Microbes Environ. Vol. 25, No. 4, 253–265, 2010 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10155 Minireview

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in within Natural Environments and Microcosms

MASAKI SHINTANI1,2, YURIKA TAKAHASHI2, HISAKAZU YAMANE2, and HIDEAKI NOJIRI2* 1Bioresource Center, Japan Collection of Microorganisms (BRC-JCM), Riken, 2–1 Hirosawa, Wako, Saitama 351–0198, Japan; and 2Biotechnology Research Center, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan (Received August 3, 2010—Accepted September 30, 2010—Published online October 21, 2010)

Over the past few decades, degradative plasmids have been isolated from capable of degrading a variety of both natural and man-made compounds. Degradative plasmids belonging to three incompatibility (Inc) groups in Pseudomonas (IncP-1, P-7, and P-9) have been well studied in terms of their replication, maintenance, and capacity for conjugative transfer. The host ranges of these plasmids are determined by replication or conjugative transfer systems. The host range of IncP-1 is broad, that of IncP-9 is intermediate, and that of IncP-7 is narrow. To understand the behavior of these plasmids and their hosts in various environments, the survivability of inocula, stability or trans- ferability, and efficiency of biodegradation in environments and microcosms have been monitored. The biodegradation and plasmid transfer in various environments have been observed for all three groups, although the kinds of trans- conjugants differed with the Inc groups. In some cases, the deletion and amplification of catabolic genes acted to reduce the production of toxic catabolic intermediates, or to increase the activity on a particular catabolic pathway. The combination of degradative genes, the plasmid backbone of each Inc group, and the host of the plasmids is key to the degraders adapting to various hosts or to heterogeneous environments. Key words: degradative plasmid, IncP plasmid, conjugative transfer, bioaugmentation

Introduction microorganisms may become attenuated (relative to the ini- tial inoculum) during growth at contaminated sites, which is Plasmids are bacterial mobile genetic elements that facili- particularly true in soil. Adequate dispersion of biodegrada- tate rapid evolution and adaptation by conjugative transfer tive microorganisms over contaminated areas is also prob- between bacterial cells in the natural environment. To date lematic. To overcome these problems, ‘gene bioaugmenta- (the end of Sept. 2010), the nucleotide sequences of 2178 plas- tion’ or ‘plasmid-mediated bioaugmentation’ (4, 7, 23, 82), mids have been determined and deposited in the NCBI data- the introduction of appropriate degradative plasmids con- base (http://www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxid tained within a microbial host, has been attempted. The =2&type=2&name=BacteriaPlasmids). Some plasmids con- rationale for this approach is that the degradative genes fer antibiotic resistance or virulence on the host bacterium, survive through incorporation into indigenous bacteria by while others—known as degradative plasmids—enhance the horizontal transfer. Additionally, plasmid transfer may result ability of the host cell to catabolize xenobiotic compounds in spatial movement of the catabolic genes through the soil, (33, 97). Here we use the term ‘xenobiotic compounds’ resulting in dissemination of the desired catabolic activity in a broad sense, to include “all compounds that are released into deeper layers. The artificial spread of specific genes in into the environment by human actions and whose concentra- the natural environment, however, may not be desirable. tions are higher than natural” (54). Over the past decade, Therefore, understanding the behavior of degradative plas- xenobiotic-degradative plasmids have been found in various mids and their hosts in natural microcosms is necessary biodegradative microorganisms isolated from geographically not only for elucidating bacterial evolution and adaptation diverse locations (25, 74, 75, 78, 113, 126). The genomes of systems, but also for evaluating the benefits and risks of some of these have been sequenced, and the capacity for bioaugmentation itself. replication, partition, and conjugative transfer were assessed Plasmids are classified into incompatibility (Inc) groups (35, 50, 52, 109). based on their replication and partition systems: two plas- Bioaugmentation is a method of removing xenobiotic mids belonging to the same Inc group are unable to coexist in compounds from contaminated sites via the introduction of one bacterial cell (i.e., they are incompatible). Members of biodegradative microorganisms. However, this method does the same group have a common plasmid backbone yielding not always successfully remove the contaminating sub- their basic features. There are at least 14 Inc groups in stances. Furthermore, the relationships among inoculum Pseudomonas, which are completely different from those of size, survival and fate of the inocula, and efficacy of con- E. coli, although some groups in each classification form a tamination removal remain unclear (32, 110). Biodegradative single Inc group; e.g., IncP-1 in the Pseudomonas plasmid classification corresponds to IncP in the Escherichia coli * Corresponding author. E-mail: [email protected]; plasmid classification (108). In this review, we use ‘Inc Tel: +81–3–5841–3064; Fax: +81–3–5841–8030. groups in Pseudomonas’ to indicate the groups IncP-1, P-2, 254 SHINTANI et al.

