The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies

2005 ◽  
Vol 386 (6) ◽  
pp. 523-534 ◽  
Author(s):  
Annette Hillebrand ◽  
Reinhild Wurm ◽  
Artur Menzel ◽  
Rolf Wagner
Keyword(s):  
Protein Complexes ◽  
Rrna Synthesis ◽  
Dna Fragments ◽  
Promoter Regions ◽  
Regulatory Regions ◽  
E Coli ◽  
Upstream Activating Sequence ◽  
Dna Elements ◽  
The Individual ◽  
Upstream Regulatory Regions

AbstractRibosomal RNAs inE. coliare transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the majorE. colirRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for therrnBandrrnDfragments and less pronounced for therrnAandrrnEoperons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.

scholarly journals Mutational Analysis of the Myxococcus xanthus Ω4400 Promoter Region Provides Insight into Developmental Gene Regulation by C Signaling

2004 ◽  
Vol 186 (3) ◽  
pp. 661-671 ◽  
Author(s):  
Deborah R. Yoder ◽  
Lee Kroos
Keyword(s):  
Dna Sequences ◽  
Promoter Region ◽  
Mutational Analysis ◽  
Myxococcus Xanthus ◽  
Regulatory Region ◽  
Cell Movement ◽  
Site Directed Mutagenesis ◽  
Promoter Regions ◽  
Regulatory Regions ◽  
The Individual

ABSTRACT Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression. One of these signals, the C signal, regulates the expression of many genes, including Ω4400, a gene identified by an insertion of Tn5 lac into the chromosome. Expression of Tn5 lac Ω4400 is reduced in csgA mutant cells, which fail to perform C signaling, and the promoter region has several sequences similar to sequences found in the regulatory regions of other C-signal-dependent genes. One such gene, Ω4403, depends absolutely on the C signal for expression, and its promoter region has been characterized previously by mutational analysis. To determine if the similar sequences within the Ω4400 and Ω4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the Ω4400 promoter region were performed. A 7-bp sequence centered at −49 bp, termed a C box, is identical in the Ω4400 and Ω4403 promoter regions, yet mutations in the individual base pairs affected expression from the two promoters very differently. Also, a single-base-pair change within a similar 5-bp element, which is centered at −61 bp in both promoter regions, had very different effects on the activities of the two promoters. Further mutational analysis showed that two regions are important for Ω4400 expression; one region, from −63 to −31 bp, is required for Ω4400 expression, and the other, from −86 to −81 bp, exerts a two- to fourfold effect on expression and is at least partially responsible for the C signal dependence of the Ω4400 promoter. Mutations in sigD and sigE, which are genes that encode σ factors, abolished and reduced Ω4400 expression, respectively. Expression of Ω4400 in actB or actC mutants correlated well with the altered levels of C signal produced in these mutants. Our results provide the first detailed analysis of an M. xanthus regulatory region that depends partially on C signaling for expression and indicate that similar DNA sequences in the Ω4400 and Ω4403 promoter regions function differently.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

scholarly journals Secretion of Anti-Plasmodium Effector Proteins from a Natural Pantoea agglomerans Isolate by Using PelB and HlyA Secretion Signals

2011 ◽  
Vol 77 (13) ◽  
pp. 4669-4675 ◽  
Author(s):  
Dawn C. Bisi ◽  
David J. Lampe
Keyword(s):  
Control Strategies ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Individual Characteristics ◽  
Pantoea Agglomerans ◽  
Effector Proteins ◽  
World Health ◽  
Content Type ◽  
E Coli ◽  
The Individual

ABSTRACTThe insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodiumspp.) in the mosquito host. We hypothesized thatPantoea agglomerans, a bacterial symbiont ofAnophelesmosquitoes, could be engineered to express and secrete anti-Plasmodiumeffector proteins, a strategy termed paratransgenesis. To this end, plasmids that include thepelBorhlyAsecretion signals from the genes of related species (pectate lyase fromErwinia carotovoraand hemolysin A fromEscherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodiumeffector proteins (SM1, anti-Pbs21, and PLA2) inP. agglomeransandE. coli.P. agglomeranssuccessfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodiumactivity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

scholarly journals HIRA stabilizes skeletal muscle lineage identity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joana Esteves de Lima ◽  
Reem Bou Akar ◽  
Léo Machado ◽  
Yuefeng Li ◽  
Bernadette Drayton-Libotte ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Lineage ◽  
Promoter Regions ◽  
Cell Fates ◽  
Regulatory Regions ◽  
Muscle Stem Cells ◽  
Adult Mice ◽  
H3k4me3 Mark ◽  
Muscle Genes ◽  
Muscle Gene

