The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

We previously showed that the product of the wbaP gene of Salmonella enterica serovar Typhimurium has two functions: it is involved in the first step of O-antigen synthesis (the galactosyltransferase [GT] function) and in a later step (the T function), first thought to be the flipping of the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic face to the periplasmic face of the cytoplasmic membrane. We now locate two wbaP(T) mutations within the first half of the wbaP gene by sequencing. Both mutants retain GT activity, although one was a frameshift mutation resulting in a stop codon 10 codons after the frameshift to give an open reading frame containing only 138 of the 476 codons in WbaP. We also show that there is a secondary translation starting within the wbaP gene resulting in the synthesis of a polypeptide with GT activity. These results indicate that the N-and C-terminal halves of WbaP are the T and GT functional domains, respectively. We now propose that the T block operates prior to the flippase function, probably at the release of undecaprenyl pyrophosphate-linked galactose from WbaP.

NAD (NAD؉ ) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD ؉ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD ؉ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD ؉ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD ؉ . We then constructed the NAD ؉ -auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD ؉ biosynthesis in cells harboring the ntt4 gene. The minimal NAD ؉ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD ؉ , while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD ؉ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD ؉ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044؋ and 9.6؋ those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.