Frequency and diversity of small cryptic plasmids in the genus Rahnella

Abstract

Background: Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids.

Results: In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified.

Conclusions: For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to different groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the importance of plasmids for lateral gene transfer (including chromosomal sequences) to distinct genera.

Figure 1

Maps of plasmids and homologous sequences. Same colours indicate homologous genes, operons or genetic elements (mrs, ssi). Larger regions exhibiting more than 85% sequence identity at the DNA level are marked with grey areas or are indicated below the sequence. Nucleotide sequence identities are given in percent. Replication and transfer origins are shown above the DNA when they are located on the sense strand and below if they are placed on the antisense strand. The plasmids pECA1039 and ColE1 as well as parts of the chromosomes from P. luminescens TT01 and E. tasmaniensis Et1/99 are shown for comparison. Abbreviations: DRs, direct repeats; mrs, multimer resolution sites; oriT, origin of transfer; oriV, origin of replication; ssi, single strand initiation site.

Figure 2

The ColE1-like plasmids of Rahnella form a sub-family. Phylogenetic trees were constructed based on RNA II (A) or the mrs (B). In both trees most ColE1-like plasmids isolated from Rahnella (shown in bold letters) formed a cluster with pECA1039, a plasmid of Pectobacterium artrosepticum. (C) Alignment of the multimer resolution sites. The ArgR, FIS, XerC and XerD binding sites are boxed and conserved A-T stretches responsible for DNA bending are underlined. The -10 and -35 boxes of the ColE1 P_cer promoter are underlined and the start of the Rcd coding region is indicated by an arrow. Nucleotides conserved in at least 50% of the sequences are shown in bold and invariant sites are marked with an asterisk.

Figure 3

ColE2 origins of replication. The thick arrow indicates the primer RNA and direction of replication in ColE2-P9. Further codes as in Fig. 2.

Figure 4

pHW121, pHW104 and pHW126 belong to different classes of plasmids replicating by the rolling circle mechanism. (A) A stretch upstream of the pHW121 repA gene is similar to replication origins of pC191/pUB110-family plasmids and the E. coli bacteriophage öX174. The experimentally determined cleavage sites of pC194 and öX174 are indicated by vertical arrows. (B) pHW104 and pHW126 are members of poorly characterised families of rolling circle plasmids. The G+C contents calculated for pAM10.6 and pM3 are based on partial sequences. (C) Evidence that pHW126 replicates via the rolling circle mechanism. Constructs containing two origins of replication of pHW126 and, as control, pHW15 were grown E. coli INVαF' for 40 generations. Subsequently DNA was isolated and analysed by restriction digestion with HindIII (similar results were obtained for digests with SalI; data not shown). The expected positions of constructs containing one or two origins are indicated by arrows. The deletion of the second origin was confirmed by sequencing (data not shown). The size of the marker bands is given in kb. (D) G+C contents of small plasmids and their hosts are correlated. The trendline was calculated from 124 enterobacterial plasmid sequences retrieved from the Genome Project Database http://www.ncbi.nlm.nih.gov/genomes. For strains with unavailable genomic G+C contents the mean value of the species according to Bergey's Manual of Systematic Bacteriology [59] was used. Plasmids from Rahnella are shown as filled circles while plasmids from other Enterobacteriaceae are shown as open circles.

Figure 5

Alignments of transfer origin (oriT) nic sites of the ColE1-superfamily (A) and the pMV158-superfamily (B). Experimentally determined nic-cleavage sites are indicated by vertical arrows. Inverted repeats involved in formation of a stem-loop-stem structure are underlined. Other codes as in Fig. 2.

Figure 6

The orf4 orf5 orf6 gene cluster of pHW4594 is not derived from its host. DNA from different Rahnella strains was digested with HindIII (right panel) and subsequently analysed with an orf5 orf6 specific probe (left panel). Two different amounts of DNA were loaded of DSM 4594^T, the host strain of pHW4596, to account for plasmid copy number (approximately 3 μg and 0.2 μg in the first and second lane, respectively). The detected band corresponded to the restriction fragment of the plasmid pHW4594 with an expected size of 1.3 kb. The same result was obtained with HpaII digested DNA (data not shown). GS, genomospecies.