GENETIC-ANALYSIS OF THE O7-POLYSACCHARIDE BIOSYNTHESIS REGION FROM THE ESCHERICHIA-COLI O7-K1 STRAIN VW187

被引:74
作者
MAROLDA, CL [1 ]
WELSH, J [1 ]
DAFOE, L [1 ]
VALVANO, MA [1 ]
机构
[1] UNIV WESTERN ONTARIO,HLTH SCI CTR,DEPT MICROBIOL & IMMUNOL,LONDON N6A 5C1,ONTARIO,CANADA
关键词
D O I
10.1128/jb.172.7.3590-3599.1990
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M.A. Valvano, and J.H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of β-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.
引用
收藏
页码:3590 / 3599
页数:10
相关论文
共 38 条
[1]   CLONAL ANALYSIS OF DESCENT AND VIRULENCE AMONG SELECTED ESCHERICHIA-COLI [J].
ACHTMAN, M ;
PLUSCHKE, G .
ANNUAL REVIEW OF MICROBIOLOGY, 1986, 40 :185-210
[2]  
BIRNBOIM HC, 1979, NUCLEIC ACIDS RES, V7, P1513
[3]  
BOULNOIS GJ, 1984, ADV MOL GENETICS, P204
[4]   CLONING PART OF THE REGION ENCODING BIOSYNTHETIC-ENZYMES FOR SURFACE-ANTIGEN (O-ANTIGEN) OF SALMONELLA-TYPHIMURIUM [J].
BRAHMBHATT, HN ;
QUIGLEY, NB ;
REEVES, PR .
MOLECULAR & GENERAL GENETICS, 1986, 203 (01) :172-176
[5]   COMPLETE PHYSICAL MAP OF THE RFB GENE-CLUSTER ENCODING BIOSYNTHETIC-ENZYMES FOR THE O-ANTIGEN OF SALMONELLA-TYPHIMURIUM LT2 [J].
BRAHMBHATT, HN ;
WYK, P ;
QUIGLEY, NB ;
REEVES, PR .
JOURNAL OF BACTERIOLOGY, 1988, 170 (01) :98-102
[6]   CONSTRUCTION AND CHARACTERIZATION OF AMPLIFIABLE MULTICOPY DNA CLONING VEHICLES DERIVED FROM P15A CRYPTIC MINIPLASMID [J].
CHANG, ACY ;
COHEN, SN .
JOURNAL OF BACTERIOLOGY, 1978, 134 (03) :1141-1156
[7]   NONCHROMOSOMAL ANTIBIOTIC RESISTANCE IN BACTERIA - GENETIC TRANSFORMATION OF ESCHERICHIA-COLI BY R-FACTOR DNA [J].
COHEN, SN ;
CHANG, ACY ;
HSU, L .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1972, 69 (08) :2110-&
[8]   2 MUTATIONS WHICH AFFECT THE BARRIER FUNCTION OF THE ESCHERICHIA-COLI K-12 OUTER-MEMBRANE [J].
COLEMAN, WG ;
LEIVE, L .
JOURNAL OF BACTERIOLOGY, 1979, 139 (03) :899-910
[9]   A TECHNIQUE FOR RADIOLABELING DNA RESTRICTION ENDONUCLEASE FRAGMENTS TO HIGH SPECIFIC ACTIVITY [J].
FEINBERG, AP ;
VOGELSTEIN, B .
ANALYTICAL BIOCHEMISTRY, 1983, 132 (01) :6-13
[10]   CONDUCTION OF NONCONJUGATIVE PLASMIDS BY F' LAC IS NOT NECESSARILY ASSOCIATED WITH TRANSPOSITION OF THE GAMMA-DELTA-SEQUENCE [J].
GOTO, N ;
SHOJI, A ;
HORIUCHI, S ;
NAKAYA, R .
JOURNAL OF BACTERIOLOGY, 1984, 159 (02) :590-596