No abstract

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

Until recently, no examples of the in-frame translation of several proteins from one gene in bacteria were known. The first one was the VirF transcription factor controlling pathogenicity development in Shigella flexneri and CobB sirtuin in Salmonella enterica. Recently, we observed synthesis of shortened protein products for YjjM (LgoR) and LeuO functioning as transcription factors in Escherichia coli. To determine the evolutionary factors that could lead to the appearance of alternative start codons, we performed phylogenetic analysis and showed that each protein had a unique phylogenetic history, and additional starting methionines appeared only in Enterobacteria. Using the Western-blot analysis of proteins synthesized from the Escherichia coli K-12 MG1655 chromosome with the his-tagged leuO gene two shortened variants of LeuO, corresponding to translation starting from Met34 and Met48 were detected. Synthesis of all three LeuO forms was inhibited in the absence of the yjjM gene, suggesting interplay of these transcription factors. The YjjM recognition motif revealed from the ChIP-seq data appeared to be very similar to that of LeuO, known previously. Taking this into account, we compared ChIP and SELEX data for LeuO and YjjM and found six common targets. At least five of them were confirmed to be under control of these regulators by qRT-PCR. Interestingly, the effects were more prominent during anaerobic growth at 37°C simulating conditions inside a host organism. Two genes, coding for the enterobactin transporter FepA, and a repressor of genes responsible for flagellar biosynthesis and virulence YjjQ, were repressed, mainly by YjjM, only in these conditions, while tsr coding for the chemotaxis receptor protein was more repressed under lower temperature and higher aeration.

Vaccines against the severe acute respiratory syndrome coronavirus 2, which have been in urgent need and development since the beginning of 2020, are aimed to induce a prominent immune system response capable of recognizing and fighting future infection. Here we analyzed the levels of IgG antibodies against the receptor-binding domain (RBD) of the viral spike protein after the administration of three types of popular vaccines, BNT162b2, mRNA-1273, or Sputnik V, using the same ELISA assay to compare their effects. An efficient immune response was observed in the majority of cases. The obtained ranges of signal values were wide, presumably reflecting specific features of the immune system of individuals. At the same time, these ranges were comparable among the three studied vaccines. The anti-RBD IgG levels after vaccination were also similar to those in the patients with moderate/severe course of the COVID-19, and significantly higher than in the individuals with asymptomatic or light symptomatic courses of the disease. No significant correlation was observed between the levels of anti-RBD IgG and sex or age of the vaccinated individuals. The signals measured at different time points for several individuals after full Sputnik V vaccination did not have a significant tendency to lower within many weeks. The rate of neutralization of the interaction of the RBD with the ACE2 receptor after vaccination with Sputnik V was on average slightly higher than in patients with a moderate/severe course of COVID-19. The importance of the second dose administration of the two-dose Sputnik V vaccine was confirmed: while several individuals had not developed detectable levels of the anti-RBD IgG antibodies after the first dose of Sputnik V, after the second dose the antibody signal became positive for all tested individuals and raised on average 5.4 fold. Finally, we showed that people previously infected with SARS-CoV-2 developed high levels of antibodies, efficiently neutralizing interaction of RBD with ACE2 after the first dose of Sputnik V, with almost no change after the second dose.