A new type of phasin characterized by the presence of a helix-hairpin-helix domain is required for normal polyhydroxybutyrate accumulation and granule organization in Caulobacter crescentus.

Bacteria frequently store excess carbon in hydrophobic granules of polyhydroxybutyrate (PHB) that in some growth conditions can occupy most of the cytoplasmic space. Different types of proteins associate to the surface of the granules, mainly enzymes involved in the synthesis and utilization of the reserve polymer and a diverse group of proteins known as phasins. Phasins have different functions, among which are regulating the size and number of the granules, modulating the activity of the granule-associated enzymes and helping in the distribution of the granules inside the cell. Caulobacter crescentus is an oligotrophic bacterium that shows several morphological and regulatory traits that allow it to grow in very nutrient-diluted environments. Under these conditions, storage compounds should be particularly relevant for survival. In this work, we show an initial proteomic characterization of the PHB granules and describe a new type of phasin (PhaH) characterized by the presence of an N-terminal hydrophobic helix followed by a helix-hairpin-helix (HhH) domain. The hydrophobic helix is required for maximal PHB accumulation and maintenance during the stationary phase while the HhH domain is involved in determining the size of the PHB granules and their distribution in the cell.

Bacterial cell wall quantification by a modified low-volume Nelson-Somogyi method and its use with different sugars.

The study of peptidoglycan-binding proteins frequently requires in vitro binding assays, in which the isolated peptidoglycan used as a substrate must be carefully quantified. Here, we describe an easy and sensitive assay for peptidoglycan quantification based on a modified Nelson-Somogyi reducing sugar assay. We report the response of this assay to different common sugars and adapt its use to peptidoglycan samples subjected to acid hydrolysis. This method showed better sensitivity than the peptidoglycan quantification method based on the acid detection of diaminopimelic acid. The method described in this work, besides being valuable in the characterization of peptidoglycan-binding proteins, is also useful for the quantification of reducing monosaccharides or polysaccharides after acid or hydrolysis.

Modulation of the Enzymatic Activity of the Flagellar Lytic Transglycosylase SltF by Rod Components and the Scaffolding Protein FlgJ in Rhodobacter sphaeroides.

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylase 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. Here, 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 toward the flagellar rod. All of 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.

The periplasmic component of the DctPQM TRAP-transporter is part of the DctS/DctR sensory pathway in Rhodobacter sphaeroides.

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.

The CtrA Regulon of Rhodobacter sphaeroides Favors Adaptation to a Particular Lifestyle.

Activation of the two-component system formed by CckA, ChpT, and CtrA (kinase, phosphotransferase, and response regulator, respectively) in Rhodobacter sphaeroides does not occur under the growth conditions commonly used in the laboratory. However, it is possible to isolate a gain-of-function mutant in CckA that turns the system on. Using massive parallel transcriptome sequencing (RNA-seq), we identified 321 genes that are differentially regulated by CtrA. From these genes, 239 were positively controlled and 82 were negatively regulated. Genes encoding the Fla2 polar flagella and gas vesicle proteins are strongly activated by CtrA. Genes involved in stress responses as well as several transcriptional factors are also positively controlled, whereas the photosynthetic and CO2 fixation genes are repressed. Potential CtrA-binding sites were bioinformatically identified, leading to the proposal that at least 81 genes comprise the direct regulon. Based on our results, we ponder that the transcriptional response orchestrated by CtrA enables a lifestyle in which R. sphaeroides will effectively populate the surface layer of a water body enabled by gas vesicles and will remain responsive to chemotactic stimuli using the chemosensoring system that controls the Fla2 flagellum. Simultaneously, fine-tuning of photosynthesis and stress responses will reduce the damage caused by heat and high light intensity in this water stratum. In summary, in this bacterium CtrA has evolved to control physiological responses that allow its adaptation to a particular lifestyle instead of controlling the cell cycle as occurs in other species.IMPORTANCE 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.

Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms.

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 ß-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane ß-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 ß-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 ß-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.

Characterization of FlgP, an Essential Protein for Flagellar Assembly in Rhodobacter sphaeroides.

The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

Biochemical and Phylogenetic Study of SltF, a Flagellar Lytic Transglycosylase from Rhodobacter sphaeroides.

In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.

Architecture of divergent flagellar promoters controlled by CtrA in Rhodobacter sphaeroides.

