Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet

Abstract

The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome containing 478 CDSs and an exceptional degree of genetic novelty; 80% of predicted coding sequences cannot be ascribed a function and 60% are orphans. Of those to which function could be assigned, 40% bore greatest similarity to sequences from Pseudomonas spp, and the majority of the remainder showed similarity to other gamma-proteobacterial genera and plasmids. pQBR103 has identifiable regions presumed responsible for replication and partitioning, but despite being tra+ lacks the full complement of any previously described conjugal transfer functions. The DNA sequence provided few insights into the functional significance of plant-induced transcriptional regions, but suggests that 14% of CDSs may be expressed (11 CDSs with functional annotation and 54 without), further highlighting the ecological importance of these novel CDSs. Comparative analysis indicates that pQBR103 shares significant regions of sequence with other plasmids isolated from sugar beet plants grown at the same geographic location. These plasmid sequences indicate there is more novelty in the mobile DNA pool accessible to phytosphere pseudomonas than is currently appreciated or understood.

Figure 1

The 425 094 bp genome sequence of pQBR103 is presented in a circular plot, along with markers indicating regions of plant-specific transcription, and regions of conservation between pQBR103 and other group I plasmids, pQBR44 and pQBR47. Nine concentric circles are shown (from outer to innermost): 1, base pair coordinates. Base pair 1 of the genome has been arbitrarily defined as the replication mode of this plasmid is uncharacterized. 2-3, Annotated CDSs regions in the forward and reverse strands, respectively (functional CDSs, red: DNA associated, yellow: metabolism, pink: phage and transposon, white: environmental/survival and transmission, blue: regulators, dark green: transmembrane, grey: domain match only; pseudogene, brown: conserved hypothetical CDSs, orange: orphan CDS, light green). 4, Regions of interest (black, clockwise from 0 bp): ParAB, RulAB, potential Tra region, oriV, RepA replicon and Tn5042-like transposon; and (red) IVET regions of potential plant-induced transcriptional activity. 5-7: Microarray analyses of pQBR103 CDS distribution: probe regions in pQBR103 (blue), positive hybridization from pQBR44 (cyan) and pQBR47 (magneta). 8, GC skew. 9, GC deviation from the mean% G + C. CDS, coding sequence.

Figure 2

pQBR103 contains a putative RepA minimal replicon with tandem repeat elements separate from the putative ParAB partitioning cassette. The putative minimal replicon contains CDS383, a homologue of the plasmid replication initiator gene repA from the IncA/C-IncP3 plasmid RA1, 32 copies of a 22 bp repeat element (triangles) and two DnaA boxes (vertical lines) (338 642-341 577 bp) (top). The origin of replication would be expected to be located in the region defined by the DnaA boxes. The putative partitioning cassette contains CDS001 and 002, parA and parB homologues, respectively. Located upstream of parA is a degenerate 48 bp inverted repeat (424 970-425 069 bp) (triangles), and downstream of parB is a relatively AT-rich region (2100-2600 bp) (box) containing up to 30 copies of a 6 bp repeat (5′-TGC TTT-3′) element.

Figure 3

Components of a conjugal transfer apparatus sharing homology with classical Type IV secretion systems (T4SS) are located in the 170 292-197 117 bp region of pQBR103. Shown are the F plasmid Tra (top) (TraA, L, E, K, B, V, C, G and D), putative pQBR103 Tra region (middle) (CDSs 160-191, ~27 kb) and pTi (pTiC58) VirB/D4 (bottom) (VirB1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and VirD4) regions to scale. pQBR103 CDS in white have no recognizable role in transfer. Transfer components sharing the same functional role and sequence homology are indicated by colors. Transcription is from left to right, except for those CDSs in pQBR103 marked by a circle. Adapted from Schroder and Lanka (2005). CDS, coding sequence.

Figure 4

CGH microarray results show large regions of conservation and apparent deletions between group I plasmids. The microarray was used to test the group-I plasmids pQBR44 and pQBR47, the group-III plasmid pQBR55 and the group-IV plasmid pQBR57. The microarray used 122 pQBR103 probes, which are arranged in order along the x axis. Plasmid DNA used for hybridization was labelled with either Cy3 or Cy5-dCTP and the hybridization signal reported is the mean median fluorescence value from six replicate spots for each probe. The arrows indicate the position of strong Hg^R-probe signals for pQBR57 and pQBR55. No signals were obtained using labelled P. putida UWC1 chromosomal DNA, and the negative control probes did not hybridized to any of the labelled plasmid preparations (data not shown). CGH, comparative genomic hybridization.