Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium

被引:38
作者
Schoenhals, GJ [1 ]
Macnab, RM [1 ]
机构
[1] YALE UNIV,DEPT MOLEC BIOPHYS & BIOCHEM,NEW HAVEN,CT 06520
关键词
D O I
10.1128/jb.178.14.4200-4207.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The FlgH protein of Salmonella typhimurium, From which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG up arrow C) for lipoylation and signal peptide cleavage, We have confirmed the previous finding (M. Homma, K. Ohnishi, T. lino, and R. M. Macnab, J. Bacteriol. 169:3617-3624 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca. 25 kDa. flgH alleles with an in-frame deletion or a 3' truncation still permitted processing. The deletion permitted partial restoration of motility. in complementation tests? whereas the truncation did not, Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH. When cells transformed with a plasmid containing the flgH gene were grown in the presence of [H-3]palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used, Hook-basal body complexes from cells grown in the presence of [H-3]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PelB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form, With a low-copy-number plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type. Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated [H-3]palmitate at low levels, perhaps through acylation of the N-terminal alpha-amino group, We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body. We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.
引用
收藏
页码:4200 / 4207
页数:8
相关论文
共 43 条
[1]   PURIFICATION AND CHARACTERIZATION OF THE FLAGELLAR HOOK BASAL BODY COMPLEX OF SALMONELLA-TYPHIMURIUM [J].
AIZAWA, S ;
DEAN, GE ;
JONES, CJ ;
MACNAB, RM ;
YAMAGUCHI, S .
JOURNAL OF BACTERIOLOGY, 1985, 161 (03) :836-849
[2]   IDENTIFICATION OF ESCHERICHIA-COLI REGION-III FLAGELLAR GENE-PRODUCTS AND DESCRIPTION OF 2 NEW FLAGELLAR GENES [J].
BARTLETT, DH ;
MATSUMURA, P .
JOURNAL OF BACTERIOLOGY, 1984, 160 (02) :577-585
[3]   MUREIN-LIPOPROTEIN LINKAGE IN CELL WALL OF ESCHERICHIA-COLI [J].
BRAUN, V ;
WOLFF, H .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1970, 14 (02) :387-&
[4]  
Braun V., 1994, Bacterial cell wall, P319
[5]  
BURNETTE WN, 1981, ANAL BIOCHEM, V112, P195, DOI 10.1016/0003-2697(81)90281-5
[6]  
DEMOT R, 1994, MOL MICROBIOL, V12, P333
[7]   ATTACHMENT OF FLAGELLAR BASAL BODIES TO CELL ENVELOPE - SPECIFIC ATTACHMENT TO OUTER, LIPOPOLYSACCHARIDE MEMBRANE AND CYTOPLASMIC MEMBRANE [J].
DEPAMPHI.ML ;
ADLER, J .
JOURNAL OF BACTERIOLOGY, 1971, 105 (01) :396-&
[8]   FINE STRUCTURE AND ISOLATION OF HOOK-BASAL BODY COMPLEX OF FLAGELLA FROM ESCHERICHIA-COLI AND BACILLUS-SUBTILIS [J].
DEPAMPHI.ML ;
ADLER, J .
JOURNAL OF BACTERIOLOGY, 1971, 105 (01) :384-&
[9]   IDENTIFICATION OF A CAULOBACTER BASAL BODY STRUCTURAL GENE AND A CIS-ACTING SITE REQUIRED FOR ACTIVATION OF TRANSCRIPTION [J].
DINGWALL, A ;
GOBER, JW ;
SHAPIRO, L .
JOURNAL OF BACTERIOLOGY, 1990, 172 (10) :6066-6076
[10]  
GAN K, 1993, J BIOL CHEM, V268, P16544