BACKGROUND: Rhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several α-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent promoters located a few base pairs apart, suggesting a particular arrangement of the cis-acting sites. RESULTS: Here we characterized several control regions of the R. sphaeroides fla2 genes and analyzed in detail two regions containing the divergent promoters flgB2p-fliI2p, and fliL2p-fliF2p. Binding sites for CtrA of these promoters were identified in silico and tested by site directed mutagenesis. We conclude that each one of these promoter regions has a particular arrangement, either a single CtrA binding site for activation of fliL2p and fliF2p, or two independent sites for activation of flgB2p and fliI2p. ChIP experiments confirmed that CtrA binds to the control region containing the flgB2 and fliI2 promoters, supporting the notion that CtrA directly controls the expression of the fla2 genes. The flgB and fliI genes are syntenic and show a short intercistronic region in closely related bacterial species. We analyzed these regions and found that the arrangement of the CtrA binding sites varies considerably. CONCLUSIONS: The results in this work reveal the arrangement of the fla2 divergent promoters showing that CtrA promotes transcriptional activation using more than a single architecture.

A New Essential Cell Division Protein in Caulobacter crescentus.

Bacterial cell division is a complex process that relies on a multiprotein complex composed of a core of widely conserved and generally essential proteins and on accessory proteins that vary in number and identity in different bacteria. The assembly of this complex and, particularly, the initiation of constriction are regulated processes that have come under intensive study. In this work, we characterize the function of DipI, a protein conserved in Alphaproteobacteria and Betaproteobacteria that is essential in Caulobacter crescentus Our results show that DipI is a periplasmic protein that is recruited late to the division site and that it is required for the initiation of constriction. The recruitment of the conserved cell division proteins is not affected by the absence of DipI, but localization of DipI to the division site occurs only after a mature divisome has formed. Yeast two-hybrid analysis showed that DipI strongly interacts with the FtsQLB complex, which has been recently implicated in regulating constriction initiation. A possible role of DipI in this process is discussed.IMPORTANCE Bacterial cell division is a complex process for which most bacterial cells assemble a multiprotein complex that consists of conserved proteins and of accessory proteins that differ among bacterial groups. In this work, we describe a new cell division protein (DipI) present only in a group of bacteria but essential in Caulobacter crescentus Cells devoid of DipI cannot constrict. Although a mature divisome is required for DipI recruitment, DipI is not needed for recruiting other division proteins. These results, together with the interaction of DipI with a protein complex that has been suggested to regulate cell wall synthesis during division, suggest that DipI may be part of the regulatory mechanism that controls constriction initiation.

The Master Regulators of the Fla1 and Fla2 Flagella of Rhodobacter sphaeroides Control the Expression of Their Cognate CheY Proteins.

Rhodobacter sphaeroides is an alphaproteobacterium that has two complete sets of flagellar genes. The fla1 set was acquired by horizontal transfer from an ancestral gammaproteobacterium and is the only set of flagellar genes that is expressed during growth under standard laboratory conditions. The products of these genes assemble a single, subpolar flagellum. In the absence of the Fla1 flagellum, a gain-of-function mutation in the histidine kinase CckA turns on the expression of the fla2 flagellar genes through the response regulator CtrA. The rotation of the Fla1 and Fla2 flagella is controlled by different sets of chemotaxis proteins. Here, we show that the expression of the chemotaxis proteins that control Fla2, along with the expression of the fla2 genes, is coordinated by CtrA, whereas the expression of the chemotaxis genes that control Fla1 is mediated by the master regulators of the Fla1 system. The coordinated expression of the chemosensory proteins with their cognate flagellar genes highlights the relevance of integrating the expression of the horizontally acquired fla1 genes with a chemosensory system independently of the regulatory proteins responsible for the expression of fla2 and its cognate chemosensory system. IMPORTANCE Gene acquisition via horizontal transfer represents a challenge to the recipient organism to adjust its metabolic and genetic networks to incorporate the new material in a way that represents an adaptive advantage. In the case of Rhodobacter sphaeroides, a complete set of flagellar genes was acquired and successfully coordinated with the native flagellar system. Here we show that the expression of the chemosensory proteins that control flagellar rotation is dependent on the master regulators of their corresponding flagellar system, minimizing the use of transcription factors required to express the native and horizontally acquired genes along with their chemotaxis proteins.

Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions.

UNLABELLED: The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCE: Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled.

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides.

UNLABELLED: Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 µm and a diameter of 1.4 µm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE: R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1(-) mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism.

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.

Induction of the lateral flagellar system of Vibrio shilonii is an early event after inhibition of the sodium ion flux in the polar flagellum.

In this study, we show the induction of lateral flagella by the action of the sodium channel blocker phenamil, in the marine bacterium Vibrio shilonii, a coral pathogen that causes bleaching. We analyzed the growth and morphology of cells treated with phenamil. A time course analysis showed that after 30 min of exposure to the sodium channel blocker, lateral flagella were present and could be detected by electron microscopy. Detection of the mRNA of the master regulator (lafK) and lateral flagellin (lafA) by RT-PCR confirmed the expression of lateral flagellar genes. We show the simultaneous isolation of polar and, for the first time, lateral flagellar hook-basal bodies. This allowed us to compare the dimensions and morphological characteristics of the 2 structures.

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.

A distant homologue of the FlgT protein interacts with MotB and FliL and is essential for flagellar rotation in Rhodobacter sphaeroides.

