Evolutionary insights into the emergence of virulent Leptospira spirochetes.

Pathogenic Leptospira are spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulent Leptospira species, to explore the key phenotypic traits and genetic determinants of Leptospira virulence. Through a comprehensive approach integrating phylogenomic comparisons with in vitro and in vivo phenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the human complement system and the adaptations to avoid activation of the innate immune cells. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution in Leptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision on Leptospira pathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.

Inter-species Transcriptomic Analysis Reveals a Constitutive Adaptation Against Oxidative Stress for the Highly Virulent Leptospira Species.

Transcriptomic analyses across large scales of evolutionary distance have great potential to shed light on regulatory evolution but are complicated by difficulties in establishing orthology and limited availability of accessible software. We introduce here a method and a graphical user interface wrapper, called Annotator-RNAtor, for performing interspecies transcriptomic analysis and studying intragenus evolution. The pipeline uses third-party software to infer homologous genes in various species and highlight differences in the expression of the core-genes. To illustrate the methodology and demonstrate its usefulness, we focus on the emergence of the highly virulent Leptospira subclade known as P1+, which includes the causative agents of leptospirosis. Here, we expand on the genomic study through the comparison of transcriptomes between species from P1+ and their related P1- counterparts (low-virulent pathogens). In doing so, we shed light on differentially expressed pathways and focused on describing a specific example of adaptation based on a differential expression of PerRA-controlled genes. We showed that P1+ species exhibit higher expression of the katE gene, a well-known virulence determinant in pathogenic Leptospira species correlated with greater tolerance to peroxide. Switching PerRA alleles between P1+ and P1- species demonstrated that the lower repression of katE and greater tolerance to peroxide in P1+ species was solely controlled by PerRA and partly caused by a PerRA amino-acid permutation. Overall, these results demonstrate the strategic fit of the methodology and its ability to decipher adaptive transcriptomic changes, not observable by comparative genome analysis, that may have been implicated in the emergence of these pathogens.

Copper management strategies in obligate bacterial symbionts: balancing cost and benefit.

Bacteria employ diverse mechanisms to manage toxic copper in their environments, and these evolutionary strategies can be divided into two main categories: accumulation and rationalization of metabolic pathways. The strategies employed depend on the bacteria's lifestyle and environmental context, optimizing the metabolic cost-benefit ratio. Environmental and opportunistically pathogenic bacteria often possess an extensive range of copper regulation systems in order to respond to variations in copper concentrations and environmental conditions, investing in diversity and/or redundancy as a safeguard against uncertainty. In contrast, obligate symbiotic bacteria, such as Neisseria gonorrhoeae and Bordetella pertussis, tend to have specialized and more parsimonious copper regulation systems designed to function in the relatively stable host environment. These evolutionary strategies maintain copper homeostasis even in challenging conditions like encounters within phagocytic cells. These examples highlight the adaptability of bacterial copper management systems, tailored to their specific lifestyles and environmental requirements, in the context of an evolutionary the trade-off between benefits and energy costs.

Phylogenomics of Leptospira santarosai, a prevalent pathogenic species in the Americas.

BACKGROUND: Leptospirosis is a complex zoonotic disease mostly caused by a group of eight pathogenic species (L. interrogans, L. borgpetersenii, L. kirschneri, L. mayottensis, L. noguchii, L. santarosai, L. weilii, L. alexanderi), with a wide spectrum of animal reservoirs and patient outcomes. Leptospira interrogans is considered as the leading causative agent of leptospirosis worldwide and it is the most studied species. However, the genomic features and phylogeography of other Leptospira pathogenic species remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the genome diversity of the main pathogenic Leptospira species based on a collection of 914 genomes from strains isolated around the world. Genome analyses revealed species-specific genome size and GC content, and an open pangenome in the pathogenic species, except for L. mayottensis. Taking advantage of a new set of genomes of L. santarosai strains isolated from patients in Costa Rica, we took a closer look at this species. L. santarosai strains are largely distributed in America, including the Caribbean islands, with over 96% of the available genomes originating from this continent. Phylogenetic analysis showed high genetic diversity within L. santarosai, and the clonal groups identified by cgMLST were strongly associated with geographical areas. Serotype identification based on serogrouping and/or analysis of the O-antigen biosynthesis gene loci further confirmed the great diversity of strains within the species. CONCLUSIONS/SIGNIFICANCE: In conclusion, we report a comprehensive genome analysis of pathogenic Leptospira species with a focus on L. santarosai. Our study sheds new light onto the genomic diversity, evolutionary history, and epidemiology of leptospirosis in America and globally. Our findings also expand our knowledge of the genes driving O-antigen diversity. In addition, our work provides a framework for understanding the virulence and spread of L. santarosai and for improving its surveillance in both humans and animals.

