U.S.A.), and the RNA quality was ronment. Bergholz and others (2009) examined the induction of analyzed using an Agilent 2100 Bioanalyzer (Agilent Technolo- stress response regulons of E. coli O157:H7 in a low pH, high os- gies, Santa Clara, Calif., U.S.A.) according to the manufacturer’s molarity model apple juice medium. Genes that showed significant protocol. induction in the model apple juice included those involved in acid, osmotic, oxidative, and envelope stress responses. Thus, although Genome oligo set and microarray printing there are a few reports on gene expression profiling of bacteria in The E. coli Genome AROSTM version 2.0 oligo set (Operon food or model food systems, there have been no reported studies Biotechnologies Inc., Huntville, Ala., U.S.A.) contained 9308 70- examining gene expression of E. coli O157:H7 in beef. The aim mer oligonucleotide probes representing the genomes of 4 E. of this research was to determine the genome expression profile coli strains and 3 plasmids. The number of open reading frames of E. coli O157:H7 in ground beef extract (GBE) and compare (ORFs) or genes represented was as follows: 4269 ORFs in E. it to that in tryptic soy broth (TSB) to elucidate genes important coli K12, 5306 ORFs in E. coli O157:H7 (EDL933), 5251 ORFs for growth and survival in ground beef. This information will be in E. coli O157:H7 (Sakai), 5366 ORFs in uropathogenic E. coli useful for the development of effective strategies to control this CFT073, 3 genes in OSAK1, 10 genes in pO157_Sakai, and 97 pathogen in beef. genes in pO157_EDL933. The oligonucleotide set also included randomly generated oligo sequences to serve as negative con- Materials and Methods trols for hybridization. The oligonucleotides were resuspended in × μ Preparation of the GBE 3 SSC (standard sodium citrate) at 30 M concentration, and slides were printed at the Fox Chase Cancer Center microarray Fresh, raw, 85% lean ground beef was obtained from a local core facility onto aminosylin-coated slides (UltraGAPS, Corning, supermarket and sterilized by irradiation (25 kGy) in a 137Cs self- M: Food Microbiology Lowell, Mass., U.S.A.) at 50% humidity using a GeneMachines contained Lockheed-Georgia Co. (Marietta, Ga., U.S.A.) gamma OmniGrid 100 microarrayer (GeneMachines, San Carlos, Calif., irradiator with temperature control. The irradiation temperature & Safety ◦ U.S.A.). Blank spots (3× SSC) were used to monitor for back- was −20 C, and there were no detectable differences in the ground hybridization signal levels. Following spotting, the oligos quality of the beef after irradiation. Total of 15 mL of sterile were bound to the slide by UV cross-linking, and the slides were water were added to 50-g portions of the sterile ground beef in stored in a dry environment prior to use. a Stomacher bag (Spiral Biotech Inc., Norwood, Mass., U.S.A.) followed by pummeling in a Stomacher 400 Lab Blender (Seward Ltd., London, U.K.) for 1 min, and then massaging by hand. The Aminoallyl indirect labeling of cDNA targets liquid extract was removed, centrifuged at 2100 × g for 5 min, A total of 30 μg of total RNA and 0.5 μg random hexamer and then filtered using a 0.22 μm filter and frozen at −20 ◦C until pd(N)6 primers were mixed to a final volume of 10 μLand ◦ use. incubated at 70 C for 10 min. Reverse transcription mixture (10 μL), containing 0.5 mM dATP, dCTP, dGTP, 0.2 mM dTTP, Bacterial strain and culture conditions in GBE and TSA 0.3 mM aminoallyl-dUTP, 10 mM DTT, 1× first strand buffer, 40 U rRNasin, and 400 U SuperScript II was added to RNA E.
