Biology subjects

Nano, F. E.

Publications and source records attributed to Nano, F. E..

2 recordsLinked to original sources

bioRxiv2024

Genomic analysis of laboratory-evolved, heat-adapted Escherichia coli strains

Adaptive laboratory evolution to high incubation temperatures represents a complex evolutionary problem, and each study to date performed in Escherichia coli has resulted in a different set of mutations. We performed adaptive laboratory evolution of E. coli to heat by passaging a culture at elevated temperatures for 150 days. Throughout the adaptive evolution we expressed a set of genes that induce hyper-mutagenesis. These growth conditions yielded a strain with a maximum growth temperature approximately 2 {degrees}C above that of the parental strain. We preserved evolved isolates weekly and obtained and analyzed whole-genome sequencing data for three isolates from different time points. We identified hundreds of mutations, including mutations in components of the RNA polymerase (RpoB, RpoC and RpoD), Rho, and the heat shock proteins GroES, GroEL, DnaK, ClpB, IbpA and HslU. We compared the proteomes of the starting strain and final strain grown at 37 {degrees}C and 42.5 {degrees}C and identified changes in abundance between samples for GroESL, HslVU, DnaK, ClpB and other important proteins. This study details a distinct evolutionary route towards enhanced thermotolerance, contributes to our understanding of adaptation to heat in Escherichia coli and may provide insights into heat adaptation in other organisms.

evolutionary biology↗

bioRxiv2021

Escherichia coli recombinant expression of SARS-CoV-2 protein fragments.

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach used the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest intact CBM9 fusion protein incorporates spike protein amino acids 540-588, which is a conserved region immediately C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

microbiology↗