In this work, we describe a periplasmic protein that is essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded upstream of flgA, and its expression is dependent on the flagellar master regulator FleQ and on the class III flagellar activator FleT. Sequence comparisons suggest that this protein is a distant homologue of FlgT. We show evidence that in R. sphaeroides, FlgT interacts with the periplasmic regions of MotB and FliL and with the flagellar protein MotF, which was recently characterized as a membrane component of the flagellum in this bacterium. In addition, the localization of green fluorescent protein (GFP)-MotF is completely dependent on FlgT. The Mot(-) phenotype of flgT cells was weakly suppressed by point mutants of MotB that presumably keep the proton channel open and efficiently suppress the Mot(-) phenotype of motF and fliL cells, indicating that FlgT could play an additional role beyond the opening of the proton channel. The presence of FlgT in purified filament-hook-basal bodies of the wild-type strain was confirmed by Western blotting, and the observation of these structures under an electron microscope showed that the basal bodies from flgT cells had lost the ring that covers the LP ring in the wild-type structure. Moreover, MotF was detected by immunoblotting in the basal bodies obtained from the wild-type strain but not from flgT cells. From these results, we suggest that FlgT forms a ring around the LP ring, which anchors MotF and stabilizes the stator complex of the flagellar motor.

A novel component of the Rhodobacter sphaeroides Fla1 flagellum is essential for motor rotation.

Here we describe a novel component essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded by motF (RSP_0067), the first gene of a predicted transcriptional unit which contains two hypothetical genes. Sequence analysis indicated that MotF is a bitopic membrane-spanning protein. Protease sensitivity assays and green fluorescent protein (GFP) fusions confirmed this prediction and allowed us to conclude that the C terminus of MotF is located in the periplasmic space. Wild-type cells expressing a functional GFP-MotF fusion show a single fluorescent focus per cell. The localization of this protein in different genetic backgrounds allowed us to determine that normal localization of MotF depends on the presence of FliL and MotB. Characterization of a ΔmotF pseudorevertant strain revealed that a single nucleotide change in motB suppresses the Mot(-) phenotype of the motF mutant. Additionally, we show that MotF also becomes dispensable when other mutant alleles of motB previously isolated as second-site suppressors of ΔfliL were expressed in the motF mutant strain. These results show that MotF is a new component of the Fla1 flagellum, which together with FliL is required to promote flagellar rotation, possibly through MotB.

The C terminus of the flagellar muramidase SltF modulates the interaction with FlgJ in Rhodobacter sphaeroides.

Macromolecular structures such as the bacterial flagellum in Gram-negative bacteria must traverse the cell wall. Lytic transglycosylases are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. We have previously shown that in Rhodobacter sphaeroides SltF, the flagellar muramidase, and FlgJ, a flagellar scaffold protein, are separate entities that interact in the periplasm. In this study we show that the export of SltF to the periplasm is dependent on the SecA pathway. A deletion analysis of the C-terminal portion of SltF shows that this region is required for SltF-SltF interaction. These C terminus-truncated mutants lose the capacity to interact with themselves and also bind FlgJ with higher affinity than does the wild-type protein. We propose that this region modulates the interaction with the scaffold protein FlgJ during the assembly process.

Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing.

Acinetobacter baumannii is a common pathogen whose recent resistance to drugs has emerged as a major health problem. Ethanol has been found to increase the virulence of A. baumannii in Dictyostelium discoideum and Caenorhabditis elegans models of infection. To better understand the causes of this effect, we examined the transcriptional profile of A. baumannii grown in the presence or absence of ethanol using RNA-Seq. Using the Illumina/Solexa platform, a total of 43,453,960 reads (35 nt) were obtained, of which 3,596,474 mapped uniquely to the genome. Our analysis revealed that ethanol induces the expression of 49 genes that belong to different functional categories. A strong induction was observed for genes encoding metabolic enzymes, indicating that ethanol is efficiently assimilated. In addition, we detected the induction of genes encoding stress proteins, including upsA, hsp90, groEL and lon as well as permeases, efflux pumps and a secreted phospholipase C. In stationary phase, ethanol strongly induced several genes involved with iron assimilation and a high-affinity phosphate transport system, indicating that A. baumannii makes a better use of the iron and phosphate resources in the medium when ethanol is used as a carbon source. To evaluate the role of phospholipase C (Plc1) in virulence, we generated and analyzed a deletion mutant for plc1. This strain exhibits a modest, but reproducible, reduction in the cytotoxic effect caused by A. baumannii on epithelial cells, suggesting that phospholipase C is important for virulence. Overall, our results indicate the power of applying RNA-Seq to identify key modulators of bacterial pathogenesis. We suggest that the effect of ethanol on the virulence of A. baumannii is multifactorial and includes a general stress response and other specific components such as phospholipase C.