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.

The Histidine Kinase CckA Is Directly Inhibited by a Response Regulator-like Protein in a Negative Feedback Loop.

In alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria. IMPORTANCE The two-component systems (TCS) in bacteria in its simplest architecture consist of a histidine kinase (HK) and a response regulator (RR). In response to a specific stimulus, the HK is activated and drives phosphorylation of the RR, which is responsible of generating an adaptive response. These systems are ubiquitous among bacteria and are frequently controlled by accessory proteins. In alphaproteobacteria, the TCS formed by the HK CckA, the phosphotransferase ChpT, and the RR CtrA is widely distributed. Currently, most of the information of this system and its regulatory proteins comes from findings carried out in microorganisms where it is essential. However, this is not the case in many species, and studies of this TCS and its regulatory proteins are lacking. In this study, we found that Osp, a RR-like protein, inhibits the kinase activity of CckA in a negative feedback loop since osp expression is activated by CtrA. The inhibitory role of Osp and the similar action of the previously reported FixT protein, suggests the existence of a new group of RR-like proteins whose main function is to interact with the HK and prevent its phosphorylation.

Functional requirement of tyrosine residue 429 within CD5 cytoplasmic domain for regulation of T cell activation and survival.

CD5 has been mainly described as a negative regulator of TCR and BCR signaling and recent evidence has shown an important role for this receptor in delivering pro-survival signals. However, the molecular mechanisms underlying these processes remain unresolved. TCR crosslinking leads to phosphorylation of three tyrosine residues within the cytoplasmic tail of CD5 (Y429, Y441 and Y463) leading to the recruitment of signaling molecules like PI3K, c-Cbl and RasGAP; nevertheless, the role of these residues in T cell survival has not yet been assessed. In this study, we show that alanine-scanning mutagenesis of such tyrosine residues, either singly or in combination, leads to an increased thymocyte cell death with or without α-CD3 stimulation. Remarkably, the T-cell death observed with each individual tyrosine mutant was Caspase 3-independent. Furthermore, Y429 mutation resulted in a hyper-phosphorylation of ERK suggesting that this tyrosine residue regulates cell survival through down modulation of TCR signaling. Mutation of Y441 or Y463 did not induce hyper-responsiveness to TCR activation, indicating that they promoted T-cell survival by a TCR signal-independent pathway. Our results show that three tyrosine-based domains within CD5 cytoplasmic tail promote T-cell survival through non-overlapping mechanisms. This study also reveals that Y429 domain of CD5, previously described as a "pseudo ITAM", is functionally an ITIM domain in T cells.

The flagellar set Fla2 in Rhodobacter sphaeroides is controlled by the CckA pathway and is repressed by organic acids and the expression of Fla1.

Rhodobacter sphaeroides has two different sets of flagellar genes. Under the growth conditions commonly used in the laboratory, the expression of the fla1 set is constitutive, whereas the fla2 genes are not expressed. Phylogenetic analyses have previously shown that the fla1 genes were acquired by horizontal transfer from a gammaproteobacterium and that the fla2 genes are endogenous genes of this alphaproteobacterium. In this work, we characterized a set of mutants that were selected for swimming using the Fla2 flagella in the absence of the Fla1 flagellum (Fla2(+) strains). We determined that these strains have a single missense mutation in the histidine kinase domain of CckA. The expression of these mutant alleles in a Fla1(-) strain allowed fla2-dependent motility without selection. Motility of the Fla2(+) strains is also dependent on ChpT and CtrA. The mutant versions of CckA showed an increased autophosphorylation activity in vitro. Interestingly, we found that cckA is transcriptionally repressed by the presence of organic acids, suggesting that the availability of carbon sources could be a part of the signal that turns on this flagellar set. Evidence is presented showing that reactivation of fla1 gene expression in the Fla2(+) background strongly reduces the number of cells with Fla2 flagella.