CRISPR-Cas phage defense systems and prophages inCandidatusAccumulibacter

Abstract

Candidatus Accumulibacter is a key genus of polyphosphate-accumulating organisms (PAOs) found in laboratory- and full-scale wastewater treatment systems, mediating enhanced biological phosphorus removal (EBPR). However, little is known about their ability to resist phage infection. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in diverse Ca. Accumulibacter taxa (43 in total, including 10 newly recovered genomes). Fourty complete CRISPR loci were identified in 28 genomes, falling into 6 subtypes. The occurrence and distribution of CRISPR-Cas systems did not follow a vertical evolutionary relationship. Phylogenetic analyses of the cas genes and direct repeats (DRs) suggested that the CRISPR-Cas systems were likely acquired via horizontal gene transfer, with acquisition rates higher than those of speciation, rendering different Ca. Accumulibacter distinct adaptivity to phage predations. 2448 spacers were identified, 67 of them matched to known phages. Major differences were observed among the numbers of spacers for different Ca. Accumulibacter, showing unique phages that could be resisted by different members. A comparison of the spacers in genomes having the same cas gene but from distinct geographical locations indicated that habitat isolation may have resulted in the acquisition of different spacers by different Ca. Accumulibacter. Metagenomic analysis allowed the identification of 26 viral contigs (18 are Caudovirales members) in 6 metagenomic datasets from three lab-scale enrichment reactors, matching to 73 spacers in 10 Ca. Accumulibacter genomes in these reactors, showing the specific immunity of these Ca. Accumulibacter. Metatranscriptomic analyses showed the activity of the CRISPR-Cas system under both anaerobic and aerobic conditions. Extra efforts were made to identify prophages in Ca. Accumulibacter genomes. In total, 133 prophage regions were identified. Twenty-seven of them were classified as potentially active prophages. Three prophages (all are Caudovirales members, in DS2011, SCELSE-7IIH and SCELSE-5IIH, respectively) are readily activable. Differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to their distinct responses under phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages with combined experimental and bioinformatic methods in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.

Integrated genomics provides insights for the evolution of the polyphosphate accumulation trait ofCa.Accumulibacter

“…A maximum likelihood tree was built based on the concatenation of single-copy genes with model Q. insect+F+I+I+R4. Genomes in red are those recovered from our bioreactors (Arumugam et al, 2019; Qiu et al, 2020; Tian et al, 2022; Deng et al, 2023).…”

“…The ancestral, derived, flexible and lineage-specific genes were annotated and classified based on KEGG annotations (Kanehisa et al, 2013) of clade IIC member SCUT-2 (Deng et al, 2023) and clade IIA member UW1 (García Martín et al, 2006; Oyserman et al, 2016b). The number of different types of genes annotated in each metabolic pathway was counted with the number of each type of genes being divided by the total number of genes in the pathway.…”

“…The number of genes assigned as ancestral, derived, lineage-specific, and flexible genes in SCUT-2 and UW1. bioreactors (Arumugam et al, 2019;Qiu et al, 2020;Tian et al, 2022;Deng et al, 2023).…”

“…This process is mediated by a group of microorganisms namely polyphosphate-accumulating organisms (PAOs) (He and McMahon, 2011; Nielsen et al, 2019; Dorofeev et al, 2020; Zhang et al, 2023;). Candidatus Accumulibacter is a model genus of PAOs commonly found in lab- and full-scale EBPR systems (Seviour et al, 2003; Mao et al, 2015; Roy et al, 2021; Petriglieri et al, 2022; Deng et al, 2023). Under anaerobic conditions, Ca.…”

“…With the advance in EBPR research and sequencing techniques, increasing numbers of high-quality Ca . Accumulibacter genomes have been obtained (Arumugam et al, 2019; Rubio-Rincón et al; 2019; Qiu et al, 2020; McDaniel et al, 2021; Singleton et al, 2021; Srinivasan et al, 2021; Tian et al, 2022; Petriglieri et al, 2022; Zhang et al, 2023; Deng et al, 2023). Additionally, new PAOs and glycogen accumulating organisms (GAOs) were identified in genera phylogenetically closely related to Ca .…”