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We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Background Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination. Methodology/Principal findings Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. Conclusions WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas., Author summary Cholera affects more than 4 million people globally every year; people predominantly living in poverty or in resource-constrained conditions including political crises or natural disasters. Cholera’s typical presentation is characterized by rapid onset of acute watery diarrhea and vomiting which can progress from watery stool to shock in as little as four hours. Laboratory conditions needed for culture confirmation and strain preservation are rarely to never present in these affected areas. In fact, many cholera endemic areas in Sub-Saharan African are so remote that even treatment response alone is often challenging. Here we present the genomic analysis of DNA extracted from dried filter paper, which is a low-cost, low-tech and sustainable method. Previously this method has facilitated cholera confirmation by PCR, but we demonstrate that this method is also suitable for whole genome sequencing and subsequent strain characterization by presenting the analysis of samples from an outbreak in a remote area of Cameroon. This method will facilitate the understanding of the molecular epidemiology in cholera-prone areas, which were previously too challenging to attempt. It also introduces a method that can be used on a broader scale for diarrheal disease surveillance, including providing a window into co-infection and microbiome analyses.

A Gram-positive, non-motile, rod-shaped, non-spore-forming bacterium, designated CAAS 251T, was isolated from paper-mill effluent in Wuhan, China. The organism grew optimally at 40-42 degrees C and at pH 9.0-10.0. The major menaquinones were MK-7, MK-8 and MK-9. The predominant cellular fatty acids were anteiso-C15:0 (34.78 %), anteiso-C17:0 (25.24 %) and C16:0 (13.37 %). The G+C content of the genomic DNA was 65.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAAS 251T belongs to the genus Nesterenkonia, having sequence identities ranging from 96.0 to 97.0 % with respect to eight recognized species of the genus Nesterenkonia. Data from DNA-DNA hybridization and physiological and biochemical tests indicated that strain CAAS 251T represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia flava sp. nov. is proposed. The type strain is CAAS 251T (=CCTCC AB 207010T=JCM 14814T).

Persistent infection by specific oncogenic human papillomaviruses (HPVs) is established as the necessary cause of cervix cancer. DNA sequence differences between HPV genomes determine whether an HPV has the potential to cause cancer. Of the more than 100 HPV genotypes characterized at the genetic level, at least 15 are associated, to varying degrees, with cervical cancer. Classification based on nucleotide similarity places nearly all HPVs that infect the cervicovaginal area within the α-PV genus. Within this genus, phylogenetic trees inferred from the entire viral genome cluster all cancer-causing types together, suggesting the existence of a common ancestor for the oncogenic HPVs. However, in separate trees built from the early open reading frames (ORFs; i.e. E1, E2, E6, E7) or the late ORFs (i.e. L1, L2), the carcinogenic potential sorts with the early region of the genome, but not the late region. Thus, genetic differences within the early region specify the pathogenic potential of α-HPV infections. Since the HPV genomes are monophyletic and sites are highly correlated across the genome, diagnosis of oncogenic types and non-oncogenic types can be accomplished using any region across the genome. Here we review our current understanding of the evolutionary history of the oncogenic HPVs, in particular, we focus on the importance of viral genome heterogeneity and discuss the genetic basis for the oncogenic phenotype in some but not all α-PVs.

Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase a-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1-5 degrees C, all of them have their growth optima in the range of moderate temperatures (25-35 degrees C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas.

Four aerobic, gram-negative bacterial strains isolated from kaolin slurry used in the production of paper were subjected to a polyphasic analysis and characterization to determine their taxonomic position. Analysis of the 16S rDNA sequences of the four strains revealed that they represent a new lineage within the gamma-Proteobacteria, related to the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Luteimonas, Xylella and Rhodanobacter. Analysis of the quinone system, the polyamines, the fatty acids and the polar lipids revealed a combination of characteristics that is unique and not described for the phylogenetic relatives. The four strains contain a ubiquinone Q-8, spermidine as the major polyamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, and a fatty acid profile with predominantly iso-branched fatty acids. The G+C content of the genomic DNA was determined to be within the narrow range 67.1-68.7 mol%. Determination of DNA relatedness, as well as riboprint band patterns and amplified fragment length polymorphism profiles, clearly demonstrated that the four strains are members of a single species. Antibiotic-susceptibility patterns were identical for the four strains. Although showing a high degree of similarites in physiological and biochemical patterns, each of the four strains could be distinguished from the others on the basis of a few biochemical characteristics. On the basis of the estimates of phylogenetic relationships derived from the 16S rDNA sequence analyses, the observed chemotaxonomic characteristics and other phenotypic traits, a new genus, Thermomonas gen. nov., and species, Thermomonas haemolytica sp. nov., are proposed for the strains A50-7-3T (= DSM 13605T = LMG 19653T), B 50-7-1 (= DSM 13598 = LMG 19655), D50-7-1 (= DSM 13610 = LMG 19656) and B50-8-1 (= DSM 13599 = LMG 19654), with strain A50-7-3T as the type strain.

VERTEBRATE neurohypophysial hormones have a common structural pattern characterized by a chain of nine amino-acid residues with a disulphide bridge connecting the amino-acids in positions 1 and 6. There are usually two hormones in each species, the active principles rarely varying from one species to another within a given class. Thus oxytocin and arginine vasopressin have been chemically identified in five species of mammals belonging to the orders Primates, Artiodactyla, Perissodactyla and Cetacea. Isotocin (Ser4-Ile8-oxytocin) and vasotocin (Arg8-oxytocin) have up to now been isolated from six species of bony fish belonging to the families Gadidae, Scombridae and Cyprinidae. The structural variations which occur between one vertebrate class and another are confined to one or two amino-acid substitutions in positions 3, 4 or 8 (for a review of this subject see ref. 1).