Cieslewicz M, Vimr E
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign 61801, USA.
J Bacteriol. 1996 Jun;178(11):3212-20. doi: 10.1128/jb.178.11.3212-3220.1996.
The kps locus for biosynthesis of the capsular polysialic acid virulence factor in Escherichia coli K1 contains at least two convergently transcribed operons, designated region 1 and regions 2 plus 3. On the basis of DNA sequence analysis, kpsF appeared to be a good candidate for the first gene of region 1 (M. J. Cieslewicz, S. M. Steenbergen, and E. R. Vimr, J. Bacteriol. 175:8018-8023, 1993). A preliminary indication that kpsF is required for capsule production is the capsule-negative phenotype of an aph T insertion in the chromosomal copy of kpsF. The present communication describes the isolation and phenotypic characterization of this mutant. Although transcription through kpsF was required for capsule production, complementation analysis failed to indicate a clear requirement for the KpsF polypeptide. However, since E. coli contains at least two other open reading frames that could code for homologs of KpsF, the apparent dispensability of KpsF remains provisional. DNA sequence analysis of 1,100 bp upstream from the kpsF translational start site did not reveal any open reading frames longer than 174 nucleotides, consistent with kpsF being the first gene of region 1. Since kpsF appeared to be the first gene of a region whose gene products are required for polysialic acid transport and because capsule production is known to be thermoregulated, primer extension analyses were carried out with total RNA isolated from cells grown at permissive (37 degrees C) and nonpermissive (20 degrees C) temperatures. The results revealed a potentially complex kpsF promoter-like region that was transcriptionally silent at the nonpermissive temperature, suggesting that thermoregulation of region 1 may be exerted through variations in kpsF expression. Additional evidence supporting this conclusion was obtained by demonstrating the effects of temperature on expression of the gene kpsE, immediately downstream of kpsF. Chloramphenicol acetyltransferase assays were carried out with constructs containing the kpsF 5' untranslated region fused to a promoterless cat cassette, providing further evidence that kpsF is thermoregulated. Although the function of KpsF is unclear, primary structure analysis indicated two motifs commonly observed in regulatory proteins and homology with glucosamine synthase from Rhizobium meliloti.
大肠杆菌K1中荚膜多聚唾液酸毒力因子生物合成的kps位点包含至少两个反向转录的操纵子，分别命名为区域1和区域2加3。基于DNA序列分析，kpsF似乎是区域1第一个基因的良好候选者（M. J. 西尔斯维茨、S. M. 斯滕伯格和E. R. 维姆尔，《细菌学杂志》175:8018 - 8023，1993）。kpsF是荚膜产生所必需的一个初步迹象是kpsF染色体拷贝中aph T插入导致的荚膜阴性表型。本通讯描述了该突变体的分离和表型特征。虽然荚膜产生需要通过kpsF进行转录，但互补分析未能表明对KpsF多肽有明确需求。然而，由于大肠杆菌至少还含有另外两个可能编码KpsF同源物的开放阅读框，KpsF明显的非必需性仍属暂时情况。对kpsF翻译起始位点上游1100 bp的DNA序列分析未发现任何长度超过174个核苷酸的开放阅读框，这与kpsF是区域1的第一个基因一致。由于kpsF似乎是一个区域的第一个基因，该区域的基因产物是多聚唾液酸运输所必需的，并且已知荚膜产生受温度调节，因此对从允许温度（37℃）和非允许温度（20℃）下生长的细胞中分离的总RNA进行了引物延伸分析。结果揭示了一个潜在复杂的类似kpsF启动子的区域，该区域在非允许温度下转录沉默，这表明区域1的温度调节可能通过kpsF表达的变化来实现。通过证明温度对紧邻kpsF下游的kpsE基因表达的影响，获得了支持这一结论的更多证据。用含有与无启动子cat盒融合的kpsF 5'非翻译区的构建体进行了氯霉素乙酰转移酶测定，进一步证明kpsF受温度调节。虽然KpsF的功能尚不清楚，但一级结构分析表明在调节蛋白中常见的两个基序，并与苜蓿根瘤菌的氨基葡萄糖合酶具有同源性。
