Sequence analysis of pKF3-70 in Klebsiella pneumoniae: probable origin from R100-like plasmid of Escherichia coli

Abstract

Background: Klebsiella pneumoniae is a clinically significant species of bacterium which causes a variety of diseases. Clinical treatment of this bacterial infection is greatly hindered by the emergence of multidrug-resistant strains. The resistance is largely due to the acquisition of plasmids carrying drug-resistant as well as pathogenic genes, and its conjugal transfer facilitates the spread of resistant phenotypes.

Methodology/principal findings: The 70,057 bp plasmid pKF3-70, commonly found in Klebsiella pneumoniae, is composed of five main functional modules, including regions involved in replication, partition, conjugation, transfer leading, and variable regions. This plasmid is more similar to several Escherichia coli plasmids than any previously reported K. pneumoniae plasmids and pKF3-70 like plasmids share a common and conserved backbone sequence. The replication system of the pKF3-70 is 100% identical to that of RepFII plasmid R100 from E. coli. A beta-lactamase gene ctx-m-14 with its surrounding insertion elements (ISEcp1, truncated IS903 and a 20 bp inverted repeat sequence) may compose an active transposon which is directly bordered by two putative target repeats "ATTAC."

Conclusions/significance: The K. pneumoniae plasmid pKF3-70 carries an extended-spectrum beta-lactamase gene, ctx-m-14. The conjugative characteristic makes it a widespread plasmid among genetically relevant genera which poses significant threat to public health.

Figure 1. Structure of the replication system of pKF3-70.

RNAII: the mRNA on which RepA was transcripted; pII: promoter for RNAII; Numbers represent the coordinates (in reference to the whole plasmid sequence).

Figure 2. Alignment of parCs of pKF3-70 and R1/R100.

Points in the bottom sequence (R1/R100) represent the same nucleotides as in the top sequence. “−” represents a deletion, arrows represent tandem repeats. The framed regions represent −35 and −10 boxes of the downstream parA-parB operon. Numbers at the start and end of the sequence represent the position of this region in plasmid pKF3-70.

Figure 3. Comparative genomic analysis of conjugation and transfer leading regions of pKF3-70 and other plasmids.

(A,C) show 6 conjugation regions and 3 transfer leading regions of plasmids from different incompatibility groups, respectively. Homologous genes are shown using arrows with the same color/pattern except for arrows in gray, which indicate genes without homology. The name of each gene is provided above or below. In conjugation regions of the 4 plasmids pkF3-70, R100, F and R751, tra genes are named in corresponding capital single letters, while trb genes are in the lowercase single letters. trw genes in R388 are marked using corresponding lowercase single letter. Numbers 1–11 in the Ti conjugation region represent corresponding VirB genes. The leading region arrows are marked using ORF or gene names. Red frame in R100 of Fig. 3C shows the transposon Tn10. (B,D) show statistics for homologous genes in conjugation regions and transfer leading regions, respectively. Numbers in brackets indicate the percentage. Except for the sequence of pKF3-70, plasmid gene information was retrieved from GenBank (accession numbers, R100: AP000342; F: NC_002483; R388: BR000038; R751: NC_001735; Ti: NC_002377.).

Figure 4. The sequence of the region containing ctx-m-14.

DR, direct repeat; IRL, left inverted repeat; IRR, right inverted repeat; Tn, transposon. Truncated IS903 is represented with a rectangle; complete genes are represented with arrows and with gene names inside. Positions of the start points of EitD, IRL, IRR1, IRR2 and the end point of YadA are marked. These positions also serve as horizontal coordinates for the GC content map below. The map was generated using a window size of 80 bp.

Figure 5. EitABCD system has variable context in different genome.

(A) Arrows with the same color/label (except grey one) represent homologous genes. Grey Arrows represent genes of unknown functions. Numbers in the arrows represent the protein families classified by ACLAME database (A Classification of Mobile genetic Elements). Mobile elements are omitted and marked using “//”. (B) The tree was built based on the total nucleotide sequences of eitABCD. ColBM: pAPEC-O1-ColBM; ColV: pAPEC-O2-ColV; 70: pKF3-70; MGH: K. pneumoniae MGH; 342: K. pneumonia 342.

Figure 6. Gene-content tree and RepA tree of Enterobacteriaceae plasmids.

Reported plasmids from Enterobacteriaceae were subjected to cluster analysis based on gene content (top) or the RepA protein sequence (bottom). Plasmids are represented by their respective NCBI accession numbers.

Figure 7

(A) R100 backbone and accessory genetic elements on different plasmids. (B) Mutation rates profile of the four plasmids along the backbone sequences. Longitudinal coordinate represents mutation rates and horizon coordinate represents position in backbone sequences. This analysis use slide window of 100 bp. Red line: pKF3-70; yellow line: pO26I; blue line: pC15-1a; black line: R100. Region with shallow grey background was absolutely identical in all four plasmids and genes in the identical regions were marked.