P-3, and so on, although these plasmids do not necessarily replication in host Pseudomonas but is not functional in exist in Pseudomonas bacteria. Among the degradative E. coli or in other subdivisions of (92, 129). plasmids, IncP-1, P-2, P-7, and P-9 are present in Gram- Plasmid maintenance. An active partitioning system is negative bacteria (25, 74, 75, 78), although many others, commonly found in these Inc group plasmids, with ParA, a including plasmids from Gram-positive bacteria, probably Walker-type ATPase, ParB, a DNA-binding protein, parS, a belong to unidentified Inc groups (74, 78). In this review, ParB-binding centromere-like site (for detail, please see we focus on the degradative plasmids in Inc groups in recent review by Funnell and Slavcev (35)). The par systems Pseudomonas, because the basic features of three plasmid of IncP-1 and IncP-9 are closely related at the protein level, groups (excluding IncP-2) under laboratory conditions have but the IncP-7 par system is quite distinct (108). The been well-characterized (see below). First, the basic features ParA and ParB homologues in the IncP-1 system are IncC brought about by the plasmid backbone of three Inc groups in and KorB (1). The most likely candidate for the parS site Pseudomonas are summarized and then their behavior in a of IncP-1 plasmids is located downstream of korB (35). variety of microcosms is described. IncP-1 plasmids have been considered stable in most hosts, however; De Gelder and coworkers (22) found hosts in which Basic features of IncP-1, IncP-7 and IncP-9 plasmids the plasmid was not stable, although why remains unclear. IncP-9 plasmids also have this system (parABkorAparC is Host range of plasmids. Host range is particularly impor- located on oriV-rep upstream), although detailed functional tant when considering the behavior of degradative plasmids analyses have not been reported. Intriguingly, the oriV-rep in natural environments and microcosms. The hosts of the system of IncP-9 cannot function in E. coli unless it is degradative plasmids in the IncP-1, IncP-7 and IncP-9 accompanied by at least part of the par region in cis (88). As groups are listed in Table 1, as determined by the identifica- for the IncP-7 plasmids, products of parWAB are essential tion of isolated degrading bacteria, transconjugants obtained for their maintenance, although the parS site has not been by mating assays with single recipients, or transconjugants determined; and the function of ParW is still unclear (92). from environmental samples after inoculation of the plasmid Plasmid transfer. The conjugative transfer of a plasmid is host cells. The definition of the host range of plasmids initiated by the cleavage of DNA in the origin of transfer remains controversial. “Broad host range” plasmids have (oriT) by a protein named relaxase that covalently binds to been proposed to be able to replicate themselves and be the oriT region. Afterward, the DNA-relaxase complex is maintained stably without any selections in at least two transported into the recipient cell by an export system, the different subdivisions of Proteobacteria (108). According type IV secretion system. These events are regulated by to this definition, IncP-1 plasmids have a broad host range, two systems: Mating Pair Formation (Mpf) and DNA and IncP-7 plasmids a narrow host range (Table 1). As for Transfer and Replication (Dtr). Additionally, a coupling the IncP-9 plasmids, there are several reports that this group protein, associated with the cell membrane, actively pumps is found in β-Proteobacteria (Table 1), though their stability the DNA into the recipient cell (33). A plasmid that carries without selection markers was not been assessed. In this all the gene sets needed for its conjugative transfer is called review, we recognize the IncP-9 plasmids as having an “self-transmissible”. Small plasmids only carry the gene set ‘intermediate’ host range. Because the host ranges of plas- for Dtr (mob for mobilizable), and require a conjugative mids are limited by replication (maintenance) or conjugative system to mobilize them. Both IncP-1 and IncP-9 plasmids transfer systems, these features of the three Inc groups are have two DNA regions encoding Dtr and Mpf systems, Tra1 described in more detail below. (tra genes for Dtr system) and Tra2 (trb genes for Mpf) on Plasmid replication. All of the plasmids in the three Inc IncP-1 (only traF is located in the Tra1 region, the product of groups contain replicons that depend on at least one Rep which is involved in the Mpf system) and tra (Dtr) and mpf protein and a replication origin (oriV) with Rep-binding sites (Mpf) genes of IncP-9 plasmids. The products of genes for (iterons). The Rep-oriV system of IncP-1 plasmids is TrfA the Mpf system of these two Inc groups show similarities to and oriV (oriV is located on trfA downstream), and trfA each other (51). On the other hand, IncP-7 plasmids carry the encodes two in-frame products, TrfA-1 (TrfA44) and TrfA-2 equivalent gene sets (tra for Dtr, and trh for Mpf, note that (TrfA33) (1, 108). The length of the essential oriV region and trhF, traF, and traD were renamed trhP, trhF, and traG, the necessity of TrfA44 can alter in different hosts (1). The 129), although they show more similarity to the systems replication system of IncP-1 plasmids may have flexibility found from the plasmids of Enterobacteriaceae than to in different hosts resulting in a broad host range. IncP-9 those of IncP-1 or IncP-9 plasmids (56, 129). As for the plasmids have long been known to replicate in a wide range Dtr system, Garcillán-Barcia and coworkers proposed six of bacteria of the genus Pseudomonas, but many are unstable families of MOB based on a classification of the essential and replication-defective at higher temperatures in other taxa protein for transfer machinery, relaxases (38). Interestingly, such as E. coli (10). The oriV-rep fragment (oriV is located the Dtr systems of the three Inc group plasmids belong on rep upstream) of minimal replicon of the IncP-9 plasmid to distinct families; IncP-1 (TraI) to MOBP, IncP-7 (TraI) pMT3 is sufficient for replication in P. putida but not E. coli to MOBH, and IncP-9 (TraC) to MOBF (38). The location because of a difference in rep promoter activity between of the oriT of IncP-1 and IncP-9 plasmids has already these two hosts (88). The amount of Rep protein is suggested been determined experimentally (39, 81), whereas that of to be important for the replication of IncP-9 plasmids (88). IncP-7 is still not clear, although the putative oriT region The oriV-repA (oriV is located on repA upstream) system is was suggested (56, 129). The transcriptional regulation of also found in the IncP-7 plasmids, and sufficient for genes for conjugative transfer on IncP-1 plasmids has been Behavior of Degradative Plasmids within Microcosms 255

Table 1. Host ranges of IncP-1, IncP-9, and IncP-7 degradative plasmids.

