scholarly journals A- and T-Tract-Mediated Intrinsic Curvature in Native DNA between the Binding Site of the Upstream Activator NtrC and the nifLA Promoter of Klebsiella pneumoniaeFacilitates Transcription

1999 ◽  
Vol 181 (17) ◽  
pp. 5296-5302 ◽  
Author(s):  
Amrita Kaur Cheema ◽  
Nirupam Roy Choudhury ◽  
H. K. Das
Keyword(s):  
Integration Host Factor ◽  
Dna Looping ◽  
Dna Curvature ◽  
Intrinsic Curvature ◽  
E Coli ◽  
Dna Base ◽  
Dna Fragment ◽  
The One ◽  
Rna Polymerase Holoenzyme

ABSTRACT The nif promoters of Klebsiella pneumoniaemust be activated by proteins bound to upstream sequences which are thought to interact with the ς54-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the nifLA promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the Escherichia coli lacZ gene of the DNA fragment containing the nifLA promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in K. pneumoniae and E. coliwas monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from thenifLA promoter of K. pneumoniae.

scholarly journals Involvement of the Escherichia coli O157:H7(pO157) ecf Operon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs

2005 ◽  
Vol 73 (4) ◽  
pp. 2367-2378 ◽  
Author(s):  
Jang W. Yoon ◽  
Ji Youn Lim ◽  
Yong H. Park ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Membrane Structure ◽  
Lipid A ◽  
Transferase Activity ◽  
Bacterial Membrane ◽  
Dna Curvature ◽  
Bacterial Survival ◽  
Genetic Redundancy ◽  
E Coli

ABSTRACT Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

scholarly journals High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli

2015 ◽  
Vol 112 (23) ◽  
pp. 7177-7182 ◽  
Author(s):  
Nicole A. Becker ◽  
L. James Maher
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Dna Looping ◽  
Chromosomal Protein ◽  
Bent Dna ◽  
E Coli ◽  
Dna Loop ◽  
Eukaryotic Chromatin ◽  
Negative Supercoiling

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

scholarly journals Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Russell K. Karls ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroidescytochrome c 2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterizedEscherichia coli heat shock (ς32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coliEς32 −35 consensus sequence (G34T) increasedcycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς38) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).

scholarly journals Microbial production of multiple short-chain primary amines via retrobiosynthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dong In Kim ◽  
Tong Un Chae ◽  
Hyun Uk Kim ◽  
Woo Dae Jang ◽  
Sang Yup Lee
Keyword(s):  
Short Chain ◽  
Primary Amines ◽  
Biosynthetic Pathways ◽  
E Coli ◽  
Wide Range ◽  
Selection Step ◽  
Fed Batch Culture ◽  
The One

AbstractBio-based production of many chemicals is not yet possible due to the unknown biosynthetic pathways. Here, we report a strategy combining retrobiosynthesis and precursor selection step to design biosynthetic pathways for multiple short-chain primary amines (SCPAs) that have a wide range of applications in chemical industries. Using direct precursors of 15 target SCPAs determined by the above strategy, Streptomyces viridifaciens vlmD encoding valine decarboxylase is examined as a proof-of-concept promiscuous enzyme both in vitro and in vivo for generating SCPAs from their precursors. Escherichia coli expressing the heterologous vlmD produces 10 SCPAs by feeding their direct precursors. Furthermore, metabolically engineered E. coli strains are developed to produce representative SCPAs from glucose, including the one producing 10.67 g L−1 of iso-butylamine by fed-batch culture. This study presents the strategy of systematically designing biosynthetic pathways for the production of a group of related chemicals as demonstrated by multiple SCPAs as examples.

The expression ofnifBgene fromHerbaspirillum seropedicaeis dependent upon the NifA and RpoN proteins

2006 ◽  
Vol 52 (12) ◽  
pp. 1199-1207 ◽  
Author(s):  
Fabiane G.M Rego ◽  
Fábio O Pedrosa ◽  
Leda S Chubatsu ◽  
M Geoffrey Yates ◽  
Roseli Wassem ◽  
...  
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Integration Host Factor ◽  
Lacz Fusion ◽  
Herbaspirillum Seropedicae ◽  
Band Shift ◽  
E Coli ◽  
Low Levels ◽  
Ihf Protein

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a –24/–12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.Key words: Herbaspirillum seropedicae, nif, nitrogen fixation, NifA, RpoN.

scholarly journals Analysis of a “Split-and-Stuttering” Module of an Assembly Line Polyketide Synthase

2021 ◽  
Author(s):  
Katarina M. Guzman ◽  
Kai P. Yuet ◽  
Stephen R. Lynch ◽  
Corey W. Liu ◽  
Chaitan Khosla
Keyword(s):  
Assembly Line ◽  
Polyketide Synthase ◽  
Catalytic Properties ◽  
E Coli ◽  
Elongation Cycle ◽  
Depth Analysis ◽  
Number Of Cycles ◽  
The One

AbstractNotwithstanding the “one-module-one-elongation-cycle” paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as “stuttering”, have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all trans-configured triene moiety in the polyketide product. Here we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the “split-and-stuttering” module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.TOC/Abstract Graphic

scholarly journals The loopometer: a quantitative in vivo assay for DNA-looping proteins

2021 ◽  
Author(s):  
Nan Hao ◽  
Adrienne E Sullivan ◽  
Keith E Shearwin ◽  
Ian B Dodd
Keyword(s):  
Binding Sites ◽  
Dna Looping ◽  
Lacz Reporter Gene ◽  
Effective Distance ◽  
E Coli ◽  
In Vivo Assay ◽  
Reporter Gene Analysis ◽  
Phage Proteins ◽  
Laci Repressor

Abstract Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.

scholarly journals Identification and Characterization of thefis Operon in Enteric Bacteria

1998 ◽  
Vol 180 (22) ◽  
pp. 5932-5946 ◽  
Author(s):  
Michael B. Beach ◽  
Robert Osuna
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
E Coli

ABSTRACT The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, andProteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens,E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence forH. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) precedingfis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihfbinding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

STUDIES IN CONGENITAL GENERALIZED LIPODYSTROPHY

1973 ◽  
Vol 72 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Oddmund Søvik ◽  
Svein Oseid
Keyword(s):  
Biological Activity ◽  
Insulin Response ◽  
Epididymal Adipose Tissue ◽  
Large Individual ◽  
Immunoreactive Insulin ◽  
List Type ◽  
Glucose Load ◽  
Congenital Generalized Lipodystrophy ◽  
The One

ABSTRACT The biological activity of plasma insulin from 4 cases of congenital generalized lipodystrophy has been studied, using rat diaphragm and epididymal adipose tissue in vivo. The results are compared with previous data on plasma immunoreactive insulin obtained in these patients. 2 of the 4 cases exhibited unusually high biological insulin activities during the fasting state as well as after an intravenous (iv) glucose load. In the fat pad assay activities as high as 10 000 μU insulin per ml were observed. During childhood the biological insulin activities were generally high, although there were large individual variations. However, in the one case studied after the age of puberty, the insulin response to a glucose load was negligible. Taken together, the biological and immunological activities observed strongly suggest the presence of pancreatic insulin in these patients. It appears that the circulating insulin has a fully biological activity. The decreasing insulin activities after cessation of growth are in agreement with the appearance of frank diabetes at this time.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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