AbstractThe epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

2021 ◽  
Author(s):  
Allyson E. Shea ◽  
Jolie A. Stocki ◽  
Stephanie D. Himpsl ◽  
Sara N. Smith ◽  
Harry L. T. Mobley
Keyword(s):  
Ex Vivo ◽  
Cell Adherence ◽  
Upec Strain ◽  
E Coli ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Strain Cft073 ◽  
The Individual ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Preslaughter Hide Status of Cattle and the Microbiology of Carcasses

1997 ◽  
Vol 60 (12) ◽  
pp. 1502-1508 ◽  
Author(s):  
JOYCE VAN DONKERSGOED ◽  
KLAUS W. F. JERICHO ◽  
HEIDI GROGAN ◽  
BEN THORLAKSON
Keyword(s):  
Bacterial Count ◽  
Control Points ◽  
Bacterial Counts ◽  
Negatively Associated ◽  
Surface Wetness ◽  
E Coli ◽  
Line Speed ◽  
Critical Control Points ◽  
Plant Personnel ◽  
The Individual

An assessment was made of the association between tag (mud, bedding, and manure) attached to hides of beef cattle at slaughter and bacterial deposition on carcasses. A total of 624 carcasses from 52 lots of cattle in southern Alberta from January to June 1996 were studied at a high-line-speed abattoir (HLSP) which processed 285 carcasses per h and at a slow-line-speed abattoir (SLSP) which processed 135 carcasses per h. Tag was quantitatively assessed on the belly, legs, and sides of 12 carcasses per lot by the same project worker (lot tag score) and for each incoming lot of cattle by plant personnel (plant lot tag score). Swabs (approximately 10 by 10 cm) were taken from the medial rump and sacrum immediately after hide removal and from the brisket and top of shoulder after carcass splitting. These samples were pooled for each carcass and aerobic mesophilic bacteria, coliforms, and Escherichia coli were enumerated. The lot bacterial count was calculated by averaging the individual bacterial results of the 12 carcasses in a lot. At the HLSP, the lot side scores and the plant lot tag scores were negatively associated (P < 0.05) with the aerobic bacteria, coliforms, and E. coli. Counts were lower when tag was shaven off of the hides or when the line speed was slowed, but the reductions in counts were less than 0.5 log10/cm2. At the SLSP, the lot belly score was negatively associated (P < 0.003) with the aerobic bacterial counts. Neither the lot tag score nor the plant lot tag score were associated (P > 0.05) with the bacterial counts. Surface wetness of the hides was weakly (P < 0.05) associated with coli forms and E. coli counts. This study indicates that there is no consistent association between lot tag scores, plant lot tag scores, and bacterial contamination of carcasses. Changes in bacterial counts when associated with lot tag scores, plant lot tag scores, surface wetness of hides, line speed, or shaving off of tag were generally less than 0.5 log10/cm2. Thus, these variables are individually assessed as control points, but not critical control points of HACCP plans for the prevailing beef slaughter processes (including line speed adjustment at the HLSP) at the two plants studied.

scholarly journals Making Soup: Preparing and Validating Molecular Simulations of the Bacterial Cytoplasm

2019 ◽  
Author(s):  
Leandro Oliveira Bortot ◽  
Zahedeh Bashardanesh ◽  
David van der Spoel
Keyword(s):  
Large Scale ◽  
Biological Properties ◽  
Computational Power ◽  
E Coli ◽  
Solvent Models ◽  
Explicit Solvent ◽  
Computational Modeling And Simulation ◽  
Dynamics Simulations ◽  
The Individual ◽  
Bacterial Cytoplasm

