scholarly journals Strong spurious transcription likely a cause of DNA insert bias in typical metagenomic clone libraries

2015 ◽  
Author(s):  
Kathy N Lam ◽  
Trevor C Charles
Keyword(s):  
Gene Product ◽  
Transcriptional Activity ◽  
Gc Content ◽  
Cosmid Library ◽  
Clone Libraries ◽  
Consensus Sequences ◽  
E Coli ◽  
Gc Bias ◽  
Sigma 70 ◽  
Sequence Bias

Background: Clone libraries provide researchers with a powerful resource with which to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, as well as allowed the mining of novel enzymes for specific functions of interest. These libraries are often constructed by cloning large-inserts (~30 kb) into a cosmid or fosmid vector. Recently, there have been reports of GC bias in fosmid metagenomic clone libraries, and it was speculated that the bias may be a result of fragmentation and loss of AT-rich sequences during the cloning process. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role in library bias. Results: To explore the possible mechanisms responsible for sequence bias in clone libraries, and in particular whether fragmentation is involved, we constructed a cosmid clone library from a human microbiome sample, and sequenced DNA from three different steps of the library construction process: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final constructed cosmid library, and we provide strong evidence that the sequence bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after the DNA is introduced into E. coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for consensus promoters and found that rpoD/sigma-70 promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the reference genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found that the bias appears to be more closely correlated with the number of rpoD/sigma-70 consensus sequences in the genome than with simple GC content. Conclusions: The GC bias of metagenomic clone libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter consensus sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/sigma-70 consensus-like in E. coli may lead to plasmid instability or loss of insert DNA. Our results suggest that despite widespread use of E. coli to propagate foreign DNA, the effects of in vivo transcriptional activity may be under-appreciated. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

scholarly journals Epidemiological and phylogenetic analysis reveals Flavobacteriaceae as potential ancestral source of tigecycline resistance gene tet(X)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rong Zhang ◽  
Ning Dong ◽  
Zhangqi Shen ◽  
Yu Zeng ◽  
Jiauyue Lu ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Resistance Gene ◽  
Gc Content ◽  
Zhejiang Province ◽  
Bacterial Strains ◽  
Gram Negative ◽  
E Coli ◽  
Transmission Mechanisms ◽  
Low Prevalence ◽  
Potential Origin

Abstract Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994–2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X).

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1763-1770 ◽  
Author(s):  
Ryszard Zielke ◽  
Aleksandra Sikora ◽  
Rafał Dutkiewicz ◽  
Grzegorz Wegrzyn ◽  
Agata Czyż
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Product ◽  
Uv Irradiation ◽  
Vibrio Harveyi ◽  
Bacterial Species ◽  
Wild Type ◽  
E Coli ◽  
Stimulation Of

CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA + strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

scholarly journals Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus

2007 ◽  
Vol 189 (24) ◽  
pp. 8871-8879 ◽  
Author(s):  
Zhibiao Fu ◽  
Niles P. Donegan ◽  
Guido Memmi ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Environmental Stress Response ◽  
Binding Studies ◽  
Cell Growth Arrest ◽  
Consensus Sequences ◽  
E Coli ◽  
Endoribonuclease Activity

ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

The rho gene product expressed in E. Coli is a substrate of botulinum ADP-ribosyltransferase C3

1989 ◽  
Vol 158 (1) ◽  
pp. 209-213 ◽  
Author(s):  
Klaus Aktories ◽  
Ulrich Braun ◽  
Sigrid Rösener ◽  
Ingo Just ◽  
Alan Hall
Keyword(s):  
Gene Product ◽  
E Coli ◽  
Adp Ribosyltransferase

The vsr gene product of E. coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease

Nature ◽  
1991 ◽  
Vol 353 (6346) ◽  
pp. 776-778 ◽  
Author(s):  
Frank Hennecke ◽  
Harald Kolmar ◽  
Kerstin Bründl ◽  
Hans-Joachim Fritz
Keyword(s):  
Gene Product ◽  
E Coli ◽  
Dna Mismatch ◽  
K 12

scholarly journals Am E. coli gene product required for λ site-specific recombination

Cell ◽  
1980 ◽  
Vol 20 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Harvey I. Miller ◽  
David I. Friedman
Keyword(s):  
Gene Product ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli

scholarly journals Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

2019 ◽  
Vol 19 (2) ◽  
pp. 149-158
Author(s):  
Olfa Mega ◽  
Cece Sumantri ◽  
Irma Isnafia Arief ◽  
Cahyo Budiman
Keyword(s):  
Molecular Weight ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Gc Content ◽  
Whole Genome Sequence ◽  
Protease Gene ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Index Value

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico.  pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively.  Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C.  As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

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