Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches.

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

Diverse and abundant phages exploit conjugative plasmids.

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

Efficient and Robust Search of Microbial Genomes via Phylogenetic Compression.

Comprehensive collections approaching millions of sequenced genomes have become central information sources in the life sciences. However, the rapid growth of these collections makes it effectively impossible to search these data using tools such as BLAST and its successors. Here, we present a technique called phylogenetic compression, which uses evolutionary history to guide compression and efficiently search large collections of microbial genomes using existing algorithms and data structures. We show that, when applied to modern diverse collections approaching millions of genomes, lossless phylogenetic compression improves the compression ratios of assemblies, de Bruijn graphs, and k-mer indexes by one to two orders of magnitude. Additionally, we develop a pipeline for a BLAST-like search over these phylogeny-compressed reference data, and demonstrate it can align genes, plasmids, or entire sequencing experiments against all sequenced bacteria until 2019 on ordinary desktop computers within a few hours. Phylogenetic compression has broad applications in computational biology and may provide a fundamental design principle for future genomics infrastructure.

Diverse and abundant phages exploit conjugative plasmids.

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally-encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 64 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

Prophages encode phage-defense systems with cognate self-immunity.

Temperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically.

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid.

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.

Non-Coding RNA Analysis Using the Rfam Database.

Rfam is a database of non-coding RNA families in which each family is represented by a multiple sequence alignment, a consensus secondary structure, and a covariance model. Using a combination of manual and literature-based curation and a custom software pipeline, Rfam converts descriptions of RNA families found in the scientific literature into computational models that can be used to annotate RNAs belonging to those families in any DNA or RNA sequence. Valuable research outputs that are often locked up in figures and supplementary information files are encapsulated in Rfam entries and made accessible through the Rfam Web site. The data produced by Rfam have a broad application, from genome annotation to providing training sets for algorithm development. This article gives an overview of how to search and navigate the Rfam Web site, and how to annotate sequences with RNA families. The Rfam database is freely available at http://rfam.org. © 2018 by John Wiley & Sons, Inc.

Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families.

The Rfam database is a collection of RNA families in which each family is represented by a multiple sequence alignment, a consensus secondary structure, and a covariance model. In this paper we introduce Rfam release 13.0, which switches to a new genome-centric approach that annotates a non-redundant set of reference genomes with RNA families. We describe new web interface features including faceted text search and R-scape secondary structure visualizations. We discuss a new literature curation workflow and a pipeline for building families based on RNAcentral. There are 236 new families in release 13.0, bringing the total number of families to 2687. The Rfam website is http://rfam.org.