Inc group Bacterial class Bacterial order Bacterial family Genus Species Plasmida Reference IncP-1 α-Proteobacteria Rhizobiales Brucellaceae Ochrobactrum anthropi pC1g 37 Ochrobactrum tritici pC1g 37 Ochrobacterium sp. pJP4h 5 Rhizobiaceae Agrobacterium tumefaciensb pC1g, pJP4h 29, 37 Agrobacterium sp. pJP4h 5 Ensifer sp. pJP4h 5 Rhizobium sp. pJP4h 5, 29 Rhodobacterales Rhodobacteraceae Rhodobacter sphaeroidesc pJP4i 29 Sphingobacteriales Sphingobacteriaceae Sphingobacterium sp. RP4::Tn4371i 123 Blastomonas natatoria pC1g 37 β-Proteobacteria Burkholderiales Alcaligenaceae Achromobacter xylosoxidans pA81j, pEST4011j 46, 123 Achromobacter sp. pC1g, pSS60j 14, 37 Alcaligenes denitrificans RP4::Tn4371i 122 Alcaligenes sp. pBRC60k, pJP4h 5, 34 Burkholderiaceae Burkholderia caryophylli pJP4k 28 Burkholderia cepaciad pBRC60k, pIJB1j 34, 127 Burkholderia glatheie pJP4g,k 28, 36 Burkholderia hospita pJP4g 36 Burkholderia terricola pJP4g 36 Burkholderia sp. pJP4g,h, pPS12–1j 2, 9, 36 Cupriavidus campinensis pJP4g 36 Cupriavidus necatorg pC1g, pENH91j, pJP4j 29, 37, 77 Comamonadaceae Brachymonas denitrificans pC1g 37 Comamonadaceae bacterium pNB2h 4 Comamonas testosteroni pNB2h, pTSAj 4, 7, 48 Commamonas sp. pJP4g, RP4::Tn4371i 5, 26 Delftia acidovorans pBRC60k, pC1i, pUO1j 37, 49, 99, 100 Delftia tsuruhatensis pNB2h 4 Delftia sp. pJP4h 5 Variovorax paradoxus pJP4h 5 γ-Proteobacteria Aeromonadales Aeromonadaceae Aeromonas sp. pC1g 37 Enterobacteriales Enterobacteriaceae Enterobacter agglomerans RP4::Tn4371i 26 Escherichia coli pC1g, pJP4i, RP4::Tn4371i 26, 28, 37 Klebsiella sp. pC1g, pJP4h 5, 37 Serratia fonticola pC1g 37 Moraxellaceae Acinetobacter calcoaceticus pJP4k 29 Acinetobacter johnsonii RP4::Tn4371i 123 Acinetobacter sp. pBRC60k 34 Mollexella sp. pBRC60k 34 RP4::Tn4371i 26 Pseudomonas corrugata. RP4::Tn4371i 26 Pseudomonas fluorescens pJP4k, RP4::Tn4371i 29, 123 Pseudomonas putida pAC25j, pBRC60k, pC1l, pJP4l, pNB2l, 4, 5, 6, 7, 16, 34, 37, RP4::Tn4371l 123 Pseudomonas sp. pADP-1j, pBRC60k, pJP4h 5, 34, 57 Xanthomonadales Xanthomonadaceae Stenotrophomonas maltophilia pJP4g 36 Stenotrophomonas sp. pJP4h 5 Xanthomonas sp. pJP4h 5 IncP-9 β-Proteobacteria Burkholderiales Alcaligenaceae Alcaligenes sp. pSAHj 45 Comamonadaceae Hydrogenophaga palleronii pWW0o 67 γ-Proteobacteria Enterobacteriales Enterobacteriaceae Escherichia coli pWW0k 10, 84 Erwinia chrysanthemi pWW0k 84 Erwinia sp. pWW0h 62 Serratia ficaria pWW0o 67 Serratia liquefaciens pWW0o 67 Pseudomonadales Pseudomonadaceae pNL31j, pWW0h 10, 84, 89 Pseudomonas aureofaciens pOV17j 89 Pseudomonas fluorescens pNAH20j, pNL22j, pNL60j, pWW0k 41, 84, 89 Pseudomonas putida NAH7j, NPL-1j, pBS240j, pBS243j, pBS265j, 15, 24, 39, 41, 43, 88, pBS267j, pBS268j, pBS1141j, pBS1181j, pBS1191j, 87, 101, 104, 124 pDTG1j, pNAH20k, pNF143j, pNL4j, pNL25j, pSN11j, pSVS15j, pWW0j, SAL1j Pseudomonas stutzeri pWW0k 84 Pseudomonas sp. p8Cj, p15Cj, pNL29j, pWW0h 43, 62 IncP-7 γ-Proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas aeruginosa pCAR1k,m 95, 106 Pseudomonas chlororaphis pCAR1k,m 90 Pseudomonas fluorescens pCAR1k,m, pDK1k, pFME4j, pFME5j, pNK33j, 43, 90, 95, 106 pNK43j, pOS18j, pOS19j Pseudomonas putida pAK5j, pCAR1k,m, pDK1j, pWW53j,n 43, 91, 106, 128, 129 Pseudomonas resinovorans pCAR1j,m 56 Pseudomonas stutzeri pCAR1k,m 90 Pseudomonas sp. pCAR1h,m, pND6-1j,n 55, 93 Xanthomonadales Xanthomonadaceae Stenotrophomonas maltophila pCAR1h,m 93 a Substrates of the host of each degradative plasmid: pADP-1, atrazine; pBS265, pBS267, pBS268, ε-caprolactam; pBRC60, pEST4011, pIJB1, pJP4, 2,4-Dichlorophenoxyacetic acid; NAH7, NPL-1, p8C, p15C, pBS240, pBS243, pBS1141, pBS1181, pBS1191, pC1, pDTG1, pNK33, pNK43, pNF143, pOS18, pOS19, pFME4, pFME5, pAK5, pNAH20, pNL4, pNL22, pNL25, pNL29, pNL31, pNL60, pOV17, pSN11, naphthalane, pA81, (halo)aromatic acid; pA8, chlorobenzoate; pSS60, 4-chlorobenzoate; pAC25, pENH91: 3- cholorobenzoate, pPS12–1, tetrachlorobenzene, pTSA, p-toluenesulfonic acid, pDK1, pSVS15, pWW0, pWW53, toluene/xylene, SAL1, salicylate, pCAR1, carbazole. RP4::Tn4371, biphenyl (IncP-1 plasmid RP4 carrying 59-kb biphenyl catabolic transposon Tn4371) (104). b Including the previous host name Rhizobium radiobacter (29). c Previous name was Rhodopseudomonas sphaeroides (29). d Including the previous host name Pseudomonas cepacia (34). e Including the previous host name Pseudomonas glathei (28). f Previous name was Ralstonia eutropha, but currently re-identified as Cupriavidus necator (120). g A combination of genotypic and phenotypic information was employed to identify the transconjugants (36, 37). h Partial sequences of 16S rRNA genes were determined to identify the transconjugants (2, 4, 5, 7). i The transconjugant properties were identified to the species level with BIOLOG system (26, 28, 123). j The plasmid is isolated from the species. k Transconjugants were obtained by mating assays with single recipients l The inoculated strain to determine the plasmid host range (4) m pCAR1 and pCAR2 were previously identified as pCAR1.1, and pCAR1.2 due to a single nucleotide substitution, but we recognize them as being the same plasmid and refer to them as pCAR1 in this paper. n This plasmid has lost genes essential for conjugative transfer. o Transconjugants were detected by the expression of green fluorescens (67). 