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The Conserved Polarity Factor PodJ1 Impacts Multiple Cell Envelope-Associated Functions in Sinorhizobium meliloti

ABSTRACT: Although diminutive in size, bacteria possess highly diverse and spatially confined cellular structures. Two related alpha-proteobacteria, Sinorhizobium meliloti and Caulobacter crescentus, serve as models for investigating the genetic basis of morphologic variations. S. meliloti, a symbiont of leguminous plants, synthesizes multiple flagella and no prosthecae, whereas C. crescentus, a freshwater bacterium, has a single polar flagellum and stalk. The podJ gene, originally identified in C. crescentus for its role in polar organelle development, is split into two juxtaposed open reading frames, podJ1 and podJ2, in S. meliloti. Deletion of podJ1 interferes with flagellar motility, exopolysaccharide production, cell envelope integrity, cell division, and normal morphology, but not symbiosis. As in C. crescentus, the S. meliloti PodJ1 protein appears to act as a polarity beacon and localizes to the newer cell pole. Microarray analysis indicates that podJ1 affects the expression of at least 129 genes, the majority of which correspond to observed mutant phenotypes. The combination of phenotypic characterization, microarray analysis, and suppressor identification suggests that PodJ1 controls a core set of conserved elements, including flagellar and pili genes, the signaling proteins PleC and DivK, and the TacA transcriptional activator, while alternate downstream targets have evolved to suit the distinct lifestyles of individual species.

ORGANISM(S): Medicago truncatula Sinorhizobium meliloti

PROVIDER: GSE31325 | GEO | 2012/08/10

SECONDARY ACCESSION(S): PRJNA146015

REPOSITORIES: GEO

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Project description:Alphaproteobacteria stand out for their complex cell cycles, which are often regulated by the DivJ/PleC-DivK-DivL-CckA-ChpT-CtrA pathway. DivJ and PleC set up the polarity of the cell, thereby eventually leading to differential activation of the DNA-binding response regulator CtrA in the two nascent daughter cells. CtrA regulates replication and transcription of many genes, thereby ensuring that processes such as motility and cell division take place at the appropriate cell cycle stage. The cell cycle of the stalked budding alphaproteobacterium Hyphomonas neptunium culminates in an asymmetric cell division at the stalk-bud junction. Here, we investigate the role of the pathway from DivJ and PleC down to CtrA in this recently established model organism. Even though DivJ and PleC are localized to opposite poles, suggesting they are involved in polarity establishment inH. neptunium, DivJ, PleC and the other components of the upstream pathway (DivK and PleD) are not essential for cell cycle regulation. In contrast, the downstream part of the pathway starting from DivL is essential and involved in the regulation of important functions such as replication inhibition, cell division and motility, as shown by the identification of the (direct) regulon of CtrA. The overlap between the regulons of DivJ and PleC, DivK and CtrA is only partial, demonstrating that additional factors feeding into the pathway must be present in H. neptunium. Furthermore, unlike in other alphaproteobacteria, the regulation of CtrA throughout the cell cycle does not take place at the level of CtrA abundance in H. neptunium. All in all, the DivL-CckA-ChpT-CtrA pathway plays an essential role in the regulation of the complicated cell cycle ofH. neptunium, but several proteins feeding into CtrA remain undiscovered. The in-depth analysis of CtrA regulation in this stalked budding organism leads to hypotheses that might also hold in well-established model organisms such as Caulobacter crescentus.

Project description:In α-proteobacteria, strict regulation of cell cycle progression is necessary for the specific cellular differentiation required for adaptation to diverse environmental niches. The symbiotic lifestyle of Sinorhizobium meliloti requires a drastic cellular differentiation that includes genome amplification. To achieve polyploidy, the S. meliloti cell cycle program must be altered to uncouple DNA replication from cell division. In the α-proteobacterium Caulobacter crescentus, cell cycle regulated transcription plays an important role in control of cell cycle progression but this has not been demonstrated in other α-proteobacteria. Here we describe a robust method for synchronizing cell growth that enabled the first global analysis of S. meliloti cell cycle regulated gene expression. The objective of the microarray-based cell cycle gene expression analysis was to determine which genes were transcriptionally regulated as a funciton of a cell cycle in order to understand the contribution of transcriptional regulation to the timing of cell cycle events. The microarray analysis identified 462 genes with cell cycle regulated transcripts, including several key cell cycle regulators, and genes involved in motility, attachment, and cell division. Only 28% of the 462 S. meliloti cell cycle regulated genes were also transcriptionally cell cycle regulated in C. crescentus. Furthermore, CtrA and DnaA binding motif analysis revealed little overlap between the cell cycle-dependent regulons of CtrA and DnaA in S. meliloti and C. crescentus. The predicted S. meliloti cell cycle regulon of CtrA, but not that of DnaA, was strongly conserved in more closely related α-proteobacteria with similar ecological niches as S. meliloti, suggesting that the CtrA cell cycle regulatory network may control functions of central importance to the specific lifestyles of α-proteobacteria. Gene expression levels were quantified in synchronously growing S. meliloti cultures across 8 time points and compared to gene expression levels in unsynchronized culture via a two-color custom agilent array

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The Conserved Polarity Factor PodJ1 Impacts Multiple Cell Envelope-Associated Functions in Sinorhizobium meliloti

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