IDENTIFICATION OF ALGF IN THE ALGINATE BIOSYNTHETIC GENE-CLUSTER OF PSEUDOMONAS-AERUGINOSA WHICH IS REQUIRED FOR ALGINATE ACETYLATION

被引:106
作者
FRANKLIN, MJ
OHMAN, DE
机构
[1] UNIV TENNESSEE CTR HLTH SCI,DEPT MICROBIOL & IMMUNOL,MEMPHIS,TN 38163
[2] VET AFFAIRS MED CTR,MEMPHIS,TN 38163
关键词
D O I
10.1128/JB.175.16.5057-5065.1993
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Mucoid strains of Pseudomonas aeruginosa produce a high-molecular-weight exopolysaccharide called alginate that is modified by the addition of O-acetyl groups. To better understand the acetylation process, a gene involved in alginate acetylation called algF was identified in this study. We hypothesized that a gene involved in alginate acetylation would be located within the alginate biosynthetic gene cluster at 34 min on the P. aeruginosa chromosome. To isolate algF mutants, a procedure for localized mutagenesis was developed to introduce random chemical mutations into the P. aeruginosa alginate biosynthetic operon on the chromosome. For this, a DNA fragment containing the alginate biosynthetic operon and adjacent argF gene in a gene replacement cosmid vector was utilized. The plasmid was packaged in vivo into lambda phage particles, mutagenized in vitro with hydroxylamine, transduced into Escherichia coli, and mobilized to an argF auxotroph of P. aeruginosa FRD. Arg+ recombinants coinherited the mutagenized alginate gene cluster and were screened for defects in alginate acetylation by testing for increased sensitivity to an alginate lyase produced by Klebsiella aerogenes. Alginates from recombinants which showed increased sensitivity to alginate lyase were tested for acetylation by a colorimetric assay and infrared spectroscopy. Two algF mutants that produced alginates reduced more than sixfold in acetyl groups were obtained. The acetylation defect was complemented in trans by a 3.8-kb XbaI-BamHI fragment from the alginate gene cluster when placed in the correct orientation under a trc promoter. By a merodiploid analysis, the algF gene was further mapped to a region directly upstream of algA by examining the polar effects of Tn501 insertions. By gene replacement, DNA with a Tn501 insertion directly upstream of algA was recombined with the chromosome of mucoid strain FRD1. The resulting strain, FRD1003, was nonmucoid because of the polar effect of the transposon on the downstream algA gene. By providing algA in trans under the tac promoter, FRD1003 produced nonacetylated alginate, indicating that the transposon was within or just upstream of algF. These results demonstrated that algF, a gene involved in alginate acetylation, is located directly upstream of algA.
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页码:5057 / 5065
页数:9
相关论文
共 36 条
[1]  
BALTIMORE RS, 1982, J INFECT DIS, V141, P238
[2]   THE DIFFUSION OF BETA-LACTAM ANTIBIOTICS THROUGH MIXED GELS OF CYSTIC FIBROSIS-DERIVED MUCIN AND PSEUDOMONAS-AERUGINOSA ALGINATE [J].
BOLISTER, N ;
BASKER, M ;
HODGES, NA ;
MARRIOTT, C .
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, 1991, 27 (03) :285-293
[3]   GENETIC-ANALYSIS OF THE ALGINATE BIOSYNTHETIC GENE-CLUSTER OF PSEUDOMONAS-AERUGINOSA SHOWS EVIDENCE OF AN OPERONIC STRUCTURE [J].
CHITNIS, CE ;
OHMAN, DE .
MOLECULAR MICROBIOLOGY, 1993, 8 (03) :583-590
[4]  
CHITNIS CE, 1990, J BACTERIOL, V172, P294
[5]   NUCLEOTIDE-SEQUENCE AND EXPRESSION OF THE ALGE GENE INVOLVED IN ALGINATE BIOSYNTHESIS BY PSEUDOMONAS-AERUGINOSA [J].
CHU, L ;
MAY, TB ;
CHAKRABARTY, AM ;
MISRA, TK .
GENE, 1991, 107 (01) :1-10
[6]   LOCALIZATION OF O-ACETYL GROUPS OF BACTERIAL ALGINATE [J].
DAVIDSON, IW ;
SUTHERLAND, IW ;
LAWSON, CJ .
JOURNAL OF GENERAL MICROBIOLOGY, 1977, 98 (FEB) :603-606
[7]   GENE ALGD CODING FOR GDPMANNOSE DEHYDROGENASE IS TRANSCRIPTIONALLY ACTIVATED IN MUCOID PSEUDOMONAS-AERUGINOSA [J].
DERETIC, V ;
GILL, JF ;
CHAKRABARTY, AM .
JOURNAL OF BACTERIOLOGY, 1987, 169 (01) :351-358
[8]   PRODUCTION AND CHARACTERIZATION OF SLIME POLYSACCHARIDE OF PSEUDOMONAS-AERUGINOSA [J].
EVANS, LR ;
LINKER, A .
JOURNAL OF BACTERIOLOGY, 1973, 116 (02) :915-924
[9]   REPLICATION OF AN ORIGIN-CONTAINING DERIVATIVE OF PLASMID RK2 DEPENDENT ON A PLASMID FUNCTION PROVIDED IN TRANS [J].
FIGURSKI, DH ;
HELINSKI, DR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1979, 76 (04) :1648-1652
[10]   USE OF A GENE REPLACEMENT COSMID VECTOR FOR CLONING ALGINATE CONVERSION GENES FROM MUCOID AND NONMUCOID PSEUDOMONAS-AERUGINOSA STRAINS - ALGS CONTROLS EXPRESSION OF ALGT [J].
FLYNN, JL ;
OHMAN, DE .
JOURNAL OF BACTERIOLOGY, 1988, 170 (07) :3228-3236