Project description:Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Nevertheless, K+ homeostasis remains poorly studied in bacteria. Here we describe the regulation of potassium uptake systems in the oligotrophic -proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by essentially using two K+ transporters to maintain potassium homeostasis, the low affinity Kup and the high affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by improving the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABC expression, suggest that the inner membrane sensor regulatory component KdpD works as a kinase in early stages of growth and as a phosphatase to regulate transition into stationary phase. Our data also show that KdpE is not only phosphorylated by KdpD but also by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome and identified the direct targets of KdpE. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.
Project description:Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Nevertheless, K+ homeostasis remains poorly studied in bacteria. Here we describe the regulation of potassium uptake systems in the oligotrophic -proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by essentially using two K+ transporters to maintain potassium homeostasis, the low affinity Kup and the high affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by improving the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABC expression, suggest that the inner membrane sensor regulatory component KdpD works as a kinase in early stages of growth and as a phosphatase to regulate transition into stationary phase. Our data also show that KdpE is not only phosphorylated by KdpD but also by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome and identified the direct targets of KdpE. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.
Project description:Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Here we describe the regulation of potassium uptake systems in the oligotrophic alpha-proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by mainly using two K+ transporters to maintain potassium homeostasis, the low affinity Kup and the high affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by increasing the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABCDE expression, suggest that the inner membrane sensor regulatory component KdpD mainly works as a phosphatase to limit the growth when cells reach late exponential phase. Our data therefore suggest that KdpE is phosphorylated by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.
Project description:This SuperSeries is composed of the following subset Series: GSE25996: Expression data from Caulobacter crescentus starved for carbon GSE25997: Expression data from Caulobacter crescentus (syn. C. vibrioides) swarmer and stalked cells starved for carbon GSE25998: Expression data from WT, DSigT and DSigU Caulobacter crescentus (syn. C. vibrioides) starved for carbon Refer to individual Series
Project description:Investigation of whole genome gene expression level changes in a Caulobacter crescentus NA1000 dcdnL mutant, compared to the wild-type strain. In bacteria, transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Our microarray experiment demonstrates how cdnL deletion affects the transcriptome of Caulobacter crescentus.
Project description:Investigation of whole genome gene expression level changes in a Caulonacter crescentus NA1000 Plac::CCNA_00382 (ccrM) mutant, compared to the wild-type strain. The mutations engineered into this strain cause the CcrM DNA methyltransferase to be overexpressed and the chromosome to be constitutively methylated at the adenine at GANTC motifs. References of strains: CcrMOE: Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. WT: Marks, M.E., Castro-Rojas, C.M., Teiling, C., Du, L., Kapatral, V., Walunas, T.L. and Crosson, S. (2010) The genetic basis of laboratory adaptation in Caulobacter crescentus. J Bacteriol, 192, 3678-3688; Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. A six chip study using total RNA recovered from three separate wild-type cultures of Caulonacter crescentus NA1000 and three separate cultures of a triple mutant strain, Caulonacter crescentus NA1000 Plac::CCNA_00382 (ccrM), in which the ccrM gene coding for a DNA methyltransferase methylating the adenine in GANTC motifs is truncated and its product inactive. Each chip measures the expression level of 3933 genes from Caulobacter crescentus NA1000 with 3 probes per gene and with three-fold technical redundancy.
Project description:Investigation of whole genome gene expression level changes in a Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM) mutant, compared to the wild-type strain. The mutations engineered into this strain render it incapable of methylating its genome on the adenine at GANTC motifs. References for strains : WT: Marks, M.E., Castro-Rojas, C.M., Teiling, C., Du, L., Kapatral, V., Walunas, T.L. and Crosson, S. (2010) The genetic basis of laboratory adaptation in Caulobacter crescentus. J Bacteriol, 192, 3678-3688; Collier, J. and Shapiro, L. (2009) Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol, 191, 5706-5716. DccrM: Gonzalez, D. and Collier, J. (2013) DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus. Mol Microbiol, 88, 203-218. A six chip study using total RNA recovered from three separate wild-type cultures of Caulobacter crescentus NA1000 and three separate cultures of a triple mutant strain, Caulobacter crescentus NA1000 delta-CCNA_00382 (ccrM), in which the ccrM gene coding for a DNA methyltransferase methylating the adenine in GANTC motifs is truncated and its product inactive. Each chip measures the expression level of 3933 genes from Caulobacter crescentus NA1000 with 3 probes per gene and with three-fold technical redundancy.
Project description:Caulobacter crescentus is an alphaproteobacterium that divides assymetrically. Each cell cycle results in the production of a motile flagellated cell and a sessile cell called the swamer cell and the stalked cell, respectively. The flagellar filament is composed of thousands polymerized flagellins. We showed that glycosylation of flagellins is required for the assembly of the flagellum. This glycosylation is performed by soluble FlmG glycosyltransferases that transfer nonulosonic acids (pseudaminic acid or legionaminic acid) directly to the flagellins. Such glycosylation system is also present in a close relative of Caulobacter crescentus, Brevundimonas subvibrioides. The project is to identify the site of glycosylation and the potential sugar added on this site.