Load more

Recommended publications
  • Transcription Regulation of the Expression of the Plasmid Encoded Toxin of Enteroaggregative Escherichia Coli
    TRANSCRIPTION REGULATION OF THE EXPRESSION OF THE PLASMID ENCODED TOXIN OF ENTEROAGGREGATIVE ESCHERICHIA COLI by Jacob Henry Duddy A thesis submitted to The University of Birmingham for the degree of MASTER OF PHILOSOPHY February 2013 School of Biosciences The University of Birmingham University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The pathogenic properties of Enteroaggregative Escherichia coli strain 042 results from the synchronised expression of virulence factors, which include the Plasmid Encoded Toxin. Pet is a member of the serine protease autotransporter of the Enterobacteriaceae family and contributes to infection by cleaving α-fodrin, disrupting the actin cytoskeleton of host cells. The expression of Pet is induced by global transcription factor CRP with further enhancement by the nucleoid associated protein Fis. This study identifies the residues of RNA polymerase, Fis and CRP required for the induction of transcription, thereby clarifying the mechanism of activation employed by the transcription factors. Fis activates transcription from the Fis binding site via a direct interaction with RNA polymerase, facilitated by protein specific determinants. This interaction is dependent on the position of the Fis binding site on the DNA and it subsequent orientation on the helical face of the DNA.
    [Show full text]
  • The Absence of Effect of Gid Or Mioc Transcription on the Initiation of Chromosomal Replication in Escherichia Coli (DNA Replication͞oric͞transcriptional Activation)
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12497–12502, November 1997 Genetics The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli (DNA replicationyoriCytranscriptional activation) DAVID B. BATES*†,ERIK BOYE‡,TSUNEAKI ASAI†§¶, AND TOKIO KOGOMA*†§i Departments of *Biology and §Cell Biology, and †Cancer Center, University of New Mexico, Albuquerque, NM 87131; and ‡Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway Edited by Donald R. Helinski, University of California at San Diego, La Jolla, CA, and approved September 15, 1997 (received for review June 23, 1997) ABSTRACT Despite the widely accepted view that tran- scription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC. Furthermore, neither gid nor mioC tran- scription is required in cells deficient in the histone-like proteins Fis or IHF. However, oriC that is sufficiently im- paired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of mioC and especially gid is needed to activate FIG. 1. The minimal oriC and surrounding transcription. The position of the six DnaA boxes R1–R5 and M; 13-mer repeats L, M, oriC only under suboptimal conditions. We suggest that either and R; A1T-rich cluster; and binding sites for IHF and Fis proteins the rifampicin-sensitive step of initiation is some other tran- are indicated. Large arrows represent location and direction of major scription occurring from promoter(s) within oriC,orthe promoters near oriC.
    [Show full text]
  • Journal of Microbiological Methods a Proteome-Wide Screen to Identify Transcription Factors Interacting with the Vibrio Cholerae
    Journal of Microbiological Methods 165 (2019) 105702 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Note A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter T ⁎ ⁎ Julio C. Ayala1, Jorge A. Benitez , Anisia J. Silva Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology, 720 Westview Dr., SW, Atlanta, GA 30310, USA ABSTRACT We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR. Isolation of regulatory mutants has played a major role in increasing with agitation at 37 °C to stationary phase. Culture media were sup- our understanding of the regulation of gene expression (Shuman and plemented with ampicillin (Amp, 100 μg/mL), kanamycin (Km, 25 μg/ Silhavy, 2003). There are cases, however, in which the above approach mL), polymyxin B (PolB, 100 units/mL) or L-arabinose (0.2%) as re- can encounter significant obstacles. For instance, the methods used to quired. Chromosomal deletions of fis and/or hapR were created in strain genetically manipulate model organisms are not always equally effec- C7258ΔlacZ by allelic exchange as described previously (Wang et al., tive in less studied microorganisms; some regulatory mutations can be 2011; Wang et al., 2012; Wang et al., 2014). Briefly, genomic DNA highly pleiotropic, often be deleterious, unstable and difficult to select.
