Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Role of FliL in Swarmer Cell Differentiation

ABSTRACT: Proteus mirabilis is an opportunistic pathogen that causes urinary tract infections in immunocompromised patients. Flagellar motility coupled with the ability to differentiate from short swimmer cells to highly elongated swarmer cells constitute key parts of the infection process. The mechanism by which the cells transduce the signal to differentiate remains unknown, but is believed to involve inhibition of flagellar rotation. Recent work has implicated FliL as a critical component of the signal transduction process, possibly through interactions with the motor proteins. The objective in the current study was to define the molecular mechanism underlying the cellâs ability to perceive a surface and begin the differentiation process, with a specific emphasis as to the role of FliL. Thus, the deep-sequencing technology of RNA-Seq was combined with genetic analysis to further examine the role of FliL in swarmer cell differentiation. RNA-Seq analysis of wild-type swarmer cells confirmed prior reports of genes involved in the differentiation process while concomittantly identifying novel genes potentially involved in swarming (including PMI1628, PMI0714-0716, and PMI0735, PMI0737, PMI0739, and PMI0740-0734). In comparison, RNA-Seq analysis of a fliL mutant showed that nearly all genes associated with the flagellar cascade and chemotaxis are repressed. A phenotypic, genetic, and transcriptomic characterization was then performed on spontaneous revertants of the fliL mutant, in which motility was restored yet still resulted in cells that differentiated under non-inducing (i.e. broth) conditions. Sequencing revealed perturbations in the C-terminus of FliL in both the mutant and revertant. Collectively, the data suggest that FliL senses the torque applied to the motor proteins- such as is encountered by a solid surface - and relays this signal to the master flagellar regulator FlhD4C2, triggering the expression of genes involved in the flagellar cascade. Transcriptomic comparison between wild-type Proteus mirabilis swarmer cells and a fliL mutant via RNA-Seq.

ORGANISM(S): Proteus mirabilis

SUBMITTER: Kathleen Cusick

PROVIDER: E-GEOD-30810 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation.

Cusick Kathleen K Lee Yi-Ying YY Youchak Brian B Belas Robert R

Journal of bacteriology 20111111 2

Proteus mirabilis is a dimorphic, motile bacterium often associated with urinary tract infections. Colonization of urinary tract surfaces is aided by swarmer cell differentiation, which is initiated by inhibition of flagellar rotation when the bacteria first contact a surface. Mutations in fliL, encoding a flagellar structural protein with an enigmatic function, result in the inappropriate production of differentiated swarmer cells, called pseudoswarmer cells, under noninducing conditions, indic ...[more]

PMID: 22081397

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Project description:Proteus mirabilis is an opportunistic pathogen that causes urinary tract infections in immunocompromised patients. Flagellar motility coupled with the ability to differentiate from short swimmer cells to highly elongated swarmer cells constitute key parts of the infection process. The mechanism by which the cells transduce the signal to differentiate remains unknown, but is believed to involve inhibition of flagellar rotation. Recent work has implicated FliL as a critical component of the signal transduction process, possibly through interactions with the motor proteins. The objective in the current study was to define the molecular mechanism underlying the cellâs ability to perceive a surface and begin the differentiation process, with a specific emphasis as to the role of FliL. Thus, the deep-sequencing technology of RNA-Seq was combined with genetic analysis to further examine the role of FliL in swarmer cell differentiation. RNA-Seq analysis of wild-type swarmer cells confirmed prior reports of genes involved in the differentiation process while concomittantly identifying novel genes potentially involved in swarming (including PMI1628, PMI0714-0716, and PMI0735, PMI0737, PMI0739, and PMI0740-0734). In comparison, RNA-Seq analysis of a fliL mutant showed that nearly all genes associated with the flagellar cascade and chemotaxis are repressed. A phenotypic, genetic, and transcriptomic characterization was then performed on spontaneous revertants of the fliL mutant, in which motility was restored yet still resulted in cells that differentiated under non-inducing (i.e. broth) conditions. Sequencing revealed perturbations in the C-terminus of FliL in both the mutant and revertant. Collectively, the data suggest that FliL senses the torque applied to the motor proteins- such as is encountered by a solid surface - and relays this signal to the master flagellar regulator FlhD4C2, triggering the expression of genes involved in the flagellar cascade.

Project description:Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Consequently, cells in all domains of life have evolved mechanisms to repair DSBs, including through homologous recombination. Although recombination has been well characterized, the spatial organization of this process in living cells remains poorly understood. Here, we introduced site-specific DSBs in Caulobacter crescentus, and then used time-lapse microscopy to visualize the homology search, DSB repair, and the resegregation of chromosomal DNA. Even loci tethered to opposite cell poles can efficiently release, pair to enable recombination-based repair, and then resegregate to their original locations. Resegregation occurs independent of DNA replication and without disrupting global chromosome organization. Origin-proximal regions are resegregated by the same machinery, ParABS, used to segregate undamaged chromosomes following DNA replication. In contrast, origin-distal regions efficiently resegregate after a DSB independent of ParABS, and likely without dedicated segregation proteins. Instead, we propose that a physical, spring-like force drives the resegregation of origin-distal loci after DSB repair. Caulobacter cells were depleted of DnaA for 1.5 h before synchronization. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and double-strand breaks were induced for 1 h by the addition of 500 ?M vanillate. For control sample, no vanillate was added. Formadehyde (Sigma) was then added to the final concentration of 1%. Formadehyde crosslinks protein-DNA and DNA-DNA together, thereby capturing the structure of the chromosome at the time of fixation. Fixation was performed at the cell density of OD600 = 0.2. The crosslinking reactions were allowed to proceed for 30 minutes at 25 °C before quenching with 2.5 M glycine at a final concentration of 0.125 M. Fixed cells were then pelleted by centrifugation and subsequently washed twice with 1x M2 buffer (6.1 mM Na2HPO4, 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 ?M FeSO4, 0.5 mM CaCl2) before resuspending in 1x TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of 107 cells per µl. Resuspended cells were then divided into 25 µl aliquots and stored at -80 °C for no more than 2 weeks. Each Hi-C experiment was performed using two of the 25 µL aliquots. Chromosome conformation capture with next-generation seqeuncing (Hi-C) was carried out exactly as described previously (Le et al., 2013 PMID: 24158908)

Dataset Information

Role of FliL in Swarmer Cell Differentiation

Publications

Perturbation of FliL interferes with Proteus mirabilis swarmer cell gene expression and differentiation.

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