Franklin M J, Ohman D E
Department of Microbiology and Immunology, University of Tennessee, Memphis.
J Bacteriol. 1993 Aug;175(16):5057-65. doi: 10.1128/jb.175.16.5057-5065.1993.
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 effect 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.
铜绿假单胞菌的黏液型菌株产生一种名为藻酸盐的高分子量胞外多糖，该多糖会通过添加O - 乙酰基进行修饰。为了更好地理解乙酰化过程，本研究鉴定了一个参与藻酸盐乙酰化的基因algF。我们推测参与藻酸盐乙酰化的基因位于铜绿假单胞菌染色体上34分钟处的藻酸盐生物合成基因簇内。为了分离algF突变体，开发了一种局部诱变程序，将随机化学突变引入染色体上的铜绿假单胞菌藻酸盐生物合成操纵子中。为此，使用了一个在基因置换黏粒载体中包含藻酸盐生物合成操纵子和相邻argF基因的DNA片段。该质粒在体内包装成λ噬菌体颗粒，在体外用羟胺诱变，转导到大肠杆菌中，然后转移到铜绿假单胞菌FRD的argF营养缺陷型菌株中。Arg⁺重组体共同继承了诱变的藻酸盐基因簇，并通过测试对产气克雷伯菌产生的藻酸裂解酶的敏感性增加来筛选藻酸盐乙酰化缺陷。通过比色法和红外光谱法测试对藻酸裂解酶敏感性增加的重组体产生的藻酸盐的乙酰化情况。获得了两个产生的藻酸盐乙酰基减少超过六倍的algF突变体。当置于trc启动子控制下的正确方向时，来自藻酸盐基因簇的一个3.8 kb XbaI - BamHI片段通过反式互补了乙酰化缺陷。通过部分二倍体分析，通过检查Tn501插入的极性效应，将algF基因进一步定位到algA直接上游的一个区域。通过基因置换，将在algA直接上游有Tn501插入的DNA与黏液型菌株FRD1的染色体进行重组。由于转座子对下游algA基因的极性效应，产生的菌株FRD1003是非黏液型的。通过在tac启动子控制下反式提供algA，FRD1003产生了非乙酰化的藻酸盐，表明转座子位于algF内或其紧邻上游。这些结果表明，参与藻酸盐乙酰化的基因algF位于algA的直接上游。
