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The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a
J N Manch-Citron1, J Allen, M Moos
1Laboratory of Microbial Ecology, National Institute of Dental Research, Food and Drug Administration, Bethesda, Maryland 20892.
Abstract:
We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.
Insights
Researchers identified the Prevotella loescheii plaA gene, encoding a lectin-like adhesin crucial for bacterial coaggregation. This adhesin facilitates the interaction between Prevotella loescheii and Streptococcus oralis.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Prevotella loescheii interacts with other oral bacteria, contributing to the oral microbiome.
- Bacterial coaggregation is mediated by specific adhesins on the bacterial surface.
Purpose of the Study:
- To clone and sequence the Prevotella loescheii gene plaA.
- To characterize the lectin-like adhesin encoded by plaA.
- To understand the genetic basis of coaggregation between P. loescheii and Streptococcus oralis.
Main Methods:
- Construction of a P. loescheii genomic library in lambda GEM-11.
- Screening the library using a probe derived from the adhesin's N-terminal amino acid sequence.
- DNA sequencing of the plaA gene and surrounding regions.
- Polymerase chain reaction (PCR) amplification and sequencing.
- Limited proteolysis of the adhesin followed by peptide sequencing.
Main Results:
- The plaA gene was cloned and sequenced, revealing a 22-amino-acid leader sequence.
- A premature TAA termination codon was identified within the plaA gene's reading frame.
- A frameshift downstream of the terminator was necessary to access a large open reading frame.
- Sequencing of peptides confirmed the amino acid sequence derived from the large open reading frame.
- The plaA gene encodes an 89-kDa protein and contains a rho-independent terminator.
Conclusions:
- The plaA gene encodes a lectin-like adhesin involved in P. loescheii coaggregation with S. oralis.
- The gene structure includes a premature stop codon and requires a frameshift for full translation.
- The identified adhesin plays a role in interspecies bacterial interactions within the oral cavity.