De novoassembly of thePasteuria penetransgenome reveals high plasticity, host dependency, and BclA-like collagens

Abstract

12Pasteuria penetrans is a gram-positive endospore forming bacterial parasite of Meloidogyne spp. the 13 most economically damaging genus of plant parasitic nematodes globally. The obligate antagonistic 14 nature of P. penetrans makes it an attractive candidate biological control agent. However, deployment 15 of P. penetrans for this purpose is inhibited by a lack of understanding of its metabolism and the 16 molecular mechanics underpinning parasitism of the host, in particular the initial attachment of the 17 endospore to the nematode cuticle. Several attempts to assemble the genomes of species within this 18 genus have been unsuccessful. Primarily this is due to the obligate parasitic nature of the bacterium 19which makes obtaining genomic DNA of sufficient quantity and quality which is free from 20 contamination challenging. Taking advantage of recent developments in whole genome amplification, 21 long read sequencing platforms, and assembly algorithms, we have developed a protocol to generate 22 large quantities of high molecular weight genomic DNA from a small number of purified endospores. 23We demonstrate this method via genomic assembly of P. penetrans. This assembly reveals a reduced 24 genome of 2.64Mbp estimated to represent 86% of the complete sequence; its reduced metabolism 25 reflects widespread reliance on the host and possibly associated organisms. Additionally, apparent 26 expansion of transposases and prediction of partial competence pathways suggest a high degree of 27 genomic plasticity. Phylogenetic analysis places our sequence within the Bacilli, and most closely 28 related to Thermoactinomyces species. Seventeen predicted BclA-like proteins are identified which 29 may be involved in the determination of attachment specificity. This resource may be used to develop 30 in vitro culture methods and to investigate the genetic and molecular basis of attachment specificity. 31 32 33

Exploring Bacillus thuringiensis as a model for endospore adhesion and its potential to investigate adhesins in Pasteuria penetrans

“…Studies of other closely related Bacillus and Clostridium spp. suggest that the surface of the endospore is covered with a hair‐like nap which is made up of collagen‐like fibres and there is growing evidence now that Pasteuria endospores also have a similar fibrous hair‐like nap made up of glycosylated CLPs (Davies & Opperman, 2006; Orr et al, 2018) which we show here to play an important role in endospore attachment. Bioinformatics has shown that in Pasteuria CLPs are a diverse group of sequences possibly obtained through horizontal gene transfer (Srivastava et al, 2018).…”

“…We know that the P. penetrans genome contains a diverse set of genes that encode CLPs (Orr et al, 2018; Srivastava et al, 2018) and it is possible that different sets of collagen‐like adhesins are expressed on the different surfaces of the endospores. It is also likely that the treatments of endospores do not affect the convex and concave surfaces of the endospores equally, and hence produce changes in binding orientation.…”

“…Bioinformatics of genome survey sequences of P. penetrans show a diverse set of collagen‐like proteins (CLPs) the presence of which appears to be closely related to the BclA protein of Bacillus spp. (Davies & Opperman, 2006; Orr et al, 2018; Srivastava et al, 2018). It has been proposed that such BclA‐like proteins constitute the parasporal fibres of the exosporium and may interact with mucin‐like peptides present on the nematode cuticle via a Velcro‐like attachment mechanism (Davies, 2009; Phani et al, 2018).…”

The genome ofPasteuria ramosareveals a high turnover rate of collagen-like genes

“…In the P21 assembly, protein coding sequences (CDS) represent 69.98 % of the total genome (average length 878.9 bp), whereas they represent 69.93 % of the nucleotide sequence for C1, with an average gene size of 877.0 bp. For comparison, P. penetrans, a close relative, has 2,407 genes (average length 694 bp), representing 66.33 % of the genome (29). The genomes of both Pasteuria species are less dense in coding regions than related species such as Bacillus thuringiensis (83.28 %, (30)) or Thermoactinomyces sp.…”

“…The genomes of both Pasteuria species are less dense in coding regions than related species such as Bacillus thuringiensis (83. 28 (29). Notably, neither a canonical CRISPR system nor prophages were identified in the two P. ramosa genomes using CRISPRCasFinder (33) and Phaster (34,35) tools.…”