摘要:

In thedivEmutant, which has a temperature-sensitive mutation in the tRNA1Sergene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. InEscherichiacoli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in thedivEmutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutantlacZgenes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in thedivEmutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-typelacZgene, and that the defect in beta-galactosidase synthesis was accompanied by a low level oflacZmRNA. It was also found that introduction of anrne-1pnp-7 double mutation restored the expression of mutantlacZgenes with only UCA codons at position 6 or 462. A polarity suppressor mutation in therhogene had no effect on the defect inlacZgene expression in thedivEmutant. We propose a model to explain these results.Key words:divEgene, tRNA1Ser,lacZgene expression, UCA codon

ISSN:0008-4166    

DOI:10.1139/w00-019

出版商:NRC Research Press    

年代:2000

数据来源: NRC

摘要:

The salivary agglutinin-interacting adhesin P1 ofStreptococcusmutansis anchored to the cell wall via the carboxy (C) terminus, which contains a wall-associated domain, a conserved LPXTGX motif, a hydrophobic domain, and a charged tail. To further investigate the role of the C-terminal anchoring regions in cell wall sorting and anchoring, mutational analysis was performed on P1 in this study. Three truncated P1 mutants and seven site-directed mutants were generated by a polymerase chain reaction-based technique. The mutated P1 genes were returned to the P1-negativeS. mutansSM3352 for expression and localization studies by ELISA and Western immunoblotting. The results showed that P1 mutants with deletion of the hydrophobic domain and charged tail, or deletion of the charged tail alone resulted in the secretion of P1 to the culture medium. Results from cellular fractionation experiments with the truncated mutants showed that P1 was not trapped in the membrane or cytoplasm. The site-directed mutants showed normal distribution of P1 to the cell surface as compared to the wild-type. However, when cell walls prepared from the site-directed mutants were boiled with SDS, P1 could be removed readily from the mutants with Thr residue in the LPNTGV motif, altered to either Ser (T1531S) or Phe (T1531F); the mutant with Thr and Gly residues altered to two Phe residues (TG1531-1532FF), and the LPNTGV-deleted mutant (LPNTGV-). In contrast, the wild-type P1 and the other three site-directed P1 mutants (P1529V, N1530I, and G1532F) could not be removed by boiling SDS. When the cell wall P1s from the wild-type, mutants P1529V, N1530I, and G1532F were reacted with an antibody directed against the hydrophobic domain and charged tail, no reaction was detected. However, P1s from mutants T1531S, T1531F, TG1531-1532FF, and LPNTGV-were recognized by the antibody, indicating that the inability of these mutated P1s to firmly link to the cell wall was the result of failure in proteolytic cleavage of the hydrophobic domain and charged tail. In summary, the results suggest that the charged tail plays a decisive role in sorting P1 to the cell surface, while the LPXTGX motif determines the nature of P1-cell wall association. The Thr residue of the LPXTGX motif is required for enzymatic processing to link P1 to the cell wall, presumably via a covalent bond.Key words: antigen P1, cell wall proteins,Streptococcus mutans, protein anchoring, site-directed mutagenesis.

ISSN:0008-4166    

DOI:10.1139/w00-023

出版商:NRC Research Press    

年代:2000

数据来源: NRC