Domain Structure of Virulence-associated Response Regulator PhoP of Mycobacterium tuberculosis: ROLE OF THE LINKER REGION IN REGULATOR-PROMOTER INTERACTION(S)

The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met¹ to Arg¹³⁸ of PhoP constitute the minimal functional PhoPN, we identified Arg¹⁵⁰ as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.     

The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS

The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties.     

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.     

A Novel Pax-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.     

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg²⁺-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.     

Structural Basis for Interaction between Mycobacterium smegmatis Ms6564, a TetR Family Master Regulator,and Its Target DNA

Master regulators, which broadly affect expression of diverse genes, play critical roles in bacterial growth and environmental adaptation. However, the underlying mechanism by which such regulators interact with their cognate DNA remains to be elucidated. In this study, we solved the crystal structure of a broad regulator Ms6564 in Mycobacterium smegmatis and its protein-operator complex at resolutions of 1.9 and 2.5 Å, respectively. Similar to other typical TetR family regulators, two dimeric Ms6564 molecules were found to bind to opposite sides of target DNA. However, the recognition helix of Ms6564 inserted only slightly into the DNA major groove. Unexpectedly, 11 disordered water molecules bridged the interface of TetR family regulator DNA. Although the DNA was deformed upon Ms6564 binding, it still retained the conformation of B-form DNA. Within the DNA-binding domain of Ms6564, only two amino acids residues directly interacted with the bases of cognate DNA. Lys-47 was found to be essential for the specific DNA binding ability of Ms6564. These data indicate that Ms6564 can bind DNA with strong affinity but makes flexible contacts with DNA. Our study suggests that Ms6564 might slide more easily along the genomic DNA and extensively regulate the expression of diverse genes in M. smegmatis.     

Novel Polymorphisms of Nuclear Receptor SHP Associated with Functional and Structural Changes

We identified three heterozygous nonsynonymous single nucleotide polymorphisms in the small heterodimer partner (SHP, NROB2) gene in normal subjects and CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)-like patients, including two novel missense mutations (p.R38H, p.K170N) and one of the previously reported polymorphism (p.G171A). Four novel heterozygous mutations were also identified in the intron (^Intron1265T→A), 3′-untranslated region (^3′-UTR101C→G, ^3′-UTR186T→C), and promoter (^Pro-423C→T) of the SHP gene. The exonic R38H and K170N mutants exhibited impaired nuclear translocation. K170N made SHP more susceptible to ubiquitination mediated degradation and blocked SHP acetylation, which displayed lost repressive activity on its interacting partners ERRγ and HNF4α but not LRH-1. In contrast, G171A increased SHP mRNA and protein expression and maintained normal function. In general, the interaction of SHP mutants with LRH-1 and EID1 was enhanced. K170N also markedly impaired the recruitment of SHP, HNF4α, HDAC1, and HDAC3 to the apoCIII promoter. Molecular dynamics simulations of SHP showed that G171A stabilized the nuclear receptor boxes, whereas K170N promoted the conformational destabilization of all the structural elements of the receptor. This study suggests that genetic variations in SHP are common among human subjects and the Lys-170 residue plays a key role in controlling SHP ubiquitination and acetylation associated with SHP protein stability and repressive function.