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Polyphosphate Kinase Antagonizes Virulence Gene Expression in Francisella tularensis.

ABSTRACT: The alarmone ppGpp is a critical regulator of virulence gene expression in Francisella tularensis In this intracellular pathogen, ppGpp is thought to work in concert with the putative DNA-binding protein PigR and the SspA protein family members MglA and SspA to control a common set of genes. MglA and SspA form a complex that interacts with RNA polymerase (RNAP), and PigR functions by interacting with the RNAP-associated MglA-SspA complex. Prior work suggested that ppGpp indirectly exerts its regulatory effects in F. tularensis by promoting the accumulation of polyphosphate in the cell, which in turn was required for formation of the MglA-SspA complex. Here we show that in Escherichia coli, neither polyphosphate nor ppGpp is required for formation of the MglA-SspA complex but that ppGpp promotes the interaction between PigR and the MglA-SspA complex. Moreover, we show that polyphosphate kinase, the enzyme responsible for the synthesis of polyphosphate, antagonizes virulence gene expression in F. tularensis, a finding that is inconsistent with the notion that polyphosphate accumulation promotes virulence gene expression in this organism. Our findings identify polyphosphate kinase as a novel negative regulator of virulence gene expression in F. tularensis and support a model in which ppGpp exerts its positive regulatory effects by promoting the interaction between PigR and the MglA-SspA complex.IMPORTANCE In Francisella tularensis, MglA and SspA form a complex that associates with RNA polymerase to positively control the expression of key virulence genes. The MglA-SspA complex works together with the putative DNA-binding protein PigR and the alarmone ppGpp. PigR functions by interacting directly with the MglA-SspA complex, but how ppGpp exerts its effects was unclear. Prior work indicated that ppGpp acts by promoting the accumulation of polyphosphate, which is required for MglA and SspA to interact. Here we show that formation of the MglA-SspA complex does not require polyphosphate. Furthermore, we find that polyphosphate antagonizes the expression of virulence genes in F. tularensis Thus, ppGpp does not promote virulence gene expression in this organism through an effect on polyphosphate.

SUBMITTER: Rohlfing AE

PROVIDER: S-EPMC5763045 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Polyphosphate Kinase Antagonizes Virulence Gene Expression in Francisella tularensis.

Rohlfing Amy E AE Ramsey Kathryn M KM Dove Simon L SL

Journal of bacteriology 20180110 3

The alarmone ppGpp is a critical regulator of virulence gene expression in <i>Francisella tularensis</i> In this intracellular pathogen, ppGpp is thought to work in concert with the putative DNA-binding protein PigR and the SspA protein family members MglA and SspA to control a common set of genes. MglA and SspA form a complex that interacts with RNA polymerase (RNAP), and PigR functions by interacting with the RNAP-associated MglA-SspA complex. Prior work suggested that ppGpp indirectly exerts ...[more]

PMID: 29158241

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Project description:pool 16 and pool 29 arrays represent the Francisella transposon insertion mutants isolated from the spleens of two separate groups of 5 intraperitoneally infected mice. skin arrays represent the Francisella transposon insertion mutants isolated from the skin of 5 individual, subcutaneously infected mice. input arrays represent Francisella transposon insertion mutants surviving after growth in TSB broth. Detailed Materials and Methods from Weiss et al, PNAS, 2007, In vivo negative selection screen identifies genes required for Francisella virulence: Mice and Francisella infections Female C57BL/6 mice (Jackson) between 7 and 10 wk of age were kept under specific pathogen-free conditions in filter-top cages at Stanford University and provided with sterile water and food ad libitum. Experimental studies were in accordance with IACUC Guidelines. Mice were inoculated with 2x105 cfu subcutaneously in 50 ul sterile PBS or intraperitoneally (IP) in 500 ul. Spleens were harvested 2 days post-infection, homogenized, and dilutions were plated on supplemented MH agar plates containing kanamycin (infections with library) or MH plates with and without the appropriate antibiotic (competition experiments). Plates were grown overnight at 37oC, 5% CO2 and colonies were enumerated. After infections with the library, approximately 10^5 cfu were collected in sterile PBS for isolation of DNA. Competitive Index (CI) values were calculated using the formula CI = (mutant cfu in output/wild-type cfu in output)/(mutant cfu in input/wild-type cfu in input). Microarrays Genomic DNA was purified from bacterial pellets by phenol-chloroform extraction. Each DNA sample was divided in two and digested separately with BfaI or RsaI (NEB). The digested DNA was used as the template for in vitro transcription with the AmpliScribe T7-Flash transcription kit (Epicentre) following the manufacturer's protocol, except that 2 ?g of digested DNA was used, and the reaction was allowed to proceed for 12 to 16 h. Purified RNA was used in a RT reaction using Superscript II(?) (Invitrogen) and random hexamers as primers. cDNA was labeled with amino-allyl dUTP using the Klenow (exo-) enzyme. The ssDNA containing amino-allyl dUTP from the mouse output or the library input pools were labeled with Cy3 and Cy5, respectively, prior to hybridization to our Francisella microarray as described previously. Data analysis Normalized data were downloaded from the Stanford Microarray Database according to the median log2 Cy5/Cy3 (logRAT2N). Filters for feature quality, including a Cy3 net median intensity of 150 and regression correlation of >0.6, were applied. To compare data from separate IP infection experiments, each experimental sample was zero-transformed to the input/input control. Features (spots) missing values for 40% of the arrays were removed from the data set. The data sets were analyzed with the SAM program, using the two-class analysis option to identify features that consistently deviated from the input and samples across all of the arrays with a false discovery rate of 1%.

Dataset Information

Polyphosphate Kinase Antagonizes Virulence Gene Expression in Francisella tularensis.

Publications

Polyphosphate Kinase Antagonizes Virulence Gene Expression in Francisella tularensis.

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