The consensus N-myristoylation motif of a geminivirus AC4 protein is required for membrane binding and pathogenicity

Some geminiviruses encode a small protein, AC4, whose role in pathogenesis has only recently attracted attention. A few studies have shown that this protein is involved in pathogenesis and suppresses RNA silencing. Here, using Nicotiana benthamiana, we show that East African cassava mosaic Cameroon virus (EACMCV) AC4 is a pathogenicity determinant and that it suppresses the systemic phase of RNA silencing. Furthermore, confocal imaging analyses show that it binds preferentially to the plasma membrane as well as to cytosolic membranes including the perinucleus but is excluded from the nucleus. A computational examination of the AC4 protein encoded by the EACMCV, a bipartite geminivirus, shows that it encodes a consensus N-myristoylation motif and is likely posttranslationally myristoylated and palmitoylated. Replacement of Gly-2 and Cys-3 (sites of posttranslational attachment of myristic and palmatic acids, respectively) with alanine affected AC4 membrane binding and pathogenesis. Furthermore, replacement of Ile-5, a nonessential myristoylation residue, with alanine did not affect AC4 function. Together, these data indicate that EACMCV AC4 is likely dually acylated at Gly-2 and Cys-3 and that these modifications are intrinsic signals for membrane targeting and pathogenesis. This is the first report of a membrane protein to be involved in pathogenesis and RNA silencing suppression.     

High-stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD

Quinol/nitrate oxidoreductase (NarGHI) is the first enzyme involved in respiratory denitrification in prokaryotes. Although this complex in E. coli is known to operate with both ubi and menaquinones, the location and the number of quinol binding sites remain elusive. NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. To identify its location and function, we used a combination of mutagenesis, kinetics, EPR, and ENDOR spectroscopies. For the NarGHIH66Y and NarGHIH187Y mutants lacking the distal heme bD, no EPR signal of the semiquinone was observed. In contrast, a semiquinone was detected in the NarGHIH56Y mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme. The substitution by Ala of the Lys86 residue close to heme bD, which was previously proposed to be in a quinol oxidation site of NarGHI (QD), also leads to the loss of the EPR signal of the semiquinone, although both hemes are present. Enzymatic assays carried out on the NarGHIK86A mutant reveal that the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues. These observations demonstrate that the semiquinone observed in NarI is strongly associated with heme bD and that Lys86 is required for its stabilization. Overall, our results indicate that the semiquinone is located within the quinol oxidation site QD. Details of the possible binding motif of the semiquinone and mechanistic implications are discussed.     

Electronic tagging of Bluefin Tunas from the Maltese spawning ground suggests size-dependent migration dynamics

Environmental Biology of Fishes - The purse seine fishery in the Mediterranean represents about 60% of the international catch for Atlantic Bluefin Tuna (Thunnus thynnus). Yet, tagging operations...     

Extensive Genetic Variability Linked to IS26 Insertions in the fljB Promoter Region of Atypical Monophasic Variants of Salmonella enterica Serovar Typhimurium

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:− and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.