Characterization of plasmids in a human clinical strain of Lactococcus garvieae

Abstract

The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

Figure 1. Gel electrophoresis of plasmid DNA from L. garvieae 21881 and L. garvieae 8831.

Line 1, Biotools 1 kb DNA marker, line 2, L. garvieae 21881, line 3, L. garvieae 8831; C indicates the chromosomal DNA.

Figure 2. Plasmid map of pGL1 and DNA sequence similarity with other lactic bacteria plasmids.

Lactococcus lactis subsp. cremoris SK11 plasmid1, Lactococcus lactis subsp. lactis bv. diacetylactis plasmid pDBORO, Leuconostoc mesenteroides subsp. mesenteroides Y110 plasmid pTXL1 and Tetragenococcus halophilus plasmid pHDC were those exhibiting the highest coverage/similarity to pGL1. The Orfs are indicated by arrows showing the direction of transcription. The gene annotations are positioned at the midpoint of each gene. Overlapped genes mobC, mobA and orf1 are indicated in white, yellow and red respectively.

Figure 3. Plasmid map of pGL2 and DNA sequence similarity with other lactic bacteria plasmids.

Leuconostoc kimchii IMSNU 11154 plasmid LkipL48, Lactobacillus sakei plasmid pYSI8, Lactobacillus brevis plasmid pLB925A01 and Lactobacillus plantarum WCFS1 plasmid pWCFS102 were those exhibiting the highest coverage/similarity to pGL2. The Orfs are indicated by arrows showing the direction of transcription. The gene annotations are positioned at the midpoint of each gene.

Figure 4. Plasmid map of pGL3 and DNA sequence similarity with other lactic bacteria plasmids.

Lactococcus lactis plasmid pSRQ800, Lactococcus lactis subsp. lactis KF147 plasmid pKF147A, Lactococcus lactis subsp. lactis plasmid pIL7 and Lactococcus lactis subsp. cremoris plasmid pNZ4000 were those exhibiting the highest coverage/similarity to pGL3. The Orfs are indicated by arrows showing the direction of transcription. The gene annotations are positioned at the midpoint of each gene.

Figure 5. Plasmid map of pGL4 and DNA sequence similarity with other lactic bacteria plasmids.

Lactococcus lactis plasmid pSRQ900, Leuconostoc citreum KM20 plasmid pLCK4, Lactococcus lactis subsp. lactis plasmid pKP1, Lactococcus lactis subsp. cremoris A76 plasmid pQA518 and Lactococcus lactis plasmid pJW566 were those exhibiting the highest coverage/similarity to pGL4. The Orfs are indicated by arrows showing the direction of transcription. The gene annotations are positioned at the midpoint of each gene.

Figure 6. Plasmid map of pGL5 and DNA sequence similarity with other lactic bacteria plasmids.

Lactococcus lactis subsp. cremoris SK11 plasmid 3, Lactococcus lactis plasmid pGdh442, Bacillus subtilis subsp. natto plasmid pLS20 and Lactococcus lactis subsp. lactis KF147 plasmid pKF147A were those exhibiting the highest coverage/similarity to pGL5. The Orfs are indicated by arrows showing the direction of transcription. The gene annotations are positioned at the midpoint of each gene.

Figure 7. Phylogenetic trees of the lai and pox genes.

The trees are constructed on the basis of the alignment of the DNA sequences of the lai (A) and pox (B) genes. Genes from Clostridium botulinum B str. Eklund 17B and Escherichia coli str. K-12 were used as the outgroups.

Figure 8. Proposed replication regions (dso and rnaII) of rolling circle pGL2 plasmid.

The dso region is indicated in boldface. The bind locus contains four tandem distal direct repeats (DDR) which are marked with arrowheads indicating their orientation. The nic locus contains: the nick sequence (in yellow) flanked by the inverted repeat structures (in blue) and two proximal direct repeats (PDR) sequences in tandem (in green, with broken arrowheads indicating their orientation). The putative rnaII region is underlined. The predicted −10 consensus promoter sequence of rnaII is indicated in pink. The predicted transcription terminator region is indicated in italics and the inverted repeat sequences (involved in the RNA II hairpin formation) with red arrowheads indicating their orientation. The presumed ribosome-binding site (RBS) and start and stop codons of the copG gene are indicated in red. The start codon of repB is indicated in grey.

Figure 9. Proposed replication regions of theta-type plasmids pGL1, pGL3, pGL4 and pGL5.

Sequence alignment of the upstream region of repB genes of only pGL3 and pGL4 showed significant identity (78%). Indicated in yellow are the AT- rich regions. Inverted repeat structures (IR) are represented by opposing broken arrows. The 22-bp directly repeated putative iteron sequences (DR) are indicated in red, and the arrowheads indicate their orientation. Predicted −10 consensus promoter sites and presumed ribosome-binding sites are in boldface. The start codons are underlined. The DNA sequence in blue corresponds to the last nucleotides of orf30 on pGL5.

Figure 10. Amino acid sequence of the QRDRs from L. garvieae 21881.

A) GyrA, B) ParC. Sequences from L. garvieae 21881 are compared with those described for the quinolone-sensitive L. garvieae strain KL99110. The position of amino acids critical for quinolone resistance (Ser, Asp and Glu) are indicated. Identical amino acids of L. garvieae 21881 to the standard QRDR regions for E. coli are underlined.