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ChIP-chip of HA-tagged Lsr2 in Mycobacterium tuberculosis and Mycobacterium smegmatis

ABSTRACT: Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria.

ORGANISM(S): Mycolicibacterium smegmatis MC2 155 Mycolicibacterium smegmatis Mycobacterium tuberculosis H37Rv

PROVIDER: GSE18652 | GEO | 2009/11/15

SECONDARY ACCESSION(S): PRJNA121589

REPOSITORIES: GEO

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ChIP-chip of HA-tagged Lsr2 in Mycobacterium tuberculosis and Mycobacterium smegmatis

Project description:Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria. Comparison of Lsr2 chromatin-immunoprecipitated DNA sequences to total reference DNA

2010-06-19 | E-GEOD-18652 | biostudies-arrayexpress

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 sub-strates in Mycobacterium marinum

Project description:Raw and processed files in support of manuscript The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 sub-strates in Mycobacterium marinum The Virulence Lipid PDIM/PGL is essential for optimal protein secretion in Mycobacte-rium marinum WT, PDIM and complement strains lysate and culture filtrate analysis of proteomes

2023-12-21 | MSV000093713 | MassIVE

Protein kinase B-mediated phosphorylation controls binding of global transcriptional regulator Lsr2 to DNA in Mycobacterium tuberculosis

Project description:Two billion people are estimated to have latent tuberculosis infection associated with dormant Mycobacterium tuberculosis. Mycobacteria possess a great portfolio of regulatory proteins which that control transitions betweento dormancy and resuscitation, including the essential protein kinase B (PknB). Here, we established that depletion of PknB resulted in global alteration of the M. tuberculosis transcriptome, and identified and implicated the DNA-binding protein Lsr2 as a main transcriptional regulator likely responsible for these changes. Furthermore, we showed that Lsr2 was phosphorylated by PknB in vitro and phosphorylation of Lsr2 on threonine 112 was important for M. tuberculosis growth and survival in the Wayne non-replicating persistence model. Our fluorescence anisotropy and electromobility shift assays revealed demonstrated that phosphorylation prevented reduced affinity of Lsr2 binding to DNA, and ChHIP-sequencing experiments confirmed increased DNA binding by of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. According to our structural modelling studies revealed that phosphomimetic T112D mutation (T112D) to Lsr2 resulted in increased dynamics of the C-terminal part and a shift of the DNA binding loop of in the Lsr2 DNA binding domain, which disrupted prevented the DNA-protein interactions. Our findings suggest that PknB- mediated phosphorylation of Lsr2 is necessary for fine- tuning of gene expression during M. tuberculosis growth and transition to dormancy enabling M. tuberculosis to adapt and survive to cause disease.

2019-10-02 | E-MTAB-7627 | biostudies-arrayexpress

Lsr2 and its novel paralogue mediate the adjustment of Mycobacterium smegmatis to unfavorable environmental conditions

Project description:In the present study, we sought to explore the role of Lsr2 as a regulator of transcription in the saprophytic species, M. smegmatis. We used RNA-seq to compare the global transcription profiles of Δlsr2 and wild-type M. smegmatis strains in an attempt to decode Lsr2 regulatory network under optimal conditions or during hypoxia. We show that Lsr2 binds AT-rich regions and mainly acts as a gene repressor, either directly by binding promoter regions or indirectly through DNA-loop formation.

2021-04-22 | GSE169318 | GEO

Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein

Project description:The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes. ChIP-Seq of EspR in Mtb H37Rv at mid-log phase of growth. Two independent experiments were performed. Input DNA (No IP) was used as a control.

2012-04-02 | E-GEOD-35149 | biostudies-arrayexpress

Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein

2012-04-02 | GSE35149 | GEO

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus [ChIP-seq]

Project description:Mycobacterium abscessus (Mabs) is a fast-growing, non-tuberculous mycobacterium responsible for serious lung infections in patients with cystic fibrosis. This bacterium evolves during the pulmonary infection between a smooth (S) and a rough morphotype (R). We have previously isolated the lsr2 gene as being differentially expressed during the transition between Mabs-S and Mabs-R. lsr2 encodes a pleiotropic transcription factor belonging to the superfamily of nucleoid-associated proteins (NAPs), which play an essential role in the hierarchical organization of bacterial chromosomes. The present study aims to unravel the molecular role of Lsr2 in regulating expression of gene involved in the adaptation and the pathobiology of Mabs using both RNA-Seq and ChIP-seq.

2023-11-30 | GSE239870 | GEO

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus [RNA-seq]

2023-11-30 | GSE239869 | GEO

Proteomic survey of the Streptomyces coelicolor nucleoid

Project description:Nucleoid-associated proteins (NAPs) are small, highly abundant regulators with low sequence specificity and pleiotropic mutant phenotypes. They are involved in transcriptional and post-transcriptional control of gene expression, DNA protection/repair and nucleoid structuring. Discovery of the major NAPs has been largely haphazard and based with the Enterobacteriaceae, although some Actinomycete-specific NAPs (Mdp1, Lsr2, sIHF) are known. Here, we use LC-MS/MS to systematically search for novel NAPs in isolated nucleoids of the model actinomycete Streptomyces coelicolor. Based on the criteria of high abundance (emPAI) and predicted DNA-binding ability (DNAbinder) we identified a set of around twenty proteins with a good probability of being NAPs. The approach was apparently successful as the set included known major NAPs HupA, sIHF and Lsr2 as well as the global regulators BldD and CRP. It also included a number of proteins whose functions are not yet known (SCO0204, SCO3013, SCO4232, SCO4199 and SCO1839) which provide a useful set of candidates for further study.

2013-04-24 | PRD000736 | Pride

Mycobacterium tuberculosis Lsr2 is a global transcriptional regulator required for adaptation to changing oxygen levels and virulence

Project description:To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges from reactive oxygen and nitrogen stress, to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis ?lsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was as equally susceptible as the wild-type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but was not required for growth under low oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during, as well as a lag in recovery from, anaerobiosis. Gene expression profiling of ?lsr2 under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicate that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The ?lsr2 strain was capable of establishing an early infection in the Balb/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. Wild type H37Rv or lsr2 mutant strains were grown aerobically or in a Rach dormancy model in dubos tween albumin media and analyzed. Alternately, either aerobically grown or Rach model hypoxic samples were challenged with H2O2 samples and harvested 1h later. Experiments were repeated in triplicate or quadruplicate.

2014-05-24 | E-GEOD-57948 | biostudies-arrayexpress

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