Laura Camarena

Cereibacter sphaeroides is an α-proteobacteria that has two flagellar systems. Fla1 directs the assembly of a single subpolar flagellum, and Fla2 directs the assembly of multiple polar flagella. The fla2 genes are controlled by the two-component system CckA/ChpT/CtrA. In the wild-type strain, the fla2 genes are not expressed under the growth conditions commonly used in the laboratory, and thus far, their expression has only been reported in strains carrying either a gain-of-function version of CckA or a null mutation in osp, a negative regulator of CckA. In this work, the differential swimming response of two Fla2 + strains in response to the inclusion of a divalent ion in the culture medium was investigated. This analysis led to identifying a new transcriptional regulator of the XRE family, XrpA. This protein severely reduces the expression of ctrA and, consequently, the expression of the genes activated by this transcription factor. We show that XrpA binds to the control region of ctrA and chpT, suggesting that XrpA directly represses their expression. Additionally, we determined that RpoN3, one of the four RpoN paralogues of RpoN present in C. sphaeroides, and its cognate activator protein AprX are required for the expression of xrpA. XrpA is conserved in several species of Rhodobacterales and a σ⁵⁴ promoter consensus sequence is present in its control region and a homologue of AprX cooccurs with it. These results support the idea that these proteins form a novel regulatory module that controls the TCS CckA/ChpT/CtrA in C. sphaeroides and other related species.

Cereibacter sphaeroides has a quorum sensing (QS) system that has been partially characterized. Using a bioinformatic approach, six LuxR homologs and one homolog of the acylhomoserine lactone synthase were identified in this bacterium, including the previously characterized CerR and CerI proteins. This study focused on determining the roles of two LuxR homologs, CerM and CerN. CerN lacks the HTH domain and, together with CerM, controls the expression of ctrA, which is part of the TCS CckA/ChpT/CtrA. CtrA is widely conserved in alpha‐proteobacteria and regulates flagellar motility and other cellular processes. Genetic and biochemical data suggest that CerM indirectly represses ctrA expression, which is counteracted by its interaction with CerN‐AHL. A transcriptomic study identified 181 genes regulated by CerM/CerN, with a conserved sequence in their regulatory regions likely indicating the CerM binding site. This hypothesis was supported by in vitro and in vivo DNA–protein interaction assays. Our results identified a transcription factor that could connect the QS system with the regulation of the two‐component system CckA/ChpT/CtrA.

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 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.

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylases enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli. In contrast, in Rhodobacter sphaeroides, FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study we show that sltF is expressed along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF and FlgG through its C-terminal region. A deletion analysis that divides the C-terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF ensuring a timely transcription, a proper localization and a controlled enzymatic activity. Importance Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. In this work we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase towards the flagellar rod. All these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.

Rhodobacter sphaeroides is an α-proteobacterium that has the particularity of having two functional flagellar systems used for swimming. Under the growth conditions commonly used in the laboratory, a single subpolar flagellum that traverses the cell membrane, is assembled on the surface. This flagellum has been named Fla1. Phylogenetic analyses have suggested that this flagellar genetic system was acquired from an ancient γ-proteobacterium. It has been shown that this flagellum has components homologous to those present in other γ-proteobacteria such as the H-ring characteristic of the Vibrio species. Other features of this flagellum such as a straight hook, and a prominent HAP region have been studied and the molecular basis underlying these features has been revealed. It has also been shown that FliL, and the protein MotF, mainly found in several species of the family Rhodobacteraceae, contribute to remodel the amphipathic region of MotB, known as the plug, in order to allow flagellar rotation. In the absence of the plug region of MotB, FliL and MotF are dispensable. In this review we have covered the most relevant aspects of the Fla1 flagellum of this remarkable photosynthetic bacterium.

Cell motility in Alphaproteobacteria is frequently controlled by the CckA, ChpT, and CtrA two-component system. Under the growth conditions commonly used in the laboratory, ctrA is transcriptionally inactive in Rhodobacter sphaeroides , and motility depends on the Fla1 flagellar system that was acquired by a horizontal transfer event. Likely, the incorporation of this flagellar system released CtrA from the strong selective pressure of being the main motility regulator, allowing this two-component system to specialize and respond to some specific conditions. Identifying the genes that are directly regulated by CtrA could help us understand the conditions in which the products of this regulon are required. Massive parallel transcriptome sequencing (RNA-seq) revealed that CtrA orchestrates an adaptive response that contributes to the colonization of a particular environmental niche.

OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In Caulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed. IMPORTANCE Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli, suggesting a different organization of the outer membrane proteins.