    [Show full text]
  • Unexpected Properties of Srna Promoters Allow Feedback Control
    9650–9666 Nucleic Acids Research, 2016, Vol. 44, No. 20 Published online 20 July 2016 doi: 10.1093/nar/gkw642 Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system Ana¨ıs Brosse1, Anna Korobeinikova1, Susan Gottesman2 and Maude Guillier1,* 1CNRS UMR8261, Associated with University Paris Diderot, Sorbonne Paris Cite,´ Institut de Biologie Physico-Chimique, 75005 Paris, France and 2Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA Received July 19, 2015; Revised June 21, 2016; Accepted July 07, 2016 ABSTRACT This control can take place at all stages of gene expression and via a great diversity of precise molecular mechanisms. Two-component systems (TCS) and small regulatory For instance, examples of transcriptional control have RNAs (sRNAs) are both widespread regulators of been reported at the level of transcription initiation, elon- gene expression in bacteria. TCS are in most cases gation or termination, and the regulators can be DNA- or transcriptional regulators. A large class of sRNAs RNA-binding proteins, but also RNA molecules. Among act as post-transcriptional regulators of gene expres- the most widely used regulators of transcription in bacteria sion that modulate the translation and/or stability of are the two-component systems (TCS), which are typically target-mRNAs. Many connections have been recently composed of a sensor protein and its cognate response reg- unraveled between these two types of regulators, re- ulator (1). In response to specific stimuli, the sensor con- sulting in mixed regulatory circuits with poorly char- trols the phosphorylation status of the regulator by acting / acterized properties.
    [Show full text]
  • Genome-Wide Analysis of Fis Binding in Escherichia Coli Indicates a Causative Role for A-/AT-Tracts
    Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Genome-wide analysis of Fis binding in Escherichia coli indicates a causative role for A-/AT-tracts Byung-Kwan Cho,1 Eric M. Knight,1 Christian L. Barrett, and Bernhard Ø. Palsson2 Department of Bioengineering, University of California–San Diego, La Jolla, California 92093-0412, USA We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A6-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that ∼21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions—indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions.
    [Show full text]
  • Protein-Protein Interactions in the Bacterial Replisome
    SHOWCASE ON RESEARCH Protein-Protein Interactions in the Bacterial Replisome Patrick Schaeffer, Madeleine Headlam and Nick Dixon Research School of Chemistry, Australian National University, ACT 0020 Replication of DNA in all organisms proceeds in proteins, while others like the small single-stranded three stages: initiation at origins of replication, DNA (SS) DNA phages make use of subsets of host synthesis (elongation) at replication forks, and enzymes. Similar reductionist approaches have more termination. Each of these processes is mediated by recently been applied to identify many of the multiple stable or transient protein-protein and replication proteins from eukaryotes. It is clear that protein-DNA interactions involving subsets of the 30 protein functions and mechanisms are conserved in or so different replication proteins (Fig. 1). The all organisms (Table 1), though the proteins may elongation phase occurs within a large nucleoprotein have little or no recognisable sequence similarity. assembly called the replisome (1). Replisomes have Work in the last ten years has produced a good n e v e r b e e n i s o l a t e d i n t a c t f r o m c e l l s , s o a n understanding of how protein-protein interactions understanding of their structure and function has mediate replisomal DNA synthesis (Fig. 2A), and required their painstaking in vitro reassembly from high-resolution structures of many of the individual individual subunits. Mechanistic studies have made proteins (or domains thereof) and some larger extensive use of the Escherichia coli proteins, in part complexes are now available.
    [Show full text]
  • Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors
    biomolecules Review Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors Georgi Muskhelishvili 1,*, Patrick Sobetzko 2, Sanja Mehandziska 3,† and Andrew Travers 4,5 1 School of Natural Sciences, Agricultural University of Georgia, David Aghmashenebeli Alley 24, Tbilisi 0159, Georgia 2 Department of Chromosome Biology, Philipps-Universität Marburg, LOEWE-Zentrum für Synthetische Mikrobiologie, Hans-Meerwein-Straße, 35043 Marburg, Germany; [email protected] 3 School of Engineering and Science, Campus Ring 1, Jacobs University Bremen, 28759 Bremen, Germany; [email protected] 4 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK; [email protected] 5 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK * Correspondence: [email protected] † Current Address: ZAN MITREV CLINIC, Str. Bledski Dogovor no.8, 1000 Skopje, Macedonia. Abstract: The coordination of bacterial genomic transcription involves an intricate network of in- terdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeo- static regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoen- Citation: Muskhelishvili, G.; zyme. In this review article, we argue that recent observations revealing DNA topoisomerases and Sobetzko, P.; Mehandziska, S.; metabolic enzymes associated with RNAP supramolecular complex support the notion of structural Travers, A. Composition of coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental Transcription Machinery and Its device coordinating the spatiotemporal genomic transcription.
