DciA Helicase Operators Exhibit Diversity across Bacterial Phyla.

A fundamental requirement for life is the replication of an organism's DNA. Studies in Escherichia coli and Bacillus subtilis have set the paradigm for DNA replication in bacteria. During replication initiation in E. coli and B. subtilis, the replicative helicase is loaded onto the DNA at the origin of replication by an ATPase helicase loader. However, most bacteria do not encode homologs to the helicase loaders in E. coli and B. subtilis. Recent work has identified the DciA protein as a predicted helicase operator that may perform a function analogous to the helicase loaders in E. coli and B. subtilis. DciA proteins, which are defined by the presence of a DUF721 domain (termed the DciA domain herein), are conserved in most bacteria but have only been studied in mycobacteria and gammaproteobacteria (Pseudomonas aeruginosa and Vibrio cholerae). Sequences outside the DciA domain in Mycobacterium tuberculosis DciA are essential for protein function but are not conserved in the P. aeruginosa and V. cholerae homologs, raising questions regarding the conservation and evolution of DciA proteins across bacterial phyla. To comprehensively define the DciA protein family, we took a computational evolutionary approach and analyzed the domain architectures and sequence properties of DciA domain-containing proteins across the tree of life. These analyses identified lineage-specific domain architectures among DciA homologs, as well as broadly conserved sequence-structural motifs. The diversity of DciA proteins represents the evolution of helicase operation in bacterial DNA replication and highlights the need for phylum-specific analyses of this fundamental biological process. IMPORTANCE Despite the fundamental importance of DNA replication for life, this process remains understudied in bacteria outside Escherichia coli and Bacillus subtilis. In particular, most bacteria do not encode the helicase-loading proteins that are essential in E. coli and B. subtilis for DNA replication. Instead, most bacteria encode a DciA homolog that likely constitutes the predominant mechanism of helicase operation in bacteria. However, it is still unknown how DciA structure and function compare across diverse phyla that encode DciA proteins. In this study, we performed computational evolutionary analyses to uncover tremendous diversity among DciA homologs. These studies provide a significant advance in our understanding of an essential component of the bacterial DNA replication machinery.

Novel internalin P homologs in Listeria.

Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of Lm and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross-protective barriers, including the placenta. One family of virulence factors, internalins, contributes to Lm pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains that enable protein-protein interactions, allowing Lm to bind host proteins. Notably, other Listeria species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus Listeria. Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Although prior studies of InlP have begun to elucidate its role in Lm pathogenesis, there remains a lack of information regarding homologs in other Listeria species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional Listeria species. We found that Listeria ivanovii londoniensis (Liv) and Listeria seeligeri (Ls) encode InlP homologs. We also found InlP-like homologs in Listeria innocua and the recently identified species Listeria costaricensis. All newly identified homologs lack the full-length LRR6 and LRR7 domains found in Lm's InlP. These findings are informative regarding the evolution of one key Lm virulence factor, InlP, and serve as a springboard for future evolutionary studies of Lm pathogenesis as well as mechanistic studies of Listeria internalins.

Construction of a chromosome-scale long-read reference genome assembly for potato.

BACKGROUND: Worldwide, the cultivated potato, Solanum tuberosum L., is the No. 1 vegetable crop and a critical food security crop. The genome sequence of DM1-3 516 R44, a doubled monoploid clone of S. tuberosum Group Phureja, was published in 2011 using a whole-genome shotgun sequencing approach with short-read sequence data. Current advanced sequencing technologies now permit generation of near-complete, high-quality chromosome-scale genome assemblies at minimal cost. FINDINGS: Here, we present an updated version of the DM1-3 516 R44 genome sequence (v6.1) using Oxford Nanopore Technologies long reads coupled with proximity-by-ligation scaffolding (Hi-C), yielding a chromosome-scale assembly. The new (v6.1) assembly represents 741.6 Mb of sequence (87.8%) of the estimated 844 Mb genome, of which 741.5 Mb is non-gapped with 731.2 Mb anchored to the 12 chromosomes. Use of Oxford Nanopore Technologies full-length complementary DNA sequencing enabled annotation of 32,917 high-confidence protein-coding genes encoding 44,851 gene models that had a significantly improved representation of conserved orthologs compared with the previous annotation. The new assembly has improved contiguity with a 595-fold increase in N50 contig size, 99% reduction in the number of contigs, a 44-fold increase in N50 scaffold size, and an LTR Assembly Index score of 13.56, placing it in the category of reference genome quality. The improved assembly also permitted annotation of the centromeres via alignment to sequencing reads derived from CENH3 nucleosomes. CONCLUSIONS: Access to advanced sequencing technologies and improved software permitted generation of a high-quality, long-read, chromosome-scale assembly and improved annotation dataset for the reference genotype of potato that will facilitate research aimed at improving agronomic traits and understanding genome evolution.