Molecular characterization of plasmid pMbo4.6 of Moraxella bovis ATCC 10900

Abstract

We report the characterization of a small cryptic plasmid unlike any previously described from Moraxella bovis ATCC 10900, a Gram-negative bacterium belonging to the family Moraxellaceae. The complete nucleotide sequence of the plasmid pMbo4.6 was determined. The plasmid was analyzed and found to be 4658 in size with a G+C content of 38.6 mol %. Computer analysis of the sequence data revealed four major open reading frames encoding putative proteins of 10.1 (ORF1), 64.2 (ORF2), 45.7 (ORF3), and 12.1 kDa (ORF4). ORF1 and ORF2 encode proteins that show a high level of amino acid sequence similarity (44 %) with some mobilization proteins. ORF3 encodes a protein showing a relatively high amino acid sequence similarity (about 40 %) with several plasmid replication initiator proteins. Upstream of ORF3, a 320-bp intergenic region, constituting the putative origin of replication that contained an AT-rich region followed by four direct repeats, was identified. This set of repeated sequences resembles iteron structures and plays an important role in the control of plasmid replication by providing a target site for the initiation of transcription and replication factors (IHF and RepA). Several palindromic sequences, inverted repeats, and hairpin-loop structures, which might confer regulatory effects on the replication of the plasmid, were also noted. ORF4 encodes an uncharacterized protein, conserved in bacteria, belonging to the DUF497 family. Sequence analysis and structural features indicate that pMbo4.6 replicates by a theta mechanism.

Fig. 1

Genetic structure of pMbo4.6 plasmid. a Partial restriction map of the plasmid. The location of the mobilization proteins, the replication protein and the DUF497 protein are indicated. b Nucleotide sequence of the putative oriV region. The fragment includes the AT-reach region, IHF binding site, 4 directed repeats (DR, iterons; solid lines under sequence), 9 inverted repeats (IR, arrows between sequence strands), and −35 and −10 boxes of the repA gene promoter. Thin black arrows (above and below) represent the positions and orientations of the various primers (specified in Table 1) used in generating PCR products. Sequence numbering according to the pMbo4.6 sequence deposited at GenBank (Accession No. GQ998872). c Alignment of the oriV regions of the pMbo4.6 and p3ABSDF (CU468233), pABIR (EU294228), p2ABSDF (CU468232), pAC63 (JN982951), pMMD (GO904226, pMMCU2 (GQ476987), pMMCU1 (GQ342610), pAB02 (AY228470) Acinetobacter plasmids. Asterisks indicate identical nucleotides. The numbers at the left and right sides of each sequence refer to the deposited nucleotide sequence. Sequences were aligned by CLUSTAL W [31]

Fig. 1

Genetic structure of pMbo4.6 plasmid. a Partial restriction map of the plasmid. The location of the mobilization proteins, the replication protein and the DUF497 protein are indicated. b Nucleotide sequence of the putative oriV region. The fragment includes the AT-reach region, IHF binding site, 4 directed repeats (DR, iterons; solid lines under sequence), 9 inverted repeats (IR, arrows between sequence strands), and −35 and −10 boxes of the repA gene promoter. Thin black arrows (above and below) represent the positions and orientations of the various primers (specified in Table 1) used in generating PCR products. Sequence numbering according to the pMbo4.6 sequence deposited at GenBank (Accession No. GQ998872). c Alignment of the oriV regions of the pMbo4.6 and p3ABSDF (CU468233), pABIR (EU294228), p2ABSDF (CU468232), pAC63 (JN982951), pMMD (GO904226, pMMCU2 (GQ476987), pMMCU1 (GQ342610), pAB02 (AY228470) Acinetobacter plasmids. Asterisks indicate identical nucleotides. The numbers at the left and right sides of each sequence refer to the deposited nucleotide sequence. Sequences were aligned by CLUSTAL W [31]

Fig. 2

Comparison of the amino acid sequences of the Rep proteins. The numbers on the right indicate the amino acid position relative to the N-terminus. Identical residues are indicated by an asterisk; two dots denote a highly conservative substitution, one dot a conservative substitution. Sequences were aligned by CLUSTAL W [31]

Fig. 3

Specific binding of RepA protein to the replication origin. Gel mobility shift assay using 90 ng of orirep35 DNA fragment (378 bp), 109 ng of competitor DNA (94 bp) and increasing amounts of purified RepA protein (50–800 ng per binding reactions) (lanes 5–10). Lanes 1 (orirep35) and 3 (competitor DNA) are negative controls without RepA protein, lanes 2 (orirep35) and 4 (competitor DNA) contain 650 ng of RepA protein

Fig. 4

Autorepression of the repA promoter. Cultures of the MC1061 strain contained derivatives of plasmid pRS415 bearing the lacZ ⁺ reporter gene in transcriptional fusion with a minimal WT repA promoter fragment (pRSplusIR, black), or a mutated promoter (pRSminusIR, white) or an unspecific promoter of ecoVIIIM DNA methyltransferases (gray). β-Galactosidase activity in exponentially growing cells harboring appropriate plasmids was measured in the absence or presence of the RepA protein (pACYCaraRep), induced by a different L-arabinose concentration by 1 h. The values obtained in the absence of induction were assumed to be 100 %. All values are mean ± SD for at least three independent experiments