256 SHINTANI et al. investigated in detail (see recent reviews by Zechner et al. Measurements of inoculum survival and bacterial numbers (131) and Lawley et al. (52)), whereas that on IncP-9 plasmids are performed through viable counting. Changes in the con- has only been reported by Lambertsen and colleagues (51). centrations of xenobiotic compounds are quantified by It is of note that the products of the traD operon on the methods such as HPLC and GC-MS. Several techniques IncP-9 plasmid NAH7 were shown to be a host-range mod- have been used to detect plasmid transfer, as reviewed by ifier in conjugative transfer (61). The IncP-7 plasmid pCAR1 Sørensen and coworkers (98). The simplest approach is to has at least four operons related to conjugative transfer (90), identify plasmid-encoded phenotypes, including the degrada- however, a detailed analysis of its transcriptional regulation tion of xenobiotics (69, 111). lacZ and fluorescent proteins has not yet been performed. (GFP and RFP) are useful markers for the detection of plasmid transfer (17, 20, 44, 76). This method enables the Behavior of degradative plasmids in environments detection of transconjugants at the single-cell level by fluo- rescence microscopy and has also been used for determining The behavior of plasmids depends upon the basic features plasmid host range (21). The behavior of each degradative mentioned above, their capacity for replication, stability, and plasmid is described below in three sections according to conjugative transfer. In addition, the degradative capability host ranges, (i) the broad-host-range IncP-1 group, (ii) the of the host is important, which can differ according to the intermediate-host range IncP-9 group, and (iii) the narrow bacterial species and/or conditions under which it is cultured host range IncP-7 group. (e.g., 11, 21, 22, 90, 113). This is because degradative Behavior of the broad host range IncP-1 group in the capacity depends primarily on the host bacterium’s meta- environment. IncP-1 is one of the most well-studied of all bolic machinery. Indeed, degradation pathways usually com- plasmid groups (1, 86). Plasmids belonging to this group prise multiple reactions, catalyzed by enzymes encoded not have a broad host range and a high transfer frequency (25, only on the degradative plasmid, but also within chromo- 103). The transfer of these plasmids occurs not only among somal DNA. Therefore, degradation pathways, rates, and the Proteobacteria, but also between bacteria of other phyla inducers can be manipulated by altering the bacterial host (1). Note that many degradative plasmids belong to the strain and/or the plasmid. The presence of homologous or IncP-1 group, and that the nucleotide sequences of many similar degradative genes involved in the metabolism of such plasmids have been determined, e.g., pA81, pADP-1, intermediate compounds on both the plasmid and host pJP4, pEST4011, and pUO1 (46, 57, 100, 114, 121). The chromosome (i.e. more than two copies of genes exist in one core regions of IncP-1 group plasmids show high identity. cell) can also affect host survivability. Indeed, in some cases, Accessory genes are usually carried by a transposon inserted plasmid and chromosomal DNA are rearranged (including in two regions, first between the oriV and the trfA genes deletion and amplification by homologous recombination) (involved in replication initiation), and second, between the (Table 2) because some degradative genes are unnecessary, tra (Dtr) and trb (Mpf) operons (1, 86, 102). This suggests or even undesirable, for the host bacterium. In these cases, that IncP-1 is one of the vectors responsible for the dissemi- the initial pure culture may quickly become genotypically nation of accessory genes among environmental bacteria. heterogeneous due to the generation of mutants. Therefore, As shown in Table 2, DNA rearrangements on the 2,4- known DNA rearrangements on degradative plasmids are dichlorophenoxyacetic acid (2,4-D)-degradative plasmid pJP4 described herein. have been detected both in pure cultures containing 3- Plasmid behavior in natural environments and micro- chlorobenzoate and during long-term cultivation (19, 66, cosms has been monitored by measuring inoculum survival, 114; Fig. 1A). In the former case, the host bacterium lost changes in concentrations of xenobiotic compounds, and the ability to grow on 2,4-D due to a deletion of about 16 kb rates of conjugative transfer to indigenous bacteria (Table 3). of DNA that includes the tfdA gene. However, the ability of

