Improving Phylogenies Based on Average Nucleotide Identity, Incorporating Saturation Correction and Nonparametric Bootstrap Support.

Whole-genome comparisons based on average nucleotide identities (ANI) and the genome-to-genome distance calculator have risen to prominence in rapidly classifying prokaryotic taxa using whole-genome sequences. Some implementations have even been proposed as a new standard in species classification and have become a common technique for papers describing newly sequenced genomes. However, attempts to apply whole-genome divergence data to the delineation of higher taxonomic units and to phylogenetic inference have had difficulty matching those produced by more complex phylogenetic methods. We present a novel method for generating statistically supported phylogenies of archaeal and bacterial groups using a combined ANI and alignment fraction-based metric. For the test cases to which we applied the developed approach, we obtained results comparable with other methodologies up to at least the family level. The developed method uses nonparametric bootstrapping to gauge support for inferred groups. This method offers the opportunity to make use of whole-genome comparison data, that is already being generated, to quickly produce phylogenies including support for inferred groups. Additionally, the developed ANI methodology can assist the classification of higher taxonomic groups.[Average nucleotide identity (ANI); genome evolution; prokaryotic species delineation; taxonomy.].

The Constructive Black Queen hypothesis: new functions can evolve under conditions favouring gene loss.

Duplication is a major route for the emergence of new gene functions. However, the emergence of new gene functions via this route may be reduced in prokaryotes, as redundant genes are often rapidly purged. In lineages with compact, streamlined genomes, it thus appears challenging for novel function to emerge via duplication and divergence. A further pressure contributing to gene loss occurs under Black Queen dynamics, as cheaters that lose the capacity to produce a public good can instead acquire it from neighbouring producers. We propose that Black Queen dynamics can favour the emergence of new function because, under an emerging Black Queen dynamic, there is high gene redundancy spread across a community of interacting cells. Using computational modelling, we demonstrate that new gene functions can emerge under Black Queen dynamics. This result holds even if there is deletion bias due to low duplication rates and selection against redundant gene copies resulting from the high cost associated with carrying a locus. However, when the public good production costs are high, Black Queen dynamics impede the fixation of new functions. Our results expand the mechanisms by which new gene functions can emerge in prokaryotic systems.

The Patchy Distribution of Restriction⁻Modification System Genes and the Conservation of Orphan Methyltransferases in Halobacteria.

Restriction⁻modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

Low-Level Antimicrobials in the Medicinal Leech Select for Resistant Pathogens That Spread to Patients.

Fluoroquinolones (FQs) and ciprofloxacin (Cp) are important antimicrobials that pollute the environment in trace amounts. Although Cp has been recommended as prophylaxis for patients undergoing leech therapy to prevent infections by the leech gut symbiont Aeromonas, a puzzling rise in Cp-resistant (Cpr) Aeromonas infections has been reported. We report on the effects of subtherapeutic FQ concentrations on bacteria in an environmental reservoir, the medicinal leech, and describe the presence of multiple antibiotic resistance mutations and a gain-of-function resistance gene. We link the rise of CprAeromonas isolates to exposure of the leech microbiota to very low levels of Cp (0.01 to 0.04 µg/ml), <1/100 of the clinical resistance breakpoint for Aeromonas Using competition experiments and comparative genomics of 37 strains, we determined the mechanisms of resistance in clinical and leech-derived Aeromonas isolates, traced their origin, and determined that the presence of merely 0.01 µg/ml Cp provides a strong competitive advantage for Cpr strains. Deep-sequencing the Cpr-conferring region of gyrA enabled tracing of the mutation-harboring Aeromonas population in archived gut samples, and an increase in the frequency of the Cpr-conferring mutation in 2011 coincides with the initial reports of CprAeromonas infections in patients receiving leech therapy.IMPORTANCE The role of subtherapeutic antimicrobial contamination in selecting for resistant strains has received increasing attention and is an important clinical matter. This study describes the relationship of resistant bacteria from the medicinal leech, Hirudo verbana, with patient infections following leech therapy. While our results highlight the need for alternative antibiotic therapies, the rise of Cpr bacteria demonstrates the importance of restricting the exposure of animals to antibiotics approved for veterinary use. The shift to a more resistant community and the dispersion of Cpr-conferring mechanisms via mobile elements occurred in a natural setting due to the presence of very low levels of fluoroquinolones, revealing the challenges of controlling the spread of antibiotic-resistant bacteria and highlighting the importance of a holistic approach in the management of antibiotic use.

Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios.

Female members of many cephalopod species house a bacterial consortium in the accessory nidamental gland (ANG), part of the reproductive system. These bacteria are deposited into eggs that are then laid in the environment where they must develop unprotected from predation, pathogens, and fouling. In this study, we characterized the genome and secondary metabolite production of Leisingera sp. JC1, a member of the roseobacter clade (Rhodobacteraceae) of Alphaproteobacteria isolated from the jelly coat of eggs from the Hawaiian bobtail squid, Euprymna scolopes. Whole genome sequencing and MLSA analysis revealed that Leisingera sp. JC1 falls within a group of roseobacters associated with squid ANGs. Genome and biochemical analyses revealed the potential for and production of a number of secondary metabolites, including siderophores and acyl-homoserine lactones involved with quorum sensing. The complete biosynthetic gene cluster for the pigment indigoidine was detected in the genome and mass spectrometry confirmed the production of this compound. Furthermore, we investigated the production of indigoidine under co-culture conditions with Vibrio fischeri, the light organ symbiont of E. scolopes, and with other vibrios. Finally, both Leisingera sp. JC1 and secondary metabolite extracts of this strain had differential antimicrobial activity against a number of marine vibrios, suggesting that Leisingera sp. JC1 may play a role in host defense against other marine bacteria either in the eggs and/or ANG. These data also suggest that indigoidine may be partially, but not wholly, responsible for the antimicrobial activity of this squid-associated bacterium.

Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes.

The accessory nidamental gland (ANG) of the female Hawaiian bobtail squid, Euprymna scolopes, houses a consortium of bacteria including members of the Flavobacteriales, Rhizobiales, and Verrucomicrobia but is dominated by members of the Roseobacter clade (Rhodobacterales) within the Alphaproteobacteria. These bacteria are deposited into the jelly coat of the squid's eggs, however, the function of the ANG and its bacterial symbionts has yet to be elucidated. In order to gain insight into this consortium and its potential role in host reproduction, we cultured 12 Rhodobacterales isolates from ANGs of sexually mature female squid and sequenced their genomes with Illumina sequencing technology. For taxonomic analyses, the ribosomal proteins of 79 genomes representing both roseobacters and non-roseobacters along with a separate MLSA analysis of 33 housekeeping genes from Roseobacter organisms placed all 12 isolates from the ANG within two groups of a single Roseobacter clade. Average nucelotide identity analysis suggests the ANG isolates represent three genera (Leisingera, Ruegeria, and Tateyamaria) comprised of seven putative species groups. All but one of the isolates contains a predicted Type VI secretion system, which has been shown to be important in secreting signaling and/or effector molecules in host-microbe associations and in bacteria-bacteria interactions. All sequenced genomes also show potential for secondary metabolite production, and are predicted to be involved with the production of acyl homoserine lactones (AHLs) and/or siderophores. An AHL bioassay confirmed AHL production in three tested isolates and from whole ANG homogenates. The dominant symbiont, Leisingera sp. ANG1, showed greater viability in iron-limiting conditions compared to other roseobacters, possibly due to higher levels of siderophore production. Future comparisons will try to elucidate novel metabolic pathways of the ANG symbionts to understand their putative role in host development.

Bioinformatic genome comparisons for taxonomic and phylogenetic assignments using Aeromonas as a test case.

