Whole-genome sequencing analysis of two heat-evolved Escherichia coli strains.

BACKGROUND: High temperatures cause a suite of problems for cells, including protein unfolding and aggregation; increased membrane fluidity; and changes in DNA supercoiling, RNA stability, transcription and translation. Consequently, enhanced thermotolerance can evolve through an unknown number of genetic mechanisms even in the simple model bacterium Escherichia coli. To date, each E. coli study exploring this question resulted in a different set of mutations. To understand the changes that can arise when an organism evolves to grow at higher temperatures, we sequenced and analyzed two previously described E. coli strains, BM28 and BM28 ΔlysU, that have been laboratory adapted to the highest E. coli growth temperature reported to date. RESULTS: We found three large deletions in the BM28 and BM28 ΔlysU strains of 123, 15 and 8.5 kb in length and an expansion of IS10 elements. We found that BM28 and BM28 ΔlysU have considerably different genomes, suggesting that the BM28 culture that gave rise to BM28 and BM28 ΔlysU was a mixed population of genetically different cells. Consistent with published findings of high GroESL expression in BM28, we found that BM28 inexplicitly carries the groESL bearing plasmid pOF39 that was maintained simply by high-temperature selection pressure. We identified over 200 smaller insertions, deletions, single nucleotide polymorphisms and other mutations, including changes in master regulators such as the RNA polymerase and the transcriptional termination factor Rho. Importantly, this genome analysis demonstrates that the commonly cited findings that LysU plays a crucial role in thermotolerance and that GroESL hyper-expression is brought about by chromosomal mutations are based on a previous misinterpretation of the genotype of BM28. CONCLUSIONS: This whole-genome sequencing study describes genetically distinct mechanisms of thermotolerance evolution from those found in other heat-evolved E. coli strains. Studying adaptive laboratory evolution to heat in simple model organisms is important in the context of climate change. It is important to better understand genetic mechanisms of enhancing thermotolerance in bacteria and other organisms, both in terms of optimizing laboratory evolution methods for various organisms and in terms of potential genetic engineering of organisms most at risk or most important to our societies and ecosystems.

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 uses 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 of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel ß-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes.

We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.

Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche.