Project description:Caulobacter species are dimorphic Gram-negative bacteria that inhabit diverse terrestrial and aquatic ecosystems. A suite of molecular sensory systems enables these microbes to control growth, development, and reproduction in response to levels of essential elements, including carbon, nitrogen, and phosphorus. Although the enhancer binding protein NtrC and its cognate sensor histidine kinase NtrB are well-established regulators of bacterial nitrogen assimilation, their precise functions in Caulobacter metabolism and cell development remained largely undefined. Deletion of C. crescentus ntrC slowed cell growth in complex medium, while ntrB and ntrC were essential for growth when ammonium was the sole nitrogen source due to their requirement for glutamine synthase (glnA) expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring glnBA transcription, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nitrogen limitation. We further defined the NtrC regulon through transcriptomic and ChIP-seq analyses, which demonstrated the role of NtrC as both a transcriptional activator and repressor. The chromosome of C. crescentus harbors dozens of direct binding sites for NtrC, with a significant proportion located near genes involved in polysaccharide biosynthesis. Numerous NtrC binding sites align with those of GapR, an essential protein involved in chromosome organization, and MucR1, a cell cycle regulator. This observation suggests that NtrC participates in the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. These developmental defects were restored by supplementing media with glutamine or by ectopic expression of the glnBA operon. This study establishes a regulatory connection between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter.

Project description:Nitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result marine microorganisms have evolved specialized systems for responding to nitrogen stress. The highly abundant alphaproteobacterium Candidatus Pelagibacter ubique lacks the canonical GlnB, GlnD, and NtrB/NtrC genes for regulating nitrogen assimilation. A survey of 127 Alphaproteobacteria genomes found these genes to be highly represented in free-living and pathogenic organisms with large genomes and only missing in a subset of obligate intracellular organisms and other SAR11 strains. We examined global differences in mRNA and protein expression in Ca. P. ubique strain HTCC1062 during nitrogen-limited and nitrogen-replete stationary phase to understand how this thriving organism responds to nitrogen limitation. Transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT) were all elevated in nitrogen-limited cells, indicating they devote increased resources to the assimilation of nitrogenous compounds. Enzymes for assimilating amine into glutamine (GlnA) and glutamate (AspC, GltBD) were similarly up-regulated. Differential regulation of the transcriptional regulator NtrX in the two-component signaling system NtrY/NtrX was also observed, implicating it in the control of the nitrogen starvation response. Comparisons of the transcriptome and proteome suggest that Amt is post-transcriptionally repressed during nitrogen limitation, supporting previous studies that computationally identified a novel cis-acting riboswitch upstream of this gene. These observations support the conclusion that Ca. P. ubique has an unusually simple regulatory system that enables it to increase its capacity for the uptake of nitrogenous compounds in response to nitrogen limitation. Batch cultures of P. ubique were grown in a defined arificial seawater media. Three cultures were given no nitrogen amendment, and three other cultures received an excess concentration of NH3. Cultures were harvested for microarray analyses during log and stationary phase for the purpose of observing differences in gene expression related to nitrogen limitation. Proteomic analysis was conducted in parallel and is available at http://omics.pnl.gov .

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A 3´ UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria

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