Novel Self-Transmissible and Broad-Host-Range Plasmids Exogenously Captured From Anaerobic Granules or Cow Manure

Abstract

Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAβ. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these “cryptic” but broad-host-range plasmids may be important for spreading several genes as “vehicles” in a wider range of bacteria in natural environments.

PromA Plasmids Are Instrumental in the Dissemination of Linuron Catabolic Genes Between Different Genera

“…These so far cryptic plasmids have no apparent benefit to the host and are still propagated in absence of selective pressure (Fox et al, 2008). A recently discovered group of BHR plasmids are the PromA plasmids, most of which were isolated by exogenous plasmid capture (Schneiker et al, 2001;Tauch et al, 2002;Van der Auwera et al, 2009;Li et al, 2014;Thomas et al, 2017;Yanagiya et al, 2018), hence originated from unknown hosts. Exogenous plasmid isolation allows capturing conjugative as well as mobilizable plasmids from environmental microorganisms by means of biparental and triparental matings without the need to cultivate the host (Smalla et al, 2015).…”

Proma Plasmids are Instrumental in the Dissemination of Linuron Catabolic Genes between Different Genera

“…PBS-H4, although this strain lacks the dca cluster genes that are required for complete linuron mineralization (Öztürk et al, 2019). Remarkably, the catabolic PromA plasmids have a near-identical backbone to the previously-described PromA γ plasmids isolated in Japan (Yanagiya et al, 2018). It has been reported before that BHR plasmids isolated from different geographic locations can be highly-conserved (Li et al, 2016; Heuer et al, 2004; Chen et al, 2015b), however, some degree of divergence in the backbone structures of PromA plasmid groups were reported before (Li et al, 2014).…”

“…(Öztürk et al, 2019) (Figure 1). Indeed, RepA-based phylogenetic analysis showed that these plasmids all belong to the PromA γ group, together with the plasmids pSN1104-11 and pSN1104-34 (Yanagiya et al, 2018) (Figure 2).…”

“…These so far cryptic plasmids have no apparent benefit to the host and are still propagated in absence of selective pressure (Fox et al, 2008). A recently discovered group of BHR plasmids are the PromA plasmids, most of which were isolated by exogenous plasmid capture (Thomas et al, 2017; Van der Auwera et al, 2009; Schneiker et al, 2001; Yanagiya et al, 2018; Li et al, 2014; Tauch et al, 2002) hence from unknown hosts, but also few derived from Proteobacterial isolates (Mela et al, 2008; Ito and Iizuka, 1971; Van der Auwera et al, 2009). With the exception of SFA231 (Li et al, 2014), pMOL98 (Van der Auwera et al, 2009) and pSB102 (Schneiker et al, 2001) which carry heavy metal resistance-related genes, all 12 completely sequenced PromA plasmids identified to date are cryptic plasmids with no clear indication of their ecological or possible benefit for the host organism.…”

“…The catechol intermediate is further degraded to Krebs cycle intermediates by the enzymes encoded by the ccdCFDER gene cluster (Bers et al 2011, 2013). The two Variovorax PromA plasmids belong to the PromA γ subgroup together with the plasmids pSN1104-11 and pSN1104-34 (Yanagiya et al, 2018) that were exogeneously isolated from cow manure. pPBS-H4-2 is the first PromA plasmid that carries catabolic genes, i.e., it carries a stretch of DNA containing several gene clusters involved in the degradation of linuron and several IS 1071 elements, of which two border the cargo at both ends.…”