Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

The BPtpA protein from Burkholderia cenocepacia belongs to a new subclass of low molecular weight protein tyrosine phosphatases.

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.