Gase K, Ozegowski J, Malke H
Jena University, Institute for Molecular Biology, Winzerlaer Strasse 10, D-07745 Jena, Germany.
Biochim Biophys Acta. 1998 May 29;1398(1):86-98. doi: 10.1016/s0167-4781(98)00045-1.
We report the cloning, sequencing and expression analysis of the Streptococcus agalactiae strain 4755 hylB4755 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 hylB4755 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 hylB4755 together with its promoter and terminator was constructed and used to analyze the expression of the gene in Escherichia coli. In Northern hybridization experiments, hylB4755 was found to be transcribed as 3.3-kb monocistronic mRNA from its own promoter which exhibits an extended, sigma70-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 approximately 7000 working units/109 cells, with lyase activity detectable in all principle cellular locations. Zymography and Western analysis identified functional activity in TG1(pHYB100) to be associated with approximately 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 HylB4755 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.