Table 2. DNA rearrangements of degradative plasmids in ‘heterogenic’ microcosms Inc group Plasmid Host DNA rearrangement Substrate Reference IncP-1 pJP4 Ralstonia eutropha JMP134a deletion & duplication 3-CBb 19 pJP4 Ralstonia sp. TFD41 deletion & duplication 2,4-Dc 66 pADP1 Pseudomonas sp. ADP duplication Atrazine 27 IncP-1 pENH91 Alcaligenes eutrophus NH9 deletion 3-CBb 77 IncP-7 pCAR1d Pseudomonas resinovorans CA10 deletion Luria broth 119 pCAR1d Pseudomonas fluorescens Pf0-1 deletion Carbazole 106 IncP-9 pWW0 Pseudomonas putida mt-2 deletion Benzoate 125 pWW0 Pseudomonas putida mt-2 deletion Succinate-, sulfate-, ammonium-, or 31 phosphate-limited condition pWW0 Pseudomonas putida 54g deletion Various substrate 53 pWW0 Pseudomonas putida mt-2 deletion Toluene 63 a Currently re-identified as Cupriavidus necator (120). b 3-Chlorobenzoate c 2,4-Dichlorophenoxyacetic acid d pCAR1 and pCAR2 were previously identified as pCAR1.1, and pCAR1.2 due to a single nucleotide substitution, but we recognize them as being the same plasmid and refer to them as pCAR1 in this paper. Behavior of Degradative Plasmids within Microcosms 257

Table 3. Examples of monitoring the behavior of degradative plasmids in different microcosms Substrate Inc Site(s) of Inoculum(s) Donor Plasmid Plasmid Substrate i Reference group microcosm (host of plasmid) survivabilityh degradation transferj (or mineralization) IncP-1 pBRC60 Freshwater 3-CBb Alcaligenes sp. BR60 ND + + 34 ecosystem RP4::Tn4371a Soil Biphenyl Enterobacter agglomerans − ++26 DMK3 RP4::Tn4371a Soil Biphenyl Pseudomonas putida C8S3 + − +123 pJP4 Soil 2,4-Dc Alcaligenes eutrophus JMP134f ND ND + 69 pJP4 Soil 2,4-Dc Alcaligenes eutrophus JMP134f ND + + 28 pJP4 Soil 2,4-Dc Pseudomonas putida UWC3 − ++23 pJP4 Soil (greenhouse) 2,4-Dc Escherichia coli D11 + + + 70, 71 Ralstonia eutropha JMP134f +++ pJP4 Soil (column) 2,4-Dc Escherichia coli D11 + + + 72 pJP4::gfp Biofilm in flow cell 2,4-Dc Pseudomonas putida SM1443g +++2 chambers pJP4::DsRed Activated sludge 2,4-Dc Pseudomonas putida SM1443g ND ND + 5 pJP4::DsRed Batch biofilm 2,4-Dc Pseudomonas putida SM1443g +++6 pNB2::DsRed Activated sludge 3-CAd Pseudomonas putida SM1443g ND + + 4 pNB2::DsRed Model waste water 3-CAd Pseudomonas putida SM1443g +++7 treatment reactor pNB2::DsRed Semicontinuous 3-CAd Comamonas testosteroni SB3 + + + 8 activated sludge reactors pC1gfp Activated sludge 3-CAd Pseudomonas putida UWC3 ND + + 36 IncP-9 pWW0-EB62 Soil p-etylbenzoate Pseudomonas putida EEZ15 + ND + 83, 84 pWW0::gfp Biofilm (consisted BAe Pseudomonas putida R1 + ND + 18 of three species) Pseudomonas putida SM1443g + ND + pWW0::gfp Phylloplane − Pseudomonas putida KT2442g +ND+76 (leaf surface) pWW0::gfp Biofilm (consisted BAe Pseudomonas putida R1 + ND + 40 of two strains) Pseudomonas putida SM1443g + ND + pWW0::gfp Alfalfa sprout − Pseudomonas putida LM50g +++62 pWW0::gfp Biofilm BAe Pseudomonas putida +ND+67 TUM-PP12g pWW0 Aerobic granular BAe Pseudomonas putida +++68 sludge TUM-PP12g pWW0 Batch biofilm BAe Pseudomonas putida +++122 TUM-PP12g IncP-7 pCAR1 Soil Carbazole Pseudomonas putida SM1443g ND −−93 River water ND + − pCAR1 Artificial soil Carbazole Pseudomonas putida SM1443g ++− 94 microcosm Artificial environ- +++ mental water pCAR1 Artificial soil Carbazole Pseudomonas fluorescens −−−96 microcosm Pf0-1L Pseudomonas resinovorans + −− CA10L Pseudomonas chlororaphis + −− IAM1511L Artificial environ- Pseudomonas fluorescens − + − mental water Pf0-1L Pseudomonas resinovorans ++− CA10L Pseudomonas chlororaphis +++ IAM1511L a IncP-1 plasmid RP4 carrying 59-kb biphenyl catabolic transposon Tn4371 (104) b 3-Chlorobenzoate c 2,4-Dichlorophenoxyacetic acid d 3-Chloroaniline e Benzyl alcohol f Currently identified as Cupriavidus necator (120) g P. putida KT2440 derivative strains h ‘+’ indicates that donors are detected during the monitoring, whereas ‘−’ means that donors are not detected. ‘ND’ means not determined. i ‘+’ or ‘−’ indicates that substrate degradation is detected or not. ‘ND’ means not determined. j ‘+’ or ‘−’ indicates that plasmid transfer is detected or not. 258 SHINTANI et al.

