HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria.

GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.

Solution structure of the PAS domain of a thermophilic YybT protein homolog reveals a potential ligand-binding site.

The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small Per-ARNT-Sim (PAS) domain (~80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. We determined the solution structure of the PAS domain of GtYybT from Geobacillus thermodenitrificans by NMR spectroscopy to further probe its function. The solution structure confirms that PASGtYybT adopts the characteristic PAS fold composed of a five-stranded antiparallel ß sheet and a few short α-helices. One α-helix and three central ß-strands of PASGtYybT are noticeably shorter than those of the typical PAS domains. Despite the small size of the protein domain, a hydrophobic pocket is formed by the side chains of nonpolar residues stemming from the ß-strands and α-helices. A set of residues in the vicinity of the pocket and in the C-terminal region at the dimeric interface exhibits perturbed NMR parameters in the presence of heme or zinc protoporphyrin. Together, the results unveil a compact PAS domain with a potential ligand-binding pocket and reinforce the view that the PASYybT domains function as regulatory domains in the modulation of cellular cyclic di-AMP concentration.

2',3'-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening.

The recent report of 2',3'-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2',3'-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2',3'-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3'-AMP but not 2'-AMP. The catalytic efficiency (k(cat)/K(m)) of each enzyme against 2',3'-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2',3'-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3'-AMP is due to the P-O2' bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2',3'-cAMP hydrolysis is observed.

Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase.

The cyclic dinucleotide c-di-GMP is a widespread bacterial messenger molecule with potential application as a therapeutic agent for treating bacterial infection. Current enzymatic synthesis of c-di-GMP using mesophilic diguanylate cyclase (DGC) proteins suffers from low production yield due to protein instability and strong product inhibition. Here we report the overexpression and characterization of a stand-alone thermophilic diguanylate cyclase domain (tDGC) protein with enhanced thermostability. The product inhibition that severely limited production yield was significantly alleviated by mutation of a conserved residue in the putative regulatory I-site. With the mutant tDGC, we demonstrated that hundreds of milligrams of c-di-GMP can be readily prepared by using the optimized procedures for enzymatic reaction and product purification. The thermophilic enzyme will be a valuable tool for other research laboratories for c-di-GMP synthesis as well as the preparation of c-di-GMP derivatives.