    [Show full text]
  • Escherichia Coli
    Elevated Fis Expression Enhances Recombinant Protein Production in Escherichia coli Richard Richins,1 Tin Htay,1 Pauli Kallio,2 Wilfred Chen1 1Department of Chemical Engineering, University of California, Riverside, California 92521; telephone: (909) 787-2473; fax: (909) 787-3144; e-mail: [email protected]; 2Institute of Biotechnology, ETH-Honggerberg, CH-8093, Zurich, Switzerland. Received 18 October 1996; accepted 14 February 1997 Abstract: A genetic strategy to enhance recombinant cellular ppGpp has resulted in a five-fold increase in five protein production is discussed. A small DNA bending recombinant protein synthesis during fed-batch fermenta- protein, Fis, which has been shown to activate rRNA syn- thesis upon a nutrient upshift, was overexpressed in E. tion (Dedhia et al., 1997). It is clear from this example that coli strain W3110 carrying vector pUCR1. Overexpres- genetic modifications can be exploited to enhance protein sion of Fis during exponential growth was shown to ac- synthesis for recombinant cells during restricted growth. tivate rrn promoters to different extents. A 5-fold im- Fis is a 11.2 kDa, heat-stable DNA-binding protein in E. provement in chloramphenicol acetyltransferase (CAT) coli which has the ability to stimulate site-specific DNA production in cultures with elevated Fis level was ob- served in shake-flask cultivations. A similar improvement inversion reactions by binding the protein to an enhancer in the culture performance was also observed during fed- sequence and bending the DNA (Johnson et al., 1986; Gille batch fermentation; the specific CAT activity increased et al., 1991). In E. coli, the stable RNA operons have been by more than 50% during the fed-batch phase for cul- shown to be trans activated by Fis (Nilsson et al., 1990).
    [Show full text]
  • Expec) Belonging to the Highly Pathogenic Sequence Type 95 Reveals the Zoonotic Nature of Human and Avian Strains
    Functional genotyping of extraintestinal pathogenic E.coli (ExPEC) belonging to the highly pathogenic Sequence type 95 reveals the zoonotic nature of human and avian strains Inaugural-Dissertation To obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Department of Biology, Chemistry and Pharmacy Of Freie University Berlin By Nishant Nandanwar From (Bhopal, M.P., India) 2013 Time Period – November 2010 till October 2013 Supervisor – Prof. Dr. Lothar H. Wieler Institute – Institute for Microbiology and Epizootics, Robert-von-Ostertag-Str. 7-13, 14163, Berlin, Germany. 1st Reviewer: Prof. Dr. Lothar H. Wieler 2nd Reviewer: Prof. Dr. Heribert Hofer Date of Defense: 8th April 2014 Acknowledgement Above all, I wish to thank God and my parents for their abundant blessings and graces throughout my life and during my doctoral work. I would like to express my sincere thanks to Prof. Dr. Lothar Wieler for giving me the opportunity to pursue my doctorate under his supervision. I am very thankful to him for his constant support and guidance during this work, and for his constant encouragement and motivation. I would also like to thank Prof. Dr. Christa Ewers in a special way for her supervision, guidance and support right from the beginning of my work. My special thanks to Prof. Dr. Heribert Hofer, Leibniz Institute for Zoo and Wildlife Research (IZW), Berlin, for his external supervision during my doctoral studies. I would like to express my sincere gratitude to Associate Prof. Dr. Niyaz Ahmed, Department of Biotechnology & Bioinformatics, University of Hyderabad, India for his immense support and for providing me the opportunity to pursue my doctorate in Germany.