Adaptive Radioresistance of Enterohemorrhagic Escherichia coli O157:H7 Results in Genomic Loss of Shiga Toxin-Encoding Prophages.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen producing Shiga toxins (Stx1 and Stx2), which can cause hemorrhagic diarrhea and life-threatening infections. O157:H7 strain EDL933 carries prophages CP-933V and BP-933W, which encode Shiga toxin genes (stx1 and stx2, respectively). The aim of this work was to investigate the mechanisms of adaptive resistance of EHEC strain EDL933 to a typically lethal dose of gamma irradiation (1.5 kGy). Adaptive selection through six passages of exposure to 1.5 kGy resulted in the loss of CP-933V and BP-933W prophages from the genome and mutations within three genes: wrbA, rpoA, and Wt_02639 (molY). Three selected EHEC clones that became irradiation adapted to the 1.5-kGy dose (C1, C2, and C3) demonstrated increased resistance to oxidative stress, sensitivity to acid pH, and decreased cytotoxicity to Vero cells. To confirm that loss of prophages plays a role in increased radioresistance, clones C1 and C2 were exposed to bacteriophage-containing lysates. Although phage BP-933W could lysogenize C1, C2, and E. coli K-12 strain MG1655, it was not found to have integrated into the bacterial chromosome in C1-Φ and C2-Φ lysogens. Interestingly, for the E. coli K-12 lysogen (K-12-Φ), BP-933W DNA had integrated at the wrbA gene (K-12-Φ). Both C1-Φ and C2-Φ lysogens regained sensitivity to oxidative stress, were more effectively killed by a 1.5-kGy gamma irradiation dose, and had regained cytotoxicity and acid resistance phenotypes. Further, the K-12-Φ lysogen became cytotoxic, more sensitive to gamma irradiation and oxidative stress, and slightly more acid resistant. IMPORTANCE Gamma irradiation of food products can provide an effective means of eliminating bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, a significant foodborne pathogen that can cause severe disease due to the production of Stx. To decipher the mechanisms of adaptive resistance of the O157:H7 strain EDL933, we evolved clones of this bacterium resistant to a lethal dose of gamma irradiation by repeatedly exposing bacterial cells to irradiation following a growth restoration over six successive passages. Our findings provide evidence that adaptive selection involved modifications in the bacterial genome, including deletion of the CP-933V and BP-933W prophages. These mutations in EHEC O157:H7 resulted in loss of stx1 and stx2, loss of cytotoxicity to epithelial cells, and decreased resistance to acidity, critical virulence determinants of EHEC, concomitant with increased resistance to lethal irradiation and oxidative stress. These findings demonstrate that the potential adaptation of EHEC to high doses of radiation would involve elimination of the Stx-encoding phages and likely lead to a substantial attenuation of virulence.

Horizontal transfer of the rfb cluster in Leptospira is a genetic determinant of serovar identity.

Leptospira bacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis-encoding genes reside within the chromosomic rfb gene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of the rfb cluster strongly correlates with Leptospira serovar designation. Whole-genome sequencing of pathogenic L. noguchii, including strains of different serogroups, reveals that the rfb cluster undergoes extensive horizontal gene transfer. The rfb clusters from several Leptospira species disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing of Leptospira serovars, and to pinpointing specific genes within the distinct rfb clusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism of rfb transfer among Leptospira strains and species.

Synthesis & Evaluation of Novel Mannosylated Neoglycolipids for Liposomal Delivery System Applications.

Glycosylated NPs, including liposomes, are known to target various receptors involved in cellular carbohydrate transport, of which the mannoside binding receptors are attracting particular attention for their expression on various immune cells, cancers, and cells involved in maintaining central nervous system (CNS) integrity. As part of our interest in NP drug delivery, mannosylated glycoliposomal delivery systems formed from the self-assembly of amphiphilic neoglycolipids were developed, with a C12-alkyl mannopyranoside (ML-C12) being identified as a lead compoundcapable of entrapping, protecting, and improving the delivery of structurally diverse payloads. However, ML-C12 was not without limitations in both the synthesis of the glycolipids, and the physicochemical properties of the resulting glycoliposomes. Herein, the chemical syntheses of a novel series of mannosylated neoglycolipids are reported with the goal of further improving on the previous ML-C12 glyconanoparticles. The current work aimed to use a self-contingent strategy which overcomes previous synthetic limitations to produce neoglycolipids that have one exposed mannose residue, an aromatic scaffold, and two lipid tails with varied alkyl chains. The azido-ending carbohydrates and the carboxylic acid-ending lipid tails were ligated using a new one-pot modified Staudinger chemistry that differed advantageously to previous syntheses. The formation of stable neoglycoliposomes of controllable and ideal sizes (≈100-400 nm) was confirmed via dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM). Beyond chemical advantages, the present study further aimed to establish potential improvements in the biological activity of the neoglycoliposomes. Concanavalin A (Con A) agglutination studies demonstrated efficient and stable cross-linking abilities dependent on the length of the linkers and lipid tails. The efficacy of the glycoliposomes in improving cytosolic uptake was investigated using Nile Red as probe in immune and cancer cell lines. Preliminary ex vivo safety assessments showed that the mannosylated glycoliposomes are hemocompatible, and non-immunogenic. Finally, using a model peptide therapeutic, the relative entrapment capacity and plasma stability of the optimal glycoliposome delivery system was evaluated and compared to the previous neoglycoliposomes. Overall, the new lead glycoliposome showed improved biological activity over ML-C12, in addition to having several chemical benefits including the lack of stereocenters, a longer linker allowing better sugar availability, and ease of synthesis using novel one-pot modified Staudinger chemistry.

Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family.

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

Sequential markerless genetic manipulations of species from the Neisseria genus.

The development of simple and highly efficient strategies for genetic modifications is essential for postgenetic studies aimed at characterizing gene functions for various applications. We sought to develop a reliable system for Neisseria species that allows for both unmarked and accumulation of multiple genetic modifications in a single strain. In this work, we developed and validated three-gene cassettes named RPLK and RPCC, comprising of an antibiotic resistance marker for positive selection, the phenotypic selection marker lacZ or mCherry, and the counterselection gene rpsL. These cassettes can be transformed with high efficiency across the Neisseria genus while significantly reducing the number of false positives compared with similar approaches. We exemplified the versatility and application of these systems by obtaining unmarked luminescent strains (knock-in) or mutants (knock-out) in different pathogenic and commensal species across the Neisseria genus in addition to the cumulative deletion of six loci in a single strain of Neisseria elongata.

Leptospira ainlahdjerensis sp. nov., Leptospira ainazelensis sp. nov., Leptospira abararensis sp. nov. and Leptospira chreensis sp. nov., four new species isolated from water sources in Algeria.

Leptospira strains were isolated from freshwater sampled at four sites in Algeria and characterized by whole-genome sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The cells were spiral-shaped and motile. Phylogenetic and MALDI-TOF MS analyses showed that the strains can be clearly distinguished from the other described species in the genus Leptospira, therefore representing two novel species of the pathogen subclade P1 and two novel species of the saprophyte subclade S1. The names Leptospira ainlahdjerensis sp. nov. (type strain 201903070T=KIT0297T=CIP111912T), Leptospira ainazelensis sp. nov. (201903071T=KIT0298T=CIP111913T), Leptospira abararensis sp. nov. (201903074T=KIT0299T=CIP111914T) and Leptospira chreensis (201903075T=KIT0300T=CIP111915T) are proposed.

The oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain.

BACKGROUND: Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. METHODOLOGY/PRINCIPAL FINDINGS: We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. CONCLUSIONS/SIGNIFICANCE: The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

Bacterial glycocalyx integrity drives multicellular swarm biofilm dynamism.

Exopolysaccharide (EPS) layers on the bacterial cell surface are key determinants of biofilm establishment and maintenance, leading to the formation of higher-order 3D structures that confer numerous survival benefits to a cell community. In addition to a specific cell-associated EPS glycocalyx, we recently revealed that the social δ-proteobacterium Myxococcus xanthus secretes a novel biosurfactant polysaccharide (BPS) to the extracellular milieu. Together, secretion of the two polymers (EPS and BPS) is required for type IV pilus (T4P)-dependent swarm expansion via spatio-specific biofilm expression profiles. Thus the synergy between EPS and BPS secretion somehow modulates the multicellular lifecycle of M. xanthus. Herein, we demonstrate that BPS secretion functionally alters the EPS glycocalyx via destabilization of the latter, fundamentally changing the characteristics of the cell surface. This impacts motility behaviors at the single-cell level and the aggregative capacity of cells in groups via cell-surface EPS fibril formation as well as T4P production, stability, and positioning. These changes modulate the structure of swarm biofilms via cell layering, likely contributing to the formation of internal swarm polysaccharide architecture. Together, these data reveal the manner by which the combined secretion of two distinct polymers induces single-cell changes that modulate swarm biofilm communities.

Sodium Tetraphenylborate Displays Selective Bactericidal Activity against Neisseria meningitidis and N. gonorrhoeae and Is Effective at Reducing Bacterial Infection Load.

