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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.

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus , a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle–regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5′ untranslated region (5′ UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation ( ctrA ) or sequestration ( gcrA ) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti , representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales .

Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro . Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named Mrp ORP associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length Mrp ORP , as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the Mrp ORP can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.

Transcriptional activation of σ(54)-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ(54)-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ(54) factor is the nexus of a large regulatory network. Understanding the key players of σ(54)-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ(54) alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n -alkenes, was shown to oxidize saturated hydrocarbons ( n -alkanes in the range C 10 –C 21 ) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n -hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n -alkane (C 16 ) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.

Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro. Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named Mrp associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length Mrp , as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the Mrp can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.