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Background: More than 25% of Mtb proteins have unknown functions, limiting our understanding of Mtb physiology and pathogenesis. Results: The hypothetical Mtb protein Rv2179c is a new DEDD-type exoribonuclease. Conclusion: Rv2179c is the founding member of a new exoribonuclease family with broad phylogenetic distribution. Significance: Assigning RNase function to Rv2179c provides annotation for hundreds of bacterial orthologs and provides a new view of RNA processing in Mtb.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences. The TA complex VapBC29 is auto-regulatory and interacts with inverted repeat sequences in the vapBC29 promoter, whereas complexes VapBC1 and VapBC27 display no auto-regulatory properties. The difference in regulation could be due to the different properties of the VapB proteins, all of which belong to different VapB protein families. Regulation of the vapBC29 operon is specific, no cross-talk among Type II TA systems was observed. VapC29 is bacteriostatic when expressed in Mycobacterium smegmatis, whereas VapC1 and VapC27 displayed no toxicity upon expression in M. smegmatis. The shared sequence specificity of the five VapC proteins characterised is intriguing, we propose that the differences observed in regulation and toxicity is the key to understanding the role of these TA systems in the growth and persistence of Mtb.

PhoH2 proteins are found in a very diverse range of microorganisms that span bacteria and archaea. These proteins are composed of two domains: an N‐terminal PIN‐domain fused with a C‐terminal PhoH domain. Collectively this fusion functions as an RNA helicase and ribonuclease. In other genomic contexts, PINdomains and PhoHdomains are separate but adjacent suggesting association to achieve similar function. Exclusively among the mycobacteria, PhoH2 proteins are encoded in the genome with an upstream gene, phoAT, which is thought to play the role of an antitoxin (in place of the traditional VapB antitoxin that lies upstream of the 47 other PINdomains in the mycobacterial genome). This review examines PhoH2 proteins as a whole and describes the bioinformatics, biochemical, structural, and biological properties of the two domains that make up PhoH2: PIN and PhoH. We review the transcriptional regulators of phoH2 from two mycobacterial species and speculate on the function of PhoH2 proteins in the context of a Type II toxin–antitoxin system which are thought to play a role in the stress response in bacteria.

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate-and Mg 2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P2 1 , with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and R r.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were R work = 0.186 (0.271 in the highest resolution bin) and R free = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.

An ancestral bacterial pyruvate decarboxylase (with an inferred age of 1248 million years) was reconstructed through ancestral sequence reconstruction, synthesized and recombinantly expressed in E. coli. The enzyme is fully functional and its crystal structure was elucidated to 3.5 Å resolution.

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