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Bacteria are capable of evolving and diversifying very rapidly by acquiring new genetic material via horizontal gene transfer (HGT). Transformation is a widespread mechanism of HGT, which results from the capture of extracellular DNA by surface-exposed pili belonging to the type 4 filament (T4F) superfamily. How T4F—composed of major and minor pilins—interact with DNA remains poorly understood, especially in monoderm species that use a unique T4F for DNA capture, known as Com pilus or T4dP. The significance of this work is in characterizing a novel mode of DNA-binding by showing that the interface between two minor pilins, part of a tip-located complex of four pilins—found in different T4F—has been functionalized in monoderms to capture DNA. This is an evolutionary mechanism promoting the exceptional functional versatility of T4F.

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. 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.

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. Here, Anger et al. report the structure of the type 4 pilus (T4P) from Streptococcus sanguinis . They show that the T4P architecture seen in diderm bacteria – where the N-terminal α-helices of pilin subunits are partially unfolded upon polymerisation – is conserved in distant monoderm species.

Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions that permit PFOR to stay in its active state.

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 compartmentalization of respiratory components in bacterial cell growth. Respiration 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 maximization 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 compartmentalization of respiratory components in bacterial cell growth.

In piezophilic microorganisms, enzymes are optimized to perform under high hydrostatic pressure. The two major reported mechanisms responsible for such adaptation in bacterial species are changes in amino acids in the protein structure, favoring their activity and stability under high-pressure conditions, and the possible accumulation of micromolecular co-solutes in the cytoplasm. Recently, the accumulation of glutamate in the cytoplasm of piezophilic Desulfovibrio species has been reported under high-pressure growth conditions. In this study, analysis of the effect of glutamate on the enzymatic activity of the thioredoxin reductase/thioredoxin enzymatic complex of either a piezosensitive or a piezophilic microorganism confirms its role as a protective co-solute. Analysis of the thioredoxin structures suggests an adaptation both to the presence of glutamate and to high hydrostatic pressure in the enzyme from the piezophilic strain. Indeed, the presence of large surface pockets could counterbalance the overall compression that occurs at high hydrostatic pressure to maintain enzymatic activity. A lower isoelectric point and a greater dipolar moment than that of thioredoxin from the piezosensitive strain would allow the protein from the piezophilic strain to compensate for the presence of the charged amino acid glutamate to interact with its partner.

Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single ( Delta bd, Delta cox) and double deletion ( Delta coxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Delta bd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Delta bd and, to a lesser extent, Delta cox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions that permit PFOR to stay in its active state.

Thioredoxins 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 ^sup 1^H, ^sup 13^C and ^sup 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 ^sup 15^N-, ^sup 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).[PUBLICATION ABSTRACT]

Thioredoxins 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).

Thioredoxins 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 super(1)H, super(13)C and super(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 super(15)N-, super(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).