Thermoregulation of kpsF, the first region 1 gene in the kps locus for polysialic acid biosynthesis in Escherichia coli K1
Thermoregulation of kpsF, the first region 1 gene in the kps locus for polysialic acid biosynthesis in Escherichia coli K1
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DOI：
10.1128/jb.178.11.3212-3220.1996
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发表时间：
1996-06-01
影响因子：
3.2
通讯作者：
Vimr, E
中科院分区：
文献类型：
--
作者：
Cieslewicz, M;Vimr, E
在大肠杆菌K1中，用于生物合成衣壳多唾液酸毒力因子的KPS基因座至少含有两个可转换转录的操纵子，分别被指定为区域1和区域2+3。根据DNA序列分析，KPSF是区域1的第一个基因(M.J.Cieslewicz，S.M.Steenbergen和E.R.Vimr，J.细菌)的一个很好的候选基因。175：8018-8023,1993)。一个初步的迹象表明，KPSF是生产胶囊所必需的，即在KPSF的染色体拷贝中插入了一个aph T的包膜阴性表型。本通讯描述了该突变体的分离和表型特征。尽管通过KPSF转录是生产胶囊所必需的，但互补分析没有表明对KPSF多肽的明确需求。然而，由于大肠杆菌至少包含另外两个可编码KPSF同源物的开放阅读框架，KPSF表面上的可有可无仍然是暂时的。对KPSF翻译起始点上游1,100个核苷酸的DNA序列分析没有发现任何长于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，其基因产物是聚唾液酸运输所必需的，而且已知胶囊的产生受温度调节，因此对在允许(37摄氏度)和非允许(20摄氏度)温度下生长的细胞的总RNA进行了引物延伸分析。结果揭示了一个潜在的复杂的KPSF启动子样区，该区域在不允许的温度下转录沉默，这表明区域1的温度调节可能是通过KPSF表达的变化来实施的。支持这一结论的其他证据是通过证明温度对KPSF下游的kpsE基因表达的影响而获得的。氯霉素乙酰转移酶检测是用含有KPSF 5‘非翻译区的构建体融合到一个无启动子的猫盒上进行的，这进一步证明了KPSF是温度调节的。虽然KPSF的功能尚不清楚，但一级结构分析表明，KPSF在调控蛋白中有两个常见的基序，并与苜蓿根瘤菌的氨基葡萄糖合成酶同源。
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 aphT 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 vas 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.