Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG

How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.

The Type III Secretion Chaperone HpaB Controls the Translocation of Effector and Noneffector Proteins From Xanthomonas campestris pv. vesicatoria

Pathogenicity of the gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into plant cells. Effector proteins contain N-terminal T3S and translocation signals and interact with the T3S chaperone HpaB, which presumably escorts effectors to the secretion apparatus. The molecular mechanisms underlying the recognition of effectors by the T3S system are not yet understood. In the present study, we analyzed T3S and translocation signals in the type III effectors XopE2 and XopJ from X. campestris pv. vesicatoria. Both effectors contain minimal translocation signals, which are only recognized in the absence of HpaB. Additional N-terminal signals promote translocation of XopE2 and XopJ in the wild-type strain. The results of translocation and interaction studies revealed that the interaction of XopE2 and XopJ with HpaB and a predicted cytoplasmic substrate docking site of the T3S system is not sufficient for translocation. In agreement with this finding, we show that the presence of an artificial HpaB-binding site does not promote translocation of the noneffector XopA in the wild-type strain. Our data, therefore, suggest that the T3S chaperone HpaB not only acts as an escort protein but also controls the recognition of translocation signals.

Identification of MazF Homologue in Legionella pneumophila Which Cleaves RNA at the AACU Sequence

MazF is a sequence-specific endoribonuclease that is widely conserved in bacteria and archaea. Here, we found an MazF homologue (MazF-lp; LPO-p0114) in <i>Legionella pneumophila</i>.<i></i> The<i> mazF-lp</i> gene overlaps 14 base pairs with the upstream gene<i> mazE-lp</i> (MazE-lp; LPO-p0115). The induction of <i>mazF-lp</i> caused cell growth arrest, while <i>mazE-lp</i> co-induction recovered cell growth in <i>Escherichia coli</i>. In vivo<i></i> and<i></i> in vitro primer extension experiments showed that MazF-lp is a sequence-specific endoribonuclease cleaving RNA at AACU. The endoribonuclease activity of purified MazF-lp was inhibited by purified MazE-lp. We found that MazE-lp and the MazEF-lp complex specifically bind to the palindromic sequence present in the 5′-untranslated region of the <i>mazEF-lp</i> operon. MazE-lp and MazEF-lp both likely function as a repressor for the <i>mazEF-lp</i> operon and for other genes, including <i>icmR</i>, whose gene product functions as a secretion chaperone for the IcmQ pore-forming protein, by specifically binding to the palindromic sequence in 5′-UTR of these genes.