Table 1.
List of primers used in this study.
Figure 1.
Annotated map of pSMA198. Black boxes indicate the position of ori and oriT. Solid grey arrows indicate positions of functional genes.
Remaining light grey arrows indicate positions of predicted pseudogenes.
Figure 2.
Multiple sequence alignment of RepB sequences relevant to the respective protein of pSMA198.
The alignment was performed with ClustalW [23]. Double-headed arrows indicate positions of protein sequence signatures as determined by InterProScan [24], as follows: initiator Rep protein (dashed line), L. lactis RepB C-terminal (dotted line) and two consecutive winged helix-turn-helix transcription DNA-binding repressor (solid line).
Table 2.
Annotated features of pSMA198.
Figure 3.
Multiple sequence alignment of ori of pSMA198 and its related plasmids.
The alignment was performed using ClustalW [23]. Arrows indicate the position of the AT-rich region, the 22-bp direct repeat (DR) iterons and two inverted repeats (IR). The predicted leader region (−35, −10 and the RBS) and the start codon of the rep gene are underlined.
Figure 4.
Multiple sequence alignment of oriT of pSMA198 and its related plasmids.
The alignment was performed using ClustalW [23]. Arrows indicate positions of the six inverted repeats (IR) and the two directed repeats (DR).
Figure 5.
Sequence alignment of pSMA198 against pSK11b (A), pCIS8 (B) and pIL5 (C) represented in a circular fashion using Circoletto [30].
Local alignments produced by BLAST are presented using ribbons whose color corresponds to four quartiles of the alignment’s bitscore (red: top 25%, orange: second 25%, green: third 25% and blue: worst 25%). The positions of pSMA198 ori or oriT were added in order to aid orientation.
Figure 6.
Cloning plasmid pORI198 in L. lactis MG1363 and assessing its stability in this host.
Annotated map of pORI198 (A). The black box and the dark grey arrows indicate the position of the cloned pSMA198 replication backbone (ori—rep—orfX). The light grey box and arrows indicate the position of the pUC19 cloning vector. Cloning pORI198 in L. lactis (B). The presence of pORI198 in transformed L. lactis cells was verified by PCR using primers M13F/M13R that could amplify the entire cloned region of the pSMA198 replication backbone. Untransformed L. lactis cells were used as a negative control. Stability of pORI198 in L. lactis (C). The stability of plasmid pORI198 was assessed as described in the manuscript. No statistically significant differences were observed between L. lactis cells grown in GM17 in the presence and the absence of erythromycin (p>0.05), indicating a 100% stability during a 100-generation time.
Figure 7.
Sequence alignment of chromosomal regions of S. macedonicus ACA-DC 198 against pAH82 (A) and pGdh442 (B).
The alignment was performed using Kodon software (Applied Maths, Belgium). In each case, the flanking transposase gene showing high sequence similarity to SMA_p0006 of pSMA198 plasmid is underlined. Colored areas between the sequences correspond to different levels of identity that are specified within the areas.