Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

NDM-1 Introduction in Portugal through a ST11 KL105 Klebsiella pneumoniae Widespread in Europe

The changing epidemiology of carbapenem-resistant Klebsiella pneumoniae in Southern European countries is challenging for infection control, and it is critical to identify and track new genetic entities (genes, carbapenemases, clones) quickly and with high precision. We aimed to characterize the strain responsible for the first recognized outbreak by an NDM-1-producing K. pneumoniae in Portugal, and to elucidate its diffusion in an international context. NDM-1-producing multidrug-resistant K. pneumoniae isolates from hospitalized patients (2018–2019) were characterized using FTIR spectroscopy, molecular typing, whole-genome sequencing, and comparative genomics with available K. pneumoniae ST11 KL105 genomes. FT-IR spectroscopy allowed the rapid (ca. 4 h after incubation) identification of the outbreak strains as ST11 KL105, supporting outbreak control. Epidemiological information supports a community source but without linkage to endemic regions of NDM-1 producers. Whole-genome comparison with previous DHA-1-producing ST11 KL105 strains revealed the presence of different plasmid types and antibiotic resistance traits, suggesting the entry of a new strain. In fact, this ST11 KL105 clade has successfully disseminated in Europe with variable beta-lactamases, but essentially as ESBL or DHA-1 producers. We expand the distribution map of NDM-1-producing K. pneumoniae in Europe, at the expense of a successfully established ST11 KL105 K. pneumoniae clade circulating with variable plasmid backgrounds and beta-lactamases. Our work further supports the use of FT-IR as an asset to support quick infection control.

Unlocking the Complete Chloroplast Genome of a Native Tree Species from the Amazon Basin, Capirona (Calycophyllum Spruceanum, Rubiaceae), and Its Comparative Analysis with Other Ixoroideae Species

Capirona (Calycophyllum spruceanum Benth.) belongs to subfamily Ixoroideae, one of the major lineages in the Rubiaceae family, and is an important timber tree. It originated in the Amazon Basin and has widespread distribution in Bolivia, Peru, Colombia, and Brazil. In this study, we obtained the first complete chloroplast (cp) genome of capirona from the department of Madre de Dios located in the Peruvian Amazon. High-quality genomic DNA was used to construct libraries. Pair-end clean reads were obtained by PE 150 library and the Illumina HiSeq 2500 platform. The complete cp genome of C. spruceanum has a 154,480 bp in length with typical quadripartite structure, containing a large single copy (LSC) region (84,813 bp) and a small single-copy (SSC) region (18,101 bp), separated by two inverted repeat (IR) regions (25,783 bp). The annotation of C. spruceanum cp genome predicted 87 protein-coding genes (CDS), 8 ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes, and one pseudogene. A total of 41 simple sequence repeats (SSR) of this cp genome were divided into mononucleotides (29), dinucleotides (5), trinucleotides (3), and tetranucleotides (4). Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with the other six Ixoroideae species revealed that the small single copy and large single copy regions showed more divergence than inverted regions. Finally, phylogenetic analyses resolved that C. spruceanum is a sister species to Emmenopterys henryi and confirms its position within the subfamily Ixoroideae. This study reports for the first time the genome organization, gene content, and structural features of the chloroplast genome of C. spruceanum, providing valuable information for genetic and evolutionary studies in the genus Calycophyllum and beyond.

Comparative genome analysis of plant ascomycete fungal pathogens with different lifestyles reveals distinctive virulence strategies

Background Pathogens have evolved diverse lifestyles and adopted pivotal new roles in both natural ecosystems and human environments. However, the molecular mechanisms underlying their adaptation to new lifestyles are obscure. Comparative genomics was adopted to determine distinct strategies of plant ascomycete fungal pathogens with different lifestyles and to elucidate their distinctive virulence strategies. Results We found that plant ascomycete biotrophs exhibited lower gene gain and loss events and loss of CAZyme-encoding genes involved in plant cell wall degradation and biosynthesis gene clusters for the production of secondary metabolites in the genome. Comparison with the candidate effectome detected distinctive variations between plant biotrophic pathogens and other groups (including human, necrotrophic and hemibiotrophic pathogens). The results revealed the biotroph-specific and lifestyle-conserved candidate effector families. These data have been configured in web-based genome browser applications for public display (http://lab.malab.cn/soft/PFPG). This resource allows researchers to profile the genome, proteome, secretome and effectome of plant fungal pathogens. Conclusions Our findings demonstrated different genome evolution strategies of plant fungal pathogens with different lifestyles and explored their lifestyle-conserved and specific candidate effectors. It will provide a new basis for discovering the novel effectors and their pathogenic mechanisms.

Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology – triggered by highly persistent, interchangeable salt-bridges

The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCS_Spike) containing its 681_PRRAR_685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCS_Spike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCS_Spike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCS_Spike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. The current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

A Large-Scale Multiple Genome Comparison of Acidophilic Archaea (pH ≤ 5.0) Extends Our Understanding of Oxidative Stress Responses in Polyextreme Environments

Acidophilic archaea thrive in anaerobic and aerobic low pH environments (pH < 5) rich in dissolved heavy metals that exacerbate stress caused by the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide (O2−). ROS react with lipids, proteins and nucleic acids causing oxidative stress and damage that can lead to cell death. Herein, genes and mechanisms potentially involved in ROS mitigation are predicted in over 200 genomes of acidophilic archaea with sequenced genomes. These organisms are often be subjected to simultaneous multiple stresses such as high temperature, high salinity, low pH and high heavy metal loads. Some of the topics addressed include: (1) the phylogenomic distribution of these genes and what this can tell us about the evolution of these mechanisms in acidophilic archaea; (2) key differences in genes and mechanisms used by acidophilic versus non-acidophilic archaea and between acidophilic archaea and acidophilic bacteria and (3) how comparative genomic analysis predicts novel genes or pathways involved in oxidative stress responses in archaea and likely horizontal gene transfer (HGT) events.

Unlocking the Complete Chloroplast Genome of a Native Tree Species from the Amazon Basin, Capirona (Calycophyllum spruceanum Benth., Rubiaceae), and Its Comparative Analysis with Other Ixoroideae Species

Capirona (Calycophyllum spruceanum Benth.) belongs to subfamily Ixoroideae, one of de major lineages in the Rubiaceae family, and is an important timber tree, with origin in the Amazon Basin and has widespread distribution in Bolivia, Peru, Colombia, and Brazil. In this study, we obtained the first complete chloroplast (cp) genome of capirona from department of Madre de Dios located in the Peruvian Amazon. High-quality genomic DNA was used to construct librar-ies. Pair-end clean reads were obtained by PE 150 library and the Illumina HiSeq 2500 platform. The complete cp genome of C. spruceanum has a 154,480 bp in length with typical quadripartite structure, containing a large single copy (LSC) region (84,813 bp) and a small single-copy (SSC) region (18,101 bp), separated by two inverted repeat (IR) regions (25,783 bp). The annotation of C. spruceanum cp genome predicted 87 protein-coding genes (CDS), 8 ribosomal RNA (rRNA) genes, 37 transfer RNA (tRNA) genes and 01 pseudogene. A total of 41 simple sequence repeats (SSR) of this cp genome were divided into mononucleotides (29), dinucleotides (5), trinucleotides (3), and tetranucleotide (4). Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with the other six Ixoroideae species revealed that the small single copy and large single copy regions showed more divergence than invert regions. Finally, phylogenetic analysis resolved that C. spruceanum is a sister species to Emmenopterys henryi, and confirms its position within the subfamily Ixoroideae. This study reports for the first time the genome organization, gene content, and structural features of the chloroplast genome of C. spruceanum, providing valuable information for genetic and evolutionary studies in the genus Calycophyllum and beyond.

Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

A large scale, multiple genome comparison of acidophilic Archaea (pH ≤ 5.0) extends our understanding of oxidative stress responses in polyextreme environments

Acidophilic Archaea thrive in anaerobic and aerobic low pH environments (<pH 5) rich in dissolved heavy metals that exacerbate stress caused by the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (·OH) and superoxide (O2·−). ROS react with lipids, proteins and nucleic acids causing oxidative stress and damage that can lead to cell death. Herein, genes and mechanisms potentially involved in ROS mitigation are predicted in over 200 genomes of acidophilic Archaea with sequenced genomes. These organisms can be subjected to simultaneous multiple stresses such as high temperature, high salinity, low pH and high heavy metal loads. Some of the topics addressed include: (1) the phylogenomic distribution of these genes and what can this tell us about the evolution of these mechanisms in acidophilic Archaea; (2) key differences in genes and mechanisms used by acidophilic versus non-acidophilic Archaea and between acidophilic Archaea and acidophilic Bacteria and (3) how comparative genomic analysis predicts novel genes or pathways involved in oxidative stress responses in Archaea and possible Horizontal Gene Transfer (HGT) events.

BOPAL 2.0 and a study of tRNA and rRNA gene evolution in Clostridium

We present BOPAL 2.0, an improved version of the BOPAL algorithm for the evolutionary history inference of tRNA and rRNA genes in bacterial genomes. Our approach can infer complete evolutionary scenarios and ancestral gene orders on a phylogeny and considers a wide range of events such as duplications, deletions, substitutions, inversions and transpositions. It is based on the fact that tRNA and rRNA genes are often organized in operons/clusters in bacteria, and this information is used to help identify orthologous genes for each genome comparison. BOPAL 2.0 introduces new features, such as a triple-wise alignment step, context-aware singleton matching and a second pass of the algorithm. Evaluation on simulated datasets shows that BOPAL 2.0 outperforms the original BOPAL in terms of the accuracy of inferred events and ancestral genomes. We also present a study of the tRNA/rRNA gene evolution in the Clostridium genus, in which the organization of these genes is very divergent. Our results indicate that tRNA and rRNA genes in Clostridium have evolved through numerous duplications, losses, transpositions and substitutions, but very few inversions were inferred.