UNLABELLED: Prokaryotic taxonomy is the underpinning of microbiology, as it provides a framework for the proper identification and naming of organisms. The "gold standard" of bacterial species delineation is the overall genome similarity determined by DNA-DNA hybridization (DDH), a technically rigorous yet sometimes variable method that may produce inconsistent results. Improvements in next-generation sequencing have resulted in an upsurge of bacterial genome sequences and bioinformatic tools that compare genomic data, such as average nucleotide identity (ANI), correlation of tetranucleotide frequencies, and the genome-to-genome distance calculator, or in silico DDH (isDDH). Here, we evaluate ANI and isDDH in combination with phylogenetic studies using Aeromonas, a taxonomically challenging genus with many described species and several strains that were reassigned to different species as a test case. We generated improved, high-quality draft genome sequences for 33 Aeromonas strains and combined them with 23 publicly available genomes. ANI and isDDH distances were determined and compared to phylogenies from multilocus sequence analysis of housekeeping genes, ribosomal proteins, and expanded core genes. The expanded core phylogenetic analysis suggested relationships between distant Aeromonas clades that were inconsistent with studies using fewer genes. ANI values of ≥ 96% and isDDH values of ≥ 70% consistently grouped genomes originating from strains of the same species together. Our study confirmed known misidentifications, validated the recent revisions in the nomenclature, and revealed that a number of genomes deposited in GenBank are misnamed. In addition, two strains were identified that may represent novel Aeromonas species. IMPORTANCE: Improvements in DNA sequencing technologies have resulted in the ability to generate large numbers of high-quality draft genomes and led to a dramatic increase in the number of publically available genomes. This has allowed researchers to characterize microorganisms using genome data. Advantages of genome sequence-based classification include data and computing programs that can be readily shared, facilitating the standardization of taxonomic methodology and resolving conflicting identifications by providing greater uniformity in an overall analysis. Using Aeromonas as a test case, we compared and validated different approaches. Based on our analyses, we recommend cutoff values for distance measures for identifying species. Accurate species classification is critical not only to obviate the perpetuation of errors in public databases but also to ensure the validity of inferences made on the relationships among species within a genus and proper identification in clinical and veterinary diagnostic laboratories.

Inteins as indicators of gene flow in the halobacteria.

This research uses inteins, a type of mobile genetic element, to infer patterns of gene transfer within the Halobacteria. We surveyed 118 genomes representing 26 genera of Halobacteria for intein sequences. We then used the presence-absence profile, sequence similarity and phylogenies from the inteins recovered to explore how intein distribution can provide insight on the dynamics of gene flow between closely related and divergent organisms. We identified 24 proteins in the Halobacteria that have been invaded by inteins at some point in their evolutionary history, including two proteins not previously reported to contain an intein. Furthermore, the size of an intein is used as a heuristic for the phase of the intein's life cycle. Larger size inteins are assumed to be the canonical two domain inteins, consisting of self-splicing and homing endonuclease domains (HEN); smaller sizes are assumed to have lost the HEN domain. For many halobacterial groups the consensus phylogenetic signal derived from intein sequences is compatible with vertical inheritance or with a strong gene transfer bias creating these clusters. Regardless, the coexistence of intein-free and intein-containing alleles reveal ongoing transfer and loss of inteins within these groups. Inteins were frequently shared with other Euryarchaeota and among the Bacteria, with members of the Cyanobacteria (Cyanothece, Anabaena), Bacteriodetes (Salinibacter), Betaproteobacteria (Delftia, Acidovorax), Firmicutes (Halanaerobium), Actinobacteria (Longispora), and Deinococcus-Thermus-group.

Population and genomic analysis of the genus Halorubrum.

The Halobacteria are known to engage in frequent gene transfer and homologous recombination. For stably diverged lineages to persist some checks on the rate of between lineage recombination must exist. We surveyed a group of isolates from the Aran-Bidgol endorheic lake in Iran and sequenced a selection of them. Multilocus Sequence Analysis (MLSA) and Average Nucleotide Identity (ANI) revealed multiple clusters (phylogroups) of organisms present in the lake. Patterns of intein and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) presence/absence and their sequence similarity, GC usage along with the ANI and the identities of the genes used in the MLSA revealed that two of these clusters share an exchange bias toward others in their phylogroup while showing reduced rates of exchange with other organisms in the environment. However, a third cluster, composed in part of named species from other areas of central Asia, displayed many indications of variability in exchange partners, from within the lake as well as outside the lake. We conclude that barriers to gene exchange exist between the two purely Aran-Bidgol phylogroups, and that the third cluster with members from other regions is not a single population and likely reflects an amalgamation of several populations.

Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations.

Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions.