N-methyl-bacillithiol, a Novel Thiol from Anaerobic Bacteria

ABSTRACTJ. Hiras, S. V. Sharma, V. Raman, R. A. J. Tinson, et al. (mBio 9:e01603-18, 2018, https://doi.org/10.1128/mBio.01603-18) report on the identification of a novel thiol, N-methyl-bacillithiol (N-Me-BSH), in the green sulfur bacteriumChlorobium tepidum. In N-methyl-bacillithiol, the amine of the cysteine is methylated by a novelS-adenosylmethioneine transferase designated N-methyl-bacillithiol synthase A (NmbA). The Hiras et al. study is significant because it is the first report of the presence of N-Me-BSH in anaerobic bacteria.

Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein

The LRR (leucine-rich repeat)–Roc (Ras of complex proteins)–COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer–monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5′-diphosphate)-bound states and monomeric in the GTP (guanosine-5′-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR–Roc–COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the ‘Roc dimerization loop’. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

Pteridine glycosyltransferase from Chlorobium tepidum: crystallization and X-ray analysis

The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2′-yl)-β-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Å resolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c = 105.92 Å, β = 120.5°.

The LRR-Roc-COR module of theChlorobium tepidumRoco protein: crystallization and X-ray crystallographic analysis

Roco proteins are characterized by the presence of a Roc-COR supradomain harbouring GTPase activity, which is often preceded by an LRR domain. The most notorious member of the Roco protein family is the Parkinson's disease-associated LRRK2. The Roco protein from the bacteriumChlorobium tepidumhas been used as a model system to investigate the structure and mechanism of this class of enzymes. Here, the crystallization and crystallographic analysis of the LRR-Roc-COR construct of theC. tepidumRoco protein is reported. The LRR-Roc-COR crystals belonged to space groupP212121, with unit-cell parametersa= 95.6,b= 129.8,c= 179.5 Å, α = β = γ = 90°, and diffracted to a resolution of 3.3 Å. Based on the calculated Matthews coefficient, Patterson map analysis and an initial molecular-replacement analysis, one protein dimer is present in the asymmetric unit. The crystal structure of this protein will provide valuable insights into the interaction between the Roc-COR and LRR domains within Roco proteins.

The Diversity of Sulfide Oxidation and Sulfate Reduction Genes Expressed by the Bacterial Communities of the Cariaco Basin, Venezuela

Qualitative expression of dissimilative sulfite reductase (dsrA), a key gene in sulfate reduction, and sulfide:quinone oxidoreductase (sqr), a key gene in sulfide oxidation was investigated. Neither of the two could be amplified from mRNA retrieved with Niskin bottles but were amplified from mRNA retrieved by the Deep SID. Thesqrandsqr-like genes retrieved from the Cariaco Basin were related to thesqrgenes from aBradyrhizobiumsp.,Methylomicrobium alcaliphilum,Sulfurovumsp. NBC37-1,Sulfurimonas autotrophica, Thiorhodospira sibiricaandChlorobium tepidum. ThedsrAgene sequences obtained from the redoxcline of the Cariaco Basin belonged to chemoorganotrophic and chemoautotrophic sulfate and sulfur reducers belonging to the class Deltaproteobacteria (phylum Proteobacteria) and the order Clostridiales (phylum Firmicutes).