Fig. 1. (A) The 2,4-dichlorophenoxyacetate (2,4-D)-catabolic pathway in Cupriavidus strains harboring pJP4, with the 3-chlorobenzoate (3-CB)-catabolic pathway. Solid and dashed arrows indicate conversions catalyzed by the Tfd enzymes encoded on either pJP4 or the chromosome. TfdA, 2,4-D/α-ketoglutarate dioxygenase; TfdB, 2,4-dichlorophenol hydroxylase; TfdC, chlorocatechol-1,2-dioxygenase; TfdD, chloromuconate cycloisomerase; TfdE, dienelactone hydrolase; TfdF, maleylacetate reductase; BenABC, benzoate 1,2-dioxygenase; BenD, cis-dihydroxycyclohexa- 3,5-diene-1-carboxylate dehydrogenase; TCA, tricarboxylic acid. (B) Organization of the pJP4 tfd genes and insertion sequences (ISs). Black, gray, and white pentagons indicate regulatory genes, catabolic genes and other unrelated ORFs, respectively, and rectangles, ISs. Triangles indicate the positions of DNA regions containing IS remnants. this strain to grow on 3-chlorobenzoate was increased due introduced pJP4 into Escherichia coli D11, a strain that to duplication of an approximately 23-kb region that in- lacks the chromosomal genes essential for mineralization of cludes the tfdCDEF gene cluster (19, 114; Fig. 1B). These 2,4-D. The transformed strain was then inoculated into 2,4- rearrangements were mediated by homologous recombi- D-contaminated sites (70). Degradation of 2,4-D and trans- nation between the conserved regions of two copies of the conjugants of pJP4 among indigenous bacteria were insertion sequence IS1071, although one was truncated (19, detected, suggesting that plasmid transfer was responsible for 114; Fig. 1B). Similarly, DNA rearrangement on the atrazine the degradation (70). Moreover, degradation proceeded more catabolic plasmid pADP-1 was reported, also mediated by the rapidly when E. coli was used as the inoculum than when insertion sequences IS1071 and ISPps1 (27). IS1071-like C. necator was introduced (70). This demonstrates that the elements are frequently found on IncP-1 group plasmids choice of donor microorganisms is vital for efficient bioaug- (other than pJP4 and pADP-1) isolated from various environ- mentation. Goris and coworkers (36) assessed the diversity ments, e.g., the haloacetate-degradative plasmid pUO1 (99, of transconjugants of pJP4 (and pEMT1, a non-IncP-1 2,4-D 100), 3-chloroanilne-degradative plasmids (pNB1, pNB2, degradative plasmid) (112) isolated from soil samples taken pB8c, and pC1; 12), and the arylsulfonates-degradative at varying depths (23). Most transconjugants from the soil plasmid pTSA (48). These findings indicate that this IS samples belonged to β- and γ-Proteobacteria, probably potentially imparts genetic plasticity to the plasmid, such because the capacity to degrade 2,4-D can be expressed effi- as recombination between two copies of an IS, and plays an ciently in these two classes, but not in the α-Proteobacteria important role in the evolution of the catabolic activities that (36). pNB2 is another IncP-1 plasmid that carries some of IncP-1 plasmids would confer on their hosts. the genes required for 3-chloroaniline’s degradation (12), In the past two decades, many reports on the behavior although its complete nucleotide sequence has not yet been of IncP-1 plasmids in natural microcosms have been pub- determined. This plasmid is also transferable to indigenous lished (Table 3). First, the conjugative transfer of pBRC60 bacteria, and the transconjugants detected principally (3-chlorobenzoate degradation) to bacteria indigenous to a belonged to the family Comamonadaceae within the β- freshwater ecosystem was detected (34). Subsequently, the Proteobacteria, in activated sludge or biofilm reactors, in transfer of RP4::Tn4371 (biphenyl degradation, IncP-1 plas- contrast to pJP4 (4, 7, 8). Some γ-Proteobacteria are not able mid RP4 carrying 59-kb biphenyl catabolic transposon to express 3-chloroaniline-degradative enzymes efficiently Tn4371; 104) to indigenous bacteria in soil samples was (4; 7, 36), resulting in biases in the spectra of transconju- observed after the rapid disappearance of the donor strain gants. De Gelder and colleagues (21) reported that the IncP-1 (26). The behavior of pJP4 in soil, activated sludge, and bio- plasmid pB10 that carries mercury resistance genes (30, film has been well studied (Table 3). Neilson and colleagues 85) transferred preferentially to α- or γ-Proteobacteria. demonstrated the conjugative transfer of pJP4 in soil samples Transconjugants belonging to the β-Proteobacteria were not using Alcaligenes eutrophaus (now renamed as Cupriavidus detected (21). Although the effect of the reporter gene rfp necator; 120) JMP134 as a donor and Variovorax paradoxus (red fluorescent protein) inserted into the traC gene of pB10 as a recipient (69). Several reports have suggested that pJP4 could not be excluded, it seems likely that apparent host is able to move to indigenous bacteria in soil, activated range differs according to the detection method used, e.g., sludge, and biofilm (Table 3). Newby and Pepper (70) expression of a fluorescent protein, antibiotic resistance, or Behavior of Degradative Plasmids within Microcosms 259 xenobiotic degradation. Consequently, IncP-1 degradative host cells with wild-type pWW0 (toluene-degrading cells) plasmids may be transferred to a variety of bacteria in natural recovered from 5% to 100% (63). Note that these DNA microcosms, almost as though they ‘select’ the best host for rearrangements represent substrate-dependent evolution of xenobiotic degradation. the plasmid, and the specific 39-kb deletion was mediated by recombination between two identical IS1246 (39; Fig. 2B). Behavior of the intermediate host range IncP-9 plasmids in The capacity of the IncP-9 plasmid pWW0 to act as an the environment agent of bioaugmentation, and the risks thereof, have been studied extensively (Table 2). Normander and coworkers The IncP-9 group contains plasmids encoding degradative (76) demonstrated the conjugative transfer of a gfp-tagged systems for toluene/xylene (pWW0; 39), and naphthalene pWW0 on bean phylloplane (leaf surface); the relative (pDTG1; 24, pNAH20; 41, and NAH7; 101). Notably, these humidity of the phylloplane was positively correlated with plasmids commonly encode large class II transposons that both donor survivability and plasmid transfer frequency. contain degradative genes inserted into different positions of Dual-labeling of the plasmid and its host chromosome using the IncP-9 backbone (101): Tn4651 and Tn4653 (pWW0; GFP and other fluorescent proteins has facilitated monitoring 115, 116), and Tn4655 (NAH7; 101, 117). However, Tn4655 of the fate of donors and their conjugative plasmids, as well is transposable only if