    [Show full text]
  • Fis Activates the Rpos-Dependent Stationary-Phase Expression of Prop in Escherichia Coli
    JOURNAL OF BACTERIOLOGY, Sept. 1995, p. 5222–5231 Vol. 177, No. 18 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology Fis Activates the RpoS-Dependent Stationary-Phase Expression of proP in Escherichia coli JIMIN XU AND REID C. JOHNSON* Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095 Received 31 March 1995/Accepted 9 July 1995 Fis is a general nucleoid-associated protein in Escherichia coli whose expression is highly regulated with respect to growth conditions. A random collection of transposon-induced lac fusions was screened for those which give increased expression in the presence of Fis in order to isolate a ProP-LacZ protein fusion. We find that proP, which encodes a low-affinity transporter of the important osmoprotectants proline and glycine betaine, is transcribed from two promoters. proP1 is transiently induced upon subculture and is upregulated by increases in medium osmolarity. As cells enter stationary phase, a second promoter, proP2, is strongly induced. This promoter can also be induced by high medium osmolarity in exponential phase. The activity of s proP2 depends on Fis and the stationary-phase sigma factor s . In the presence of Fis, proP2 expression is increased over 50-fold, as judged by the LacZ activity of cells carrying the proP-lacZ fusion as well as by direct RNA analysis, making this the most strongly activated promoter by Fis that has been described. Two Fis binding sites centered at positions 241 (site I) and 281 (site II) with respect to the transcription initiation site of P2 have been defined by DNase I footprinting.
    [Show full text]
  • Mechanisms and Controls of DNA Replication in Bacteria
    13 Mechanisms and Controls of DNA Replication in Bacteria César Quiñones-Valles, Laura Espíndola-Serna and Agustino Martínez-Antonio Departamento de Ingeniería Genética, Cinvestav Irapuato México 1. Introduction DNA is the polymeric molecule that contains all the genetic information in a cell. This genetic information encodes the instructions to make a copy of itself, for the cellular structure, for the operative cellular machinery and also contains the regulatory signals, which determine when parts of this machinery should be on or off. The operative machinery in turn, is responsible for the cells functions either metabolically or in interactions with the environment. Part of this cellular machinery devoted to DNA metabolism is responsible for DNA replication, DNA-repair and for the regulation of gene expression. In this chapter we will focus our discussion on the mechanisms and controls that conduct DNA replication in bacteria, including the components, functions and regulation of replication machinery. Most of our discourse will consider this biological process in Escherichia coli but when possible we will compare it to other bacterial models, mainly Bacillus subtilis and Caulobacter crescentus as examples of organisms with asymmetrical cell division. In order to maintain a bacterial population it is necessary that cells divide, but before the physical division of a daughter cell from its mother, it is necessary among other check points, that the DNA has been replicated accurately. This is done by the universal semi- conservative replication process of DNA-strands, which generates two identical strand copies from their parent templates. To better understand this process it has been divided into three phases: initiation, elongation and termination of DNA replication.
    [Show full text]
  • Tracing the Phylogenetic History of the Crl Regulon Through the Bacteria and Archaea Genomes A
    Santos-Zavaleta et al. BMC Genomics (2019) 20:299 https://doi.org/10.1186/s12864-019-5619-z RESEARCH ARTICLE Open Access Tracing the phylogenetic history of the Crl regulon through the Bacteria and Archaea genomes A. Santos-Zavaleta1* , E. Pérez-Rueda2,3*, M. Sánchez-Pérez1, D. A. Velázquez-Ramírez1 and J. Collado-Vides1 Abstract Background: Crl, identified for curli production, is a small transcription factor that stimulates the association of the σS factor (RpoS) with the RNA polymerase core through direct and specific interactions, increasing the transcription rate of genes during the transition from exponential to stationary phase at low temperatures, using indole as an effector molecule. The lack of a comprehensive collection of information on the Crl regulon makes it difficult to identify a dominant function of Crl and to generate any hypotheses concerning its taxonomical distribution in archaeal and bacterial organisms. Results: In this work, based on a systematic literature review, we identified the first comprehensive dataset of 86 genes under the control of Crl in the bacterium Escherichia coli K-12; those genes correspond to 40% of the σS regulon in this bacterium. Based on an analysis of orthologs in 18 archaeal and 69 bacterial taxonomical divisions and using E. coli K- 12 as a framework, we suggest three main events that resulted in this regulon’s actual form: (i) in a first step, rpoS,a gene widely distributed in bacteria and archaea cellular domains, was recruited to regulate genes involved in ancient metabolic processes,
    [Show full text]