Biomolecular crowding affects the biophysical and biochemical behavior of macro- molecules when compared to the dilute environment present in experiments made with isolated proteins. Computational modeling and simulation are useful tools to study how crowding affects the structural dynamics and biological properties of macromolecules. As computational power increased, modeling and simulating large scale all-atom explicit solvent models of the prokaryote cytoplasm become possible. In this work, we build an atomistic model of the cytoplasm of Escherichia coli composed of 1.5 million atoms and submit it to a total of 3 μs of molecular dynamics simulations. The properties of biomolecules under crowding conditions are compared to those from simulations of the individual compounds under dilute conditions. The simulation model is found to be consistent with experimental data about the diffusion coefficient and stability of macromolecules under crowded conditions. In order to stimulate further work we provide a Python script and a set of files that enables other researchers to build their own E. coli cytoplasm models to address questions related to crowding.<br>

scholarly journals Probing E. coli SSB Protein-DNA topology by reversing DNA backbone polarity

2020 ◽  
Author(s):  
Alexander G. Kozlov ◽  
Timothy M. Lohman
Keyword(s):  
Dna Binding ◽  
Crystal Structures ◽  
Nearest Neighbor ◽  
Genome Maintenance ◽  
Binding Properties ◽  
Ssdna Binding ◽  
E Coli ◽  
Phosphodiester Linkage ◽  
The Individual ◽  
Dna Binding Properties

AbstractE. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35 mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high “unlimited”, nearest-neighbor cooperativity forming long protein clusters. The (SSB)65 mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays “limited” low cooperativity. Crystal structures of E. coli SSB and P. falciparum SSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)70 constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3’-3’ or 5’-5’. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70 with a 3’-3’ polarity switch shows decreased affinity for EcSSB in the (SSB)65 mode and lower cooperativity in the (SSB)35 mode. However, (dT)70 in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65 mode, but rather binds EcSSB in the (SSB)35 mode, with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)65 mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA.Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures of E. coli and P. falciparum SSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that both E. coli and P. falciparum SSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP

1988 ◽  
Vol 8 (3) ◽  
pp. 1206-1215
Author(s):  
C F Clarke ◽  
K Cheng ◽  
A B Frey ◽  
R Stein ◽  
P W Hinds ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Protein Complexes ◽  
P53 Protein ◽  
Immunoaffinity Column ◽  
E Coli ◽  
Shock Proteins

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

scholarly journals miR-215 Targeting Novel Genes EREG, NIPAL1 and PTPRU Regulates the Resistance to E.coli F18 in Piglets

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1053
Author(s):  
Chao-Hui Dai ◽  
Fang Wang ◽  
Shi-Qin Wang ◽  
Zheng-Chang Wu ◽  
Sheng-Long Wu ◽  
...  
Keyword(s):  
Target Genes ◽  
Core Promoter ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nucleotide Polymorphisms ◽  
Weaned Piglets ◽  
Promoter Regions ◽  
E Coli ◽  
Rnai Technology ◽  
Genes Expression

Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets’ resistance to Escherichia coli (E. coli) F18. In this study, target genes of miR-215 were identified by RNA-seq, bioinformatics analysis and dual luciferase detection. The relationship between target genes and E. coli infection was explored by RNAi technology, combined with E. coli stimulation and enzyme linked immunosorbent assay (ELISA) detection. Molecular regulating mechanisms of target genes expression were analyzed by methylation detection of promoter regions and dual luciferase activity assay of single nucleotide polymorphisms (SNPs) in core promoter regions. The results showed that miR-215 could target EREG, NIPAL1 and PTPRU genes. Expression levels of three genes in porcine intestinal epithelial cells (IPEC-J2) in the RNAi group were significantly lower than those in the negative control pGMLV vector (pGMLV-NC) group after E. coli F18 stimulation, while cytokines levels of TNF-α and IL-1β in the RNAi group were significantly higher than in the pGMLV-NC group. Variant sites in the promoter region of three genes could affect their promoter activities. These results suggested that miR-215 could regulate weaned piglets’ resistance to E. coli F18 by targeting EREG, NIPAL1 and PTPRU genes. This study is the first to annotate new biological functions of EREG, NIPAL1 and PTPRU genes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.

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