Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

Abstract

NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are wellcharacterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriT N ) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences that were important for efficient conjugation in the oriT N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT N region, between two copies of the pWW0 oriT (oriT W ) region (which is clearly different from the oriT N region), and between the oriT N and oriT W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT Ncontaining plasmid. Such a plasmid was, in the presence of the NAH7Δnic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT-relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.KEYWORDS Pseudomonas, conjugation, oriT, plasmid, relaxase C onjugative transfer of plasmids that carry various genetic traits contributes greatly to the rapid adaptation and evolution of host bacteria (1). The conjugative transfer of plasmids in Gram-negative bacteria consists of DNA transfer and replication (Dtr) and

Multiple plasmid origin-of-transfer substrates enable the spread of natural antimicrobial resistance to human pathogens

“…Contrary to the expectations based on the 1377 distinct relaxases found in the target dataset accordance with published findings showing that relaxases can function with relaxed specificity on non-cognate oriTs with~60% sequence homology 25,26,32 . Firstly, within a given Mob group, sequence homologies between an oriT hit and its closest query oriT region were above 60% within both the nic sites ( Fig 1B: ±5 bp around nic ) and the 40 bp relaxase-binding sites, as was also the case with the majority of larger sized subregions, where the median sequence homologies were well above the ones of the query dataset ( Fig 2D, Fig S2-3, Methods M4).…”

“…However, besides the possibility of yet unidentified enzymes and mobility groups 15,[20][21][22][23][24] , multiple new processes have recently been uncovered that might confer additional mobility to plasmids and involve the origin-of-transfer (oriT) DNA substrate. These include (i) broadened relaxase binding specificities to multiple different oriT sequence variants [25][26][27][28][29] , which, according to the evolutionary theory of such DNA regions 4,30,31 , indicates the possibility of plasmids carrying multiple functional secondary oriTs, and (ii) trans-mobilization of plasmids carrying oriTs triggered by relaxases from co-resident plasmids acting in trans on the non-cognate oriTs [32][33][34][35] . The latter mechanism demonstrates that oriT regions are the only elements of the conjugation machinery required in cis 21 and suggests that many plasmids classified as non-mobile due to the absence of putative relaxases may in fact be mobilizable 33 .…”

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

“…This oriT recombination was likely mediated by the relaxase of the ICE, which is able to recognize and nick this sequence. Such oriT site-specific recombination has already been demonstrated for several canonical relaxases of plasmids (belonging to the MobF, MobP, MobC, and MobM families) (4,28,29) and has been suggested to occur in ICEs of the SXT/R391 family and for ICEclc (both encoding canonical relaxases of the MobH family) (30,31). This mechanism that enables creation of hybrid ICEs could thus also be mediated by relaxases of the noncanonical MobT family, which are related to rolling circle replication initiators and are found in ICEs of the Tn916/ICESt3/ICEBs1 family, a family of ICEs widespread in streptococci (11) and other Firmicutes (5).…”

Implications of microbial adaptation for the assessment of environmental persistence of chemicals

“…The IncP plasmid group is one of the best characterized and described so called broad host range (BHR) plasmids, which are known to be responsible for the transfer of resistance and degradation genes in a wide range of hosts (De Gelder, Williams, Ponciano, Sota, & Top, 2008;Martin et al, 2015). The genes involved in the degradation of organophosphorus insecticides (Bhadbhade et al, 2002;Horne, Sutherland, Harcourt, Russell, & Oakeshott, 2002), carbaryl (Hashimoto et al, 2006;Singh, Trivedi, & Phale, 2013), toluene and naphthalene (Kishida, Inoue, Ohtsubo, Nagata, & Tsuda, 2016), chlorophenols (Ma, Quan, Yang, & Li, 2012;Trefault et al, 2004), 2,4,6-trichlorophenol (Ma et al, 2012) and atrazine (Aislabie et al, 2005;de Souza et al, 1998;Rousseaux et al, 2002) are also contained on BHR plasmids. Plasmidencoded pathways are advantageous for microbial communities because they provide genetically flexible systems and can be maintained in the population and transferred between bacterial species by conjugation (Sayler, Hooper, Layton, & King, 1990).…”