Systematic functional analysis of the Com pilus in Streptococcus sanguinis : a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria

International audienceABSTRACT Type 4 filaments (T4F) are a superfamily of versatile nanomachines, ubiquitous in prokaryotes, which use conserved multi-protein machineries to assemble and operate filamentous polymers of type 4 pilins. In the best-studied T4F, these machineries are complex, which has posed challenges to understanding the mechanisms of filament assembly and their multiple functions. Here, we report the systematic functional analysis of the Com pilus, a widespread T4F mediating DNA uptake during natural transformation in monoderm bacteria. Using Streptococcus sanguinis , we show that Com pili are bona fide type 4 pili (T4P), representing a new pilus sub-type. We show that with only eight components necessary for pilus assembly and functioning—all “core” proteins universally conserved in this superfamily—the Com pilus epitomizes a minimalistic T4F. We further demonstrate that core T4F components are sufficient for filament assembly. Paradoxically, akin to the more elaborate T4F, the Com pilus contains four minor pilins forming a complex, probably tip-located. Our results have global implications for T4F and make the Com pilus a model for elucidating fundamental processes underpinning filament assembly. IMPORTANCE Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F—the Com pilus that mediates DNA uptake in competent monoderm bacteria—and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly

1H, 13C and 15N chemical shift assignments of the thioredoxin from the obligate anaerobe Desulfovibrio vulgaris Hildenborough.

International audienceThioredoxins are ubiquitous key antioxidant enzymes which play an essential role in cell defense against oxidative stress. They maintain the redox homeostasis owing to the regulation of thiol-disulfide exchange. In the present paper, we report the full resonance assignments of (1)H, (13)C and (15)N atoms for the reduced and oxidized forms of Desulfovibrio vulgaris Hildenborough thioredoxin 1 (Trx1). 2D and 3D heteronuclear NMR experiments were performed using uniformly (15)N-, (13)C-labelled Trx1. Chemical shifts of 97% of the backbone and 90% of the side chain atoms were obtained for the oxidized and reduced form (BMRB deposits with accession number 17299 and 17300, respectively)

Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-ferredoxin oxidoreductase with pyruvate.

International audiencePyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4' amino substituent of the pyrimidine moiety of ThDP

The primary pathway for lactate oxidation in Desulfovibrio vulgaris

International audienceThe ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant 426-28 was highly disrupted on D-lactate, whereas the growth of mutant 432-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species

Structural and mechanistic insights into unusual thiol disulfide oxidoreductase.

International audienceCytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism

Structure of a heteropolymeric type 4 pilus from a monoderm bacterium

Abstract Type 4 pili (T4P) are important virulence factors, which belong to a superfamily of nanomachines ubiquitous in prokaryotes, called type 4 filaments (T4F). T4F are defined as helical polymers of type 4 pilins. Recent advances in cryo-electron microscopy (cryo-EM) led to structures of several T4F, revealing that the long N-terminal α-helix (α1) – the trademark of pilins – packs in the centre of the filaments to form a hydrophobic core. In diderm bacteria – all available bacterial T4F structures are from diderm species – a portion of α1 is melted (unfolded). Here we report that this architecture is conserved in phylogenetically distant monoderm species by determining the structure of Streptococcus sanguinis T4P. Our 3.7 Å resolution cryo-EM structure of S. sanguinis heteropolymeric T4P and the resulting full atomic model including all minor pilins highlight universal features of bacterial T4F and have widespread implications in understanding T4F biology

Study of the Thiol/Disulfide Redox Systems of the Anaerobe Desulfovibrio vulgaris Points Out Pyruvate:Ferredoxin Oxidoreductase as a New Target for Thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent Km value for Trx1 of 8.9 μm. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (Km for NADPH, 743 μm; Km for NADH, 5.6 μm), and Trx3 was unable to reduce insulin. The Km value of TR3 for Trx3 was 1.12 μm. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1C33S as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme

Clustering as a Means To Control Nitrate Respiration Efficiency and Toxicity in Escherichia coli

International audienceRespiration is a fundamental process that has to optimally respond to metabolic demand and environmental changes. We previously showed that nitrate respiration, crucial for gut colonization by enterobacteria, is controlled by polar clustering of the nitrate reductase increasing the electron flux through the complex. Here, we show that the formate dehydrogenase electron-donating complex, FdnGHI, also clusters at the cell poles under nitrate-respiring conditions. Its proximity to the nitrate reductase complex was confirmed by its identification in the interactome of the latter, which appears to be specific to the nitrate-respiring condition. Interestingly , we have identified a multiprotein complex dedicated to handle nitric oxide resulting from the enhanced activity of the electron transport chain terminated by nitrate reductase. We demonstrated that the cytoplasmic NADH-dependent nitrite reductase NirBD and the hybrid cluster protein Hcp are key contributors to regulation of the nitric oxide level during nitrate respiration. Thus, gathering of actors involved in respiration and NO homeostasis seems to be critical to balancing maximi-zation of electron flux and the resulting toxicity. IMPORTANCE Most bacteria rely on the redox activity of respiratory complexes embedded in the cytoplasmic membrane to gain energy in the form of ATP and of an electrochemical gradient established across the membrane. Nevertheless, production of harmful and toxic nitric oxide by actively growing bacteria as either an intermediate or side-product of nitrate respiration challenges how homeostasis control is exerted. Here, we show that components of the nitrate electron transport chain are clustered, likely influencing the kinetics of the process. Nitric oxide production from this respiratory chain is controlled and handled through a multiprotein complex, including detoxifying systems. These findings point to an essential role of compart-mentalization of respiratory components in bacterial cell growth

Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris

International audienceThioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2'-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time