Loading…
The Streptococcus agalactiae hylB gene encoding hyaluronate lyase: completion of the sequence and expression analysis
We report the cloning, sequencing and expression analysis of the Streptococcus agalactiae strain 4755 hylB 4755 allele, the first chromosomally-encoded streptococcal hyaluronate lyase gene to be cloned and sequenced completely. This gene lies in a region homologous to that found in S. mutans, betwee...
Saved in:
Published in: | Biochimica et biophysica acta 1998-05, Vol.1398 (1), p.86-98 |
---|---|
Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Summary: | We report the cloning, sequencing and expression analysis of the
Streptococcus agalactiae strain 4755
hylB
4755 allele, the first chromosomally-encoded streptococcal hyaluronate lyase gene to be cloned and sequenced completely. This gene lies in a region homologous to that found in
S. mutans, between the
mutX and
rmlB genes, a region involved in the synthesis of the serotype c-specific polysaccharide antigen of this organism. Sequencing of
hylB
4755 revealed a 3216-bp open reading frame that encodes a 121.2-kDa polypeptide possessing a 30-amino acid signal sequence which was theoretically predicted and experimentally confirmed. A recombinant plasmid, pHYB100, containing
hylB
4755 together with its promoter and terminator was constructed and used to analyze the expression of the gene in
Escherichia coli. In Northern hybridization experiments,
hylB
4755 was found to be transcribed as 3.3-kb monocistronic mRNA from its own promoter which exhibits an extended,
σ
70-like 10 consensus sequence. Transcript mapping by primer extension analysis placed the major transcription initiation site leading to the longest transcript 38 bp upstream of the translational initiation codon, ATG.
E. coli TG1(pHYB100) efficiently synthesized hyaluronan-cleaving enzyme activity at ∼7000 working units/10
9 cells, with lyase activity detectable in all principle cellular locations. Zymography and Western analysis identified functional activity in TG1(pHYB100) to be associated with ∼118, 110 and 94-kDa polypeptides, with the two low molecular weight species constituting the major components of the enzyme purified from the culture supernatant fluid of
S. agalactiae 4755. The 118-kDa form was shown to represent the undegraded mature enzyme, whereas the smaller species are likely to arise from proteolytic cleavage in the N-terminal part of the mature protein. The HylB
4755 protein showed extensive sequence identity to the homologous enzymes from
S. agalactiae 3502 and
S. pneumoniae characterized by others but sequence comparisons clearly show that incomplete genes truncated at their 5′ ends had been isolated from these two organisms. |
---|---|
ISSN: | 0167-4781 0006-3002 1879-2634 |
DOI: | 10.1016/S0167-4781(98)00045-1 |