RpfC (Rv1884) atomic structure shows high structural conservation within the resuscitation promoting factor catalytic domain

We report the first structure of the catalytic domain of RpfC (Rv1884), one of theresuscitation-promoting factors (RPFs) from Mycobacterium tuberculosis. The structure was solved using molecular replacement, once the space group had been correctly identified as twinned P21 rather than the apparent C2221 by searching for anomalous scattering sites in P1. The structure displays a very high degree of structural conservation with the structures of the catalytic domains of RpfB (Rv1009) and RpfE (Rv2450) already published. This structural conservation highlights the importance of the versatile domain composition of the RPF family

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis

Crystal structure of reduced MsAcg a putative nitroreductase from Mycobacterium smegmatis, and a close homologue of Mycobacterium tuberculosis Acg

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of M.tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using X-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turn out to be unusual flavin mononucleotide (FMN) binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase, such that the monomeric protein resembles a classical nitroreductase dimer, but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidised by oxygen, but without any evidence for a radical state commonly seen in oxygen sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways, as part of the bacteria's adaptation to a dormancy state