transposase is supplied by the host as the quantification of conjugative transfer (67). Detection cell. Cultivation-independent approaches to the isolation of of pWW0 transconjugants in indigenous environmental natural plasmids containing naphthalene-degradative genes bacteria together with substrate degradation has been from soil samples detected two further types of IncP-9 reported (Table 2), but the comprehensive characterization of plasmid. This suggests that IncP-9 plasmids are an important these transconjugants has not yet been undertaken. Mølbak vehicle for these genes (79). coworkers (62) reported that pWW0::gfp (IncP-9) (and also Several reports have suggested that cultivation of a the IncP-1 plasmid pKJK5::gfp) transfers to indigenous pWW0-containing host in the presence of benzyl alcohol or bacteria on alfalfa sprouts. Transconjugant strains comprised benzoate can cause partial loss of a pWW0 DNA region, and primarily the genera Erwinia (γ-Proteobacteria) and even a complete loss of the ability to degrade toluene (13, 31, Pseudomonas (γ-Proteobacteria) (62). Bacteria of the genus 53, 125; Table 2, Fig. 2A). A possible explanation for Pseudomonas gradually became predominant on the sprouts these phenomena is that chromosomally encoded enzymes regardless of donor inoculum size (62). Since the majority of are more suitable for the metabolism of benzoate than those the IncP-9 group were isolated originally from Pseudomonas encoded on pWW0. Muñoz and coworkers (63) reported that spp., this genus may be the primary host of these plasmids. In the accumulation of benzyl alcohol and benzoate caused many reports on pWW0 behavior under conditions in which deletion mutants of pWW0 upper- and meta-operons during benzyl alcohol was used as a model xenobiotic compound chemostat cultivation in the presence of toluene. The (and is capable of inducing DNA deletions), the presence of accumulated benzyl alcohol and benzoate are most likely DNA rearrangements in pWW0 was not determined. consumed by remaining host cells harboring wild-type pWW0 and partially by the deletion mutants (the host of Behavior of the narrow host range IncP-7 plasmids in the pWW0 also has benzoate-degradative gene on its chromo- environment some, Fig. 2A). After 13 days of continuous culture with a higher inlet concentration of toluene, the proportion of The nucleotide sequences of four IncP-7 plasmids have

Fig. 2. (A) The pWW0-harboring Pseudomonas putida toluene catabolic pathway. Solid and dashed arrows indicate conversions catalyzed by Xyl enzymes encoded on pWW0 and Cat enzymes encoded on the chromosome, respectively. XylMA, xylene monooxygenase; XylB, benzyl alcohol dehydrogenase; XylC, benzaldehyde dehydrogenase; XylE, catechol 2,3-dioxygenase; XylF, 2-hydroxymuconic semi-aldehyde hydrolase; XylG, 2-hydroxymuconic semi-aldehyde dehydrogenase; XylH, 4-oxalocrotonate tautomerase; XylI, 4-oxalocrotonate decarboxylase; XylJ, 2-oxo- 4-pentenoate hydratase; XylL, 1,2-dihydroxycyclohexa-3,4-diene carboxylate; XylXYZ, toluate 1,2 dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, cis, cis-muconate-lactonizing enzyme; CatC, muconolactone isomerase. (B) Organization of the pWW0 xyl genes and IS elements. Black, gray, and white pentagons indicate regulatory genes, catabolic genes, and other unrelated ORFs, respectively, and rectangles, ISs. 260 SHINTANI et al. been published: the carbazole (nitrogen-containing aromatic slower than that of P. putida KT2440 (pCAR1) due to heterocyclic organic compound)-degradative plasmid pCAR1 the accumulation of catechol, a compound toxic to the (56, 107), the naphthalene-degradative plasmid pND6-1 cells (106). In most Pseudomonas spp., catechol-degradative (55), and the toluene/xylene-degradative plasmids pWW53 (cat) genes are induced to express by the presence of the and pDK1 (128, 129). pCAR1 and pCAR2 were previously intermediates of catechol degradation: cis, cis-muconate identified as pCAR1.1 and pCAR1.2 due to a single nucle- (47). However, in the case of Pf0-1, these genes are con- otide substitution. However, we consider them to be identical tained in the benzoate-degradative operons, and their expres- and so refer to them as pCAR1 (56, 90, 91, 92, 106). Like sion is induced by benzoate (106; Fig. 3A). Therefore, pWW0, class II transposons are located within pCAR1, pCAR1 has lost either the genes necessary for converting pDK1, and pWW53, indicating that degradative genes on carbazole to catechol (car and ant), or the antR transcrip- these plasmids can transpose into other replicons in the tional regulator of the car and ant genes, thus avoiding host cell (56, 91, 118, 128, 129). Of the sequenced IncP-7 accumulation of catechol (106; Fig. 3A). The other product plasmids, pCAR1 and pDK1 possess functional conjugative of carbazole degradation, 2-hydroxypenta-2,4-dienoate, can transfer systems, whereas the others may have lost these be utilized by the host bacterium (Fig. 3A). Notably, these genes (55, 128). Considering that IncP-7 plasmids of DNA rearrangements are mediated by homologous recombi- multiple Pseudomonas spp. have been identified (Table 1, nation of four copies of ISPre1 (106). 43), most IncP-7 plasmids may have possessed transfera- Recently, the behavior of pCAR1 and its host bacteria in bility in nature. natural or artificial environments has been reported (93, 94, pCAR1 was found in P. resinovorans CA10, isolated 96). pCAR1 was able to transfer, together with carbazole- originally from activated sludge (73, 80). This plasmid degradative activity, to indigenous bacteria in natural river allows the host to convert carbazole to catechol through water. However, no transfer in soil samples was detected anthranilate via the CarABC and AntABC enzymes it (94). When carbazole was present, most transconjugants encodes (Fig. 3A). Catechol may then be metabolized by were bacteria of the genus Pseudomonas (93). Surprisingly, degradative enzymes encoded on the host chromosome. pCAR1 was also able to transfer to Stenotrophomonas Pseudomonas spp. are the principal host of pCAR1; indeed, spp. in natural river water samples, whose bacterial order bacteria of other genera are unable to replicate the plasmid (Xanthomonadales) is different from that of Pseudomonas (91), indicating a narrow host range (108). In the past 5 (Pseudomonadales) (Table 1, 93). Note that pCAR1’s trans- years, the effect of pCAR1 on host bacteria has been investi- fer from P. putida to the pCAR1-cured Stenotrophomonas gated through comparisons of chromosomal and plasmid was not detected, most likely due to the different conjuga- transcriptomes (59, 60, 95). Its transfer frequency varies tion conditions in laboratory and environmental samples across bacterial species (90). Interestingly, H-NS family (Shintani et al., unpublished data). The behavior of pCAR1 proteins encoded on pCAR1 (conserved on some IncP-7 and its host bacteria (P. putida KT2440, P. fluorescens plasmids) are key global regulators capable of altering host Pf0-1, P. resinovorans CA10dm4, and P. chlororaphis function (130). IAM1511) in artificial microcosms, inoculated with other As with other Inc group plasmids, DNA rearrangements plasmid-free bacteria, was assessed (94, 96). Rates of carba- have been detected on pCAR1 (106, 119; Table 2). The zole degradation were altered by adding pCAR1-containing growth of P. fluorescens Pf0-1 (pCAR1) is significantly species. Only P. putida was able to degrade carbazole in