Neisseria meningitidis and Neisseria gonorrhoeae, two highly related species that might have emerged from a common commensal ancestor, constitute major human threats. Vaccines are available to prevent N. meningitidis infection, whereas there are only a limited number of antibiotics available for N. gonorrhoeae Unfortunately, some strains of these species are rapidly evolving and capable of escaping human interventions. Thus, it is now urgent to develop new avenues to fight these bacteria. This study reports that a boron-based salt, sodium tetraphenylborate (NaBPh4), displays high bactericidal activity and remarkable specificity against N. meningitidis and N. gonorrhoeae Other closely related commensal species such as Neisseria lactamica, which is found in the normal flora of healthy individuals, were found to be less affected even at 5-fold higher doses of NaBPh4 This specificity was further observed when much lower sensitivity was found for more distant Neisseriaceae species (such as Neisseria elongata or Kingella oralis) and completely unrelated species. Significant boron uptake by N. meningitidis cells was observed after incubation with 5 µM NaBPh4, as measured by inductively coupled plasma mass spectrometry, suggesting that this drug candidate's target(s) could be located intracellularly or within the cell envelope. Furthermore, mutants with slightly decreased susceptibility displayed alterations in genes coding for cell envelope elements, which reduced their virulence in an animal model of infection. Finally, a single dose of NaBPh4 resulted in a significant reduction in bacterial burden in a mouse model of N. meningitidis bacteremia. Although numerous boron-containing species were previously reported for their complex biological activities, the observation of this narrow selectivity is unprecedented and of potential importance from a therapeutic standpoint.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

In bacteria, DNA methylation can be facilitated by 'orphan' DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

Hemocytes released in seawater act as Trojan horses for spreading of bacterial infections in mussels.

Global warming has been associated with increased episodes of mass mortality events in invertebrates, most notably in bivalves. Although the spread of pathogens is one of multiple factors that contribute to such mass mortality events, we don't fully understand the pathophysiological consequences of sea warming on invertebrates. In this work, we show that in temperature stress conditions, circulating hemocytes in mussels leave the hemolymph to gain access to the intervalvar fluid before being released in seawater. External hemocytes can survive for several hours in seawater before entering other mussels. When infected by bacteria, externally-infected hemocytes can enter naive mussels and promote bacterial dissemination in the host. These results reveal the existence of a new opportunistic mechanism used by pathogens to disseminate in marine ecosystems. Such mechanisms may explain how thermal anomalies triggered by global warming can favor episodic mass mortality observed in recent years in marine ecosystem.

CAPRIB: a user-friendly tool to study amino acid changes and selection for the exploration of intra-genus evolution.

BACKGROUND: The evolution of bacteria is shaped by different mechanisms such as mutation, gene deletion, duplication, or insertion of foreign DNA among others. These genetic changes can accumulate in the descendants as a result of natural selection. Using phylogeny and genome comparisons, evolutionary paths can be somehow retraced, with recent events being much easier to detect than older ones. For this reason, multiple tools are available to study the evolutionary events within genomes of single species, such as gene composition alterations, or subtler mutations such as SNPs. However, these tools are generally designed to compare similar genomes and require advanced skills in bioinformatics. We present CAPRIB, a unique tool developed in Java that allows to determine the amino acid changes, at the genus level, that correlate with phenotypic differences between two groups of organisms. RESULTS: CAPRIB has a user-friendly graphical interface and uses databases in SQL, making it easy to compare several genomes without the need for programming or thorough knowledge in bioinformatics. This intuitive software narrows down a list of amino acid changes that are concomitant with a given phenotypic divergence at the genus scale. Each permutation found by our software is associated with two already described statistical values that indicate its potential impact on the protein's function, helping the user decide which promising candidates to further investigate. We show that CAPRIB is able to detect already known mutations and uncovers many more, and that this tool can be used to question molecular phylogeny. Finally, we exemplify the utility of CAPRIB by pinpointing amino acid changes that coincided with the emergence of slow-growing mycobacteria from their fast-growing counterparts. The software is freely available at https://github.com/BactSymEvol/Caprib . CONCLUSIONS: CAPRIB is a new bioinformatics software aiming to make genus-scale comparisons accessible to all. With its intuitive graphical interface, this tool identifies key amino acid changes concomitant with a phenotypic divergence. By comparing fast and slow-growing mycobacteria, we shed light on evolutionary hotspots, such as the cytokinin pathway, that are interesting candidates for further experimentations.

RskA Is a Dual Function Activator-Inhibitor That Controls SigK Activity Across Distinct Bacterial Genera.

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal enzymatic extract offers a promising alternative for lignocellulose pretreatment of oil palm residues.

The pretreatment of biomass remains a critical requirement for bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm.