High-Throughput Sequencing Reveals Spacer Integration and Loss of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Subtype I-E System in <I>E. coli</I>

Phage therapy is a promising alternative therapy for the treatment of E. coli infection. Although the total number of phages on the earth is as high as 10 31 , the reported phages and thoroughly studied are very limited. Therefore, the continuous discovery of new phages and in-depth research will provide materials for the wide application of phage therapy in the future. In this study, a novel E. coli phage vB_EcoM_011D4 was isolated from sewage samples, and the biological characteristics were studied. Electron microscopy and homology analysis results showed that vB_EcoM_011D4 belongs to the family Myoviridae. One-step growth curve showing the latent period of vB_EcoM_011D4 was 10 min, with the burst size of 115 PFU/cell. Additionally, Phage vB_EcoM_011D4 was highly stabled under different temperatures (range 4 – 70 ℃) and pH conditions (range 6 – 10). At the same time, its genome was subjected to high-throughput sequencing and compared with the reported phages. The results of high-throughput sequencing assembly showed that vB_EcoM_011D4 is a linear, double-stranded DNA virus containing 163764 bp, with an average GC content of 40.50%, and a total of 273 open reading frames (ORFs). Genomic comparison analysis revealed that most of the ORFs were similar to Enterobacteria phage Phi1 and RB49. However, ORF147 and ORF148 putative DNA methylase family protein is less than 67% homology with already published phages. In addition, the phylogenetic analysis of terminates large subunit showed that it belongs to a new branch and shows less than 50 similarities to reported phages. There is no lysogenic, toxin or antibiotic-resistant related gene was found in the genome of vB_EcoM_011D4. In summary, vB_EcoM_011D4 is a newly discovered phage, which can be further studied for elucidating the phage diversity and it is benefits for the wide application of phage therapy.

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gndDb, a Database of Partial gnd Sequences To Assist with Analysis of Escherichia coli Communities Using High-Throughput Sequencing

Microbiology Resource Announcements ◽

10.1128/mra.00476-19 ◽

2019 ◽

Vol 8 (33) ◽

Author(s):

Adrian L. Cookson ◽

David W. Lacher ◽

Flemming Scheutz ◽

David A. Wilkinson ◽

Patrick J. Biggs ◽

...

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Sequencing ◽

Community Analysis ◽

Culture Methods ◽

Content Type ◽

E Coli ◽

Content Diversity ◽

Culture Independent ◽

Low Levels

The use of culture methods to detect Escherichia coli diversity does not provide sufficient resolution to identify strains present at low levels. Here, we target the hypervariable gnd gene and describe a database containing 534 distinct partial gnd sequences and associated O groups for use with culture-independent E. coli community analysis.

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Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions

10.21203/rs.2.21231/v3 ◽

2020 ◽

Author(s):

Lizhi Zhou ◽

Hai Yu ◽

Kaihang Wang ◽

Tingting Chen ◽

Yue Ma ◽

...

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Speed ◽

High Throughput Sequencing ◽

Recombinant Protein Expression ◽

External Pressure ◽

Universal Method ◽

Coding Region ◽

Human Papillomavirus 16 ◽

E Coli

Abstract Background: The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure.Results: The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3’ non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.Conclusion: The ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.

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