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The understanding of bacterial gene function has been greatly enhanced by recent advancements in the deep sequencing of microbial genomes. Transposon insertion sequencing methods combines next-generation sequencing techniques with transposon mutagenesis for the exploration of the essentiality of genes under different environmental conditions. We propose a model-based method that uses regularized negative binomial regression to estimate the change in transposon insertions attributable to gene-environment changes in this genetic interaction study without transformations or uniform normalization. An empirical Bayes model for estimating the local false discovery rate combines unique and total count information to test for genes that show a statistically significant change in transposon counts. When applied to RB-TnSeq (randomized barcode transposon sequencing) and Tn-seq (transposon sequencing) libraries made in strains of Caulobacter crescentus using both total and unique count data the model was able to identify a set of conditionally beneficial or conditionally detrimental genes for each target condition that shed light on their functions and roles during various stress conditions. 

Guanosine tetra‐ and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA‐based fluorescent sensors for the live‐cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp‐specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell‐to‐cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA‐based sensors can be broadly adapted to study bacterial stringent response.

 

Guanosine tetra‐ and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA‐based fluorescent sensors for the live‐cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp‐specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell‐to‐cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA‐based sensors can be broadly adapted to study bacterial stringent response.

 

CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacteriumSinorhizobium meliloti. Loss ofcbrAresults in increased levels of CtrA as well as its phosphorylation. While many of the knownCaulobacter crescentusregulators of CtrA phosphorylation and proteolysis are phylogenetically conserved withinS. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation inC. crescentus. In order to investigate whether CtrA proteolysis occurs inS. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss ofcbrAsignificantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1^D53A). Addition of CpdR1^D53Afully suppressescbrAmutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression inS. meliloti. Importantly, thecbrAmutant symbiosis defect is also suppressed in the presence of CpdR1^D53A. Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis.

IMPORTANCEThe cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. TheSinorhizobium melilotinitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis.