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Interesting sequence differences between the pilin gene inversion regions of Moraxella lacunata ATCC 17956 and
Abstract:
The bacterium Moraxella lacunata is a causative agent of human conjunctivitis and keratitis. We have previously reported construction of plasmid pMxL1, which includes a 5.9-kb fragment on which the pilin gene inversion region of M. lacunata resides. The inversion region of pMxL1 was shown to invert when pMxL1 was in an Escherichia coli host cell. In this report, we present Western immunoblot analysis using Moraxella bovis Epp63 anti-I and anti-Q pilin sera which demonstrate that pMxL1 makes pilin only when in orientation 1. The sequence of the pMxL1 plasmid containing the invertible region contains a perfect tandem repeat of 19 bp in the orientation 1 nonexpressed pilin gene at the middle of the recombination junction site. This 19-bp insert causes a frameshift and disrupts the pilin gene. The predicted amino acid sequence of this nonfunctional pilin gene (with the 19-bp repeat subtracted) bears closest resemblance to M. bovis Epp63 Q pilin sequence, although the other (functional) M. lacunata pilin encoded by pMxL1 shows slightly higher homology to Q pilin. Comparison of the pMxL1 sequence with that of the M. bovis Epp63 sequence shows two other particularly interesting differences. One is a 15-bp sequence addition found in pMxL1 at the 60-bp region previously reported as a possible M. bovis recombinational enhancer. The second is an AT deletion in pMxL1 compared with Epp63 within an open reading frame (tfpB) which results in the pMxL1 tfpB open reading frame being one-third shorter than in Epp63. The DNA sequences in these three altered regions from the M. lacunata strain from which pMxL1 was derived were amplified by polymerase chain reaction and sequenced. The parent strain was found to contain the differences seen in pMxL1. Comparison of the M.bovis and M. lacunata pilin gene amino acid sequences is also presented.
Insights
Moraxella lacunata plasmid pMxL1 produces pilin only in orientation 1 due to a frameshift mutation. Sequence analysis reveals differences in pilin genes and regulatory regions compared to Moraxella bovis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Moraxella lacunata causes human eye infections like conjunctivitis and keratitis.
- Previous work constructed plasmid pMxL1 with an M. lacunata pilin gene inversion region, which inverts in Escherichia coli.
- This inversion region was shown to invert in E. coli host cells.
Purpose of the Study:
- To investigate pilin production by M. lacunata plasmid pMxL1 in E. coli.
- To analyze the DNA sequence of the invertible region of pMxL1 and compare it with M. bovis sequences.
- To compare the amino acid sequences of M. lacunata and M. bovis pilin genes.
Main Methods:
- Western immunoblot analysis using specific antisera against M. bovis pilins.
- DNA sequencing of the pMxL1 plasmid and relevant regions from the parent M. lacunata strain.
- Polymerase chain reaction (PCR) amplification and sequencing of specific DNA regions.
Main Results:
- pMxL1 produces pilin only when in orientation 1, as confirmed by Western immunoblot analysis.
- A 19-bp tandem repeat in the nonexpressed pilin gene causes a frameshift, disrupting the gene in orientation 1.
- Sequence comparisons revealed a 15-bp insertion in pMxL1 and a shorter tfpB open reading frame compared to M. bovis Epp63. The parent strain harbored these differences.
Conclusions:
- The frameshift mutation due to the 19-bp repeat prevents functional pilin expression in one orientation.
- Sequence variations in pilin genes and regulatory regions exist between M. lacunata and M. bovis.
- The identified genetic differences in the parent strain explain the observed features of pMxL1.