Fig. 3. (A) The carbazole catabolism pathway in Pseudomonas spp. harboring pCAR1 and the benzoate catabolism pathway of Pseudomonas spp. Solid and dashed arrows indicate conversions catalyzed by the Car and Ant enzymes, encoded by pCAR1, or the Ben/Cat enzymes, encoded on the host chromosome, respectively. CarAaAcAd, carbazole 1,9a-dioxygenase; CarBaBb, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase; CarC, 2- hydroxy-6-oxo-6-(2'-aminobiphenyl)-hexa-2,4-dienoate hydrolase; CarD, 2-hydroxypenta-2,4-dienoate hydratase; CarE, 4-hydroxy-2-oxovalerate aldolase; CarF, acetaldehyde dehydrogenase (acylating); AntABC, anthranilate 1,2-dioxygenase; TCA, tricarboxylic acid. (B) Organization of the pCAR1 car and ant genes. Black, gray, and white pentagons indicate regulatory genes, catabolic genes, and other unrelated ORFs, respectively, and rectangles, ISs. A 163-bp deletion in the antA gene on pCAR1 in P. fluorescens Pf0-1 is shown. Behavior of Degradative Plasmids within Microcosms 261

Fig. 4. Comparison of the pCAR1 nucleotide sequence changes in P. fluorescens Pf0-1. pCAR1d and/or pCAR1Δ2 were detected in pure cultures (106). A black box with a white pentagon indicates ISPre1, while a black box with a gray pentagon indicates ISPre2. White and black triangles indi- cate the pair of ISPre1 predicted to mediate homologous recombination resulting in pCAR1d and/or pCAR1Δ2. The white pentagon shows the posi- tion of repA. Gray arrows denote antABC and car operons. The bidirectional arrow indicates the class II transposon Tn4676 carrying car and ant operons. The pCAR1 DNA inversion region is indicated by a bidirectional arrow with a dashed line. The white cross indicates the 163-bp deletion in the antA region (see Fig. 3).

artificial soil microcosms, and degradation rates were influ- genes on pCAR1 was also found in the artificial water enced by the soil water content (94). In contrast, carbazole microcosms (Fig. 4, Shintani et al., unpublished). In com- degradation was detected in most of the artificial water parison to the culture of Pf0-1(pCAR1) with carbazole, DNA microcosms regardless of the bacterium that carried pCAR1, rearrangements in both cases are driven toward avoiding the and P. resinovorans was the most degradation-efficient spe- accumulation of catechol to improve Pf0-1’s survivability. In cies (96). Conjugative transfer of pCAR1 was detected only contrast, antR was lost from pCAR1 in the pure culture, but in water microcosms inoculated with P. putida KT2440 not in the artificial microcosms. Absence of the antR gene or P. chlororaphis IAM1511 (94, 96). P. resinovorans com- will slow down carbazole’s degradation since it encodes a prised 100% of the transconjugants obtained from the transcriptional regulator of the car and ant genes in response artificial microcosms, but the reason for this remains unclear to anthranilate (58, 119). In the pure culture, delaying the (94, 96). The presence of Ca2+ and Mg2+ was important for degradation of carbazole is probably necessary to prevent pCAR1’s transfer when P. putida KT2440 was used as the catechol accumulation because no other catechol metabo- host (94). lizing strains were present. On the other hand, many other Considering the carbazole-degradation deficiency of Pf0-1 anthranilate (or catechol)-metabolizing bacteria were present (pCAR1), significant degradation could not be expected in in the artificial water microcosms, and therefore, the induc- artificial soil or water microcosms inoculated with this strain. tion systems for the car and ant operons must also have been Nevertheless, carbazole degradation was detected in the present. Consequently, the choice of pCAR1’s host strain or artificial water microcosms (96). Carbazole degraders were indigenous bacteria in the environment may have a key role not observed from 5 to 14 days post-inoculation (96). in determining bioaugmentation efficiency. Genetic analyses of the isolates that reappeared after 14 days showed that they were Pf0-1 (pCAR1) derivatives (Shintani Concluding remarks and perspectives et al., submitted for publication). Additionally, these strains were unable to degrade anthranilate due to a 163-bp deletion Degradative capacity is determined by the sum of the deg- in the antA gene (Fig. 3B; Shintani et al., submitted for radative plasmid and the bacterial chromosome. Given that publication). This deletion results in the accumulation of most degradative plasmids are isolated by screening strains anthranilate (instead of catechol), which is beneficial to the from environmental microcosms such as soil, rivers, ponds, host bacterium. Interestingly, the deletion is predicted to be activated sludge and wastewater treatment plants, the ‘best’ mediated by homologous recombination between conserved host-plasmid combination for the xenobiotic degradation was DNA regions, including ISPre1 (Fig. 4). The transposition of already determined by nature. In terms of the evolution of Tn4676 into the host chromosome and of ISPre1 into car Inc groups in Pseudomonas, homologous ISs on the plasmid 262 SHINTANI et al. may mediate DNA rearrangements, thus maintaining plas- Acknowledgements mid genetic plasticity for adaptation to various hosts and Part of this work was supported by the Program for the environments. From the view point of the host range of Promotion of Basic Research Activities for Innovative Biosciences replication and conjugative transfer, IncP-1 plasmids seem to (PROBRAIN) in Japan. The writing of this review was supported be the most appropriate vector for degradative genes, which by the Special Postdoctoral Researcher Program of Riken. possibly ‘choose’ the appropriate recipients within micro- cosms in response to environmental conditions (growth sub- References strate and host species). Nevertheless, considerable numbers 1. Adamczyk, M., and G. Jagura-Burdzy. 2003. 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