No abstract

Escherichia coli is a widely used host organism for recombinant technology, and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific molecule recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25–42°C, and its derivative B-95.ΔAΔfabR was better adapted to low temperatures and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin molecules sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin containing multiple azido groups.

The future conditions of Arctic sea ice and marine ecosystems are of interest not only to climate scientists, but also to economic and governmental bodies. However, the lack of widespread, year-long biogeochemical observations remains an obstacle to understanding the complicated variability of the Arctic marine biological pump. Here we show an early winter maximum of sinking biogenic flux in the western Arctic Ocean and illustrate the importance of shelf-break eddies to biological pumping from wide shelves to adjacent deep basins using a combination of year-long mooring observations and three-dimensional numerical modelling. The sinking flux trapped in the present study included considerable fresh organic material with soft tissues and was an order of magnitude larger than previous estimates. We predict that further reductions in sea ice will promote the entry of Pacific-origin biological species into the Arctic basin and accelerate biogeochemical cycles connecting the Arctic and subarctic oceans.

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N6-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNAPylCCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Respiration (= oxygen consumption) rates and electron transport system (ETS) enzyme activities in conjunction with body carbon and nitrogen composition (for respiration) or protein (for ETS) were determined for over 50 copepod species from the mesopelagic (M; 500 to 1000 m), upperbathypelagic (UB; 1000 to 2000 m) and lower-bathypelagic (LB; 2000 to 3000 m) zones of the western subarctic Pacific. Calculated specific respiration rates (SR, a fraction of body carbon respired) at in situ temperatures (3, 2 and 1.5°C for the M, UB and LB zones, respectively) were greater for the M zone copepods (mean: 1.1% body C d ). Respiration rates adjusted to those at 1°C by using a Q 10 value (2.0), and to those of specimens with 1 mg body nitrogen by using a body mass exponent ( 0.8) , showed the same depth-related decline from the M zone to the LB zone. Stepwise regression analysis revealed that stage/sex, feeding type and/or reaction speeds (as judged by the presence/absence of myelin sheath enveloping axons) of copepods were possible additional variables affecting their respiration rates and ETS activities. The reduction in respiration rates and ETS activities from the M zone to the UB or LB zone is more pronounced when respiration rate data on Arctic/Antarctic epipelagic copepods is added; the same is true for ETS activities when respiration rate data is added from copepods dominant in the subarctic Pacific. The present results are compared with those of micronektonic crustaceans and fishes reported for specimens collected from 500 to 2000 m in other regions and discussed in the light of the 'visual interactions' hypothesis. KEY WORDS: Mesopelagic · Bathypelagic · Copepods · Respiration · ETS activity · Western North PacificResale or republication not permitted without written consent of the publisher