MUCOID-TO-NONMUCOID CONVERSION IN ALGINATE-PRODUCING PSEUDOMONAS-AERUGINOSA OFTEN RESULTS FROM SPONTANEOUS MUTATIONS IN ALGT, ENCODING A PUTATIVE ALTERNATE SIGMA-FACTOR, AND SHOWS EVIDENCE FOR AUTOREGULATION
MUCOID-TO-NONMUCOID CONVERSION IN ALGINATE-PRODUCING PSEUDOMONAS-AERUGINOSA OFTEN RESULTS FROM SPONTANEOUS MUTATIONS IN ALGT, ENCODING A PUTATIVE ALTERNATE SIGMA-FACTOR, AND SHOWS EVIDENCE FOR AUTOREGULATION
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DOI：
10.1128/jb.176.21.6677-6687.1994
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发表时间：
1994-11-01
影响因子：
3.2
通讯作者：
OHMAN, DE
中科院分区：
文献类型：
--
作者：
DEVRIES, CA;OHMAN, DE
粘液样表型在引起囊性纤维化患者慢性肺部感染的铜绿假单胞菌菌株中很常见，这是由于一种称为藻酸盐的胞外多糖的过量产生所致。然而，粘液表型在体外不稳定，特别是当细胞在低氧张力下孵育时。自发转化为非粘液形式通常是由于与藻酸盐调节基因 algT 密切相关的突变（以前称为 algS）所致，该基因位于染色体上 68 分钟。我们对 algT 的序列分析表明，其 22 kDa 基因产物与细菌中的几个替代 sigma 因子具有同源性，这表明 AlgT（也称为 AlgU）直接与 RNA 聚合酶核心相互作用以激活藻酸盐基因的启动子。 AlgT 显示出与大肠杆菌的 sigma(E) 惊人的序列相似性 (79%)，这是一种参与高温基因表达的替代 sigma 因子。我们对囊性纤维化分离株 FRD 中从粘液样自发转化为非粘液样的分子基础进行的分析表明，非粘液样转化通常是由于 algT 中密码子 18 和 29 处发生的两种不同错义突变之一所致。RNase 保护测定表明，具有 algT18 和 algT29 等位基因的自发非粘液样菌株减少了四到五倍 与野生型粘液菌株相比，algT 转录物的积累有所增加。同样，与粘液样野生型菌株相比，质粒携带的 algT-cat 转录融合体在 algT18 和 algT29 背景中的活性大约低 3 倍，在 algT::Tn501 背景中活性低 20 倍。这些数据表明 algT 是自动调节的。自发的 algT 错义等位基因还导致相邻负调节因子 algN（也称为 mucB）的表达减少约五倍。 algT::Tn501 菌株中基本上不存在 algN 的转录本。因此，algT 调节 algTN 簇，并且这两个基因可能是共转录的。引物延伸分析显示 algT 转录在翻译起点上游 54 bp 处开始。尽管algT启动子与营养西格玛因子识别的启动子几乎没有相似之处，但它与algR启动子相似。这一发现表明 AlgT 可能作为 sigma 因子来激活其自身的启动子和其他藻酸盐基因的启动子。引物延伸分析还表明，在典型的非粘液菌株 PAO1 中很容易检测到 algT 转录本，这与 FRD 的 algT18 突变体中看到的微弱信号形成鲜明对比。 PAO1 中的质粒携带的 algT 基因导致粘液样表型和高水平的 algT 转录物，进一步支持 AlgT 控制其自身基因表达和藻酸盐调节子基因表达的假设。
The mucoid phenotype is common among strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis and is due to overproduction of an exopolysaccharide called alginate. However, the mucoid phenotype is unstable in vitro, especially when the cells are incubated under low oxygen tension. Spontaneous conversion to the nonmucoid form is typically due to mutations (previously called algS) that are closely linked to the alginate regulatory gene algT, located at 68 min on the chromosome. Our sequence analysis of algT showed that its 22-kDa gene product shares homology with several alternate sigma factors in bacteria, suggesting that AlgT (also known as AlgU) interacts directly with RNA polymerase core to activate the promoters of alginate genes. AlgT showed striking sequence similarity (79%) to sigma(E) of Escherichia coli, an alternate sigma factor involved in high-temperature gene expression. Our analysis of the molecular basis for spontaneous conversion from mucoid to nonmucoid, in the cystic fibrosis isolate FRD, revealed that nonmucoid conversion was often due to one of two distinct missense mutations in algT that occurred at codons 18 and 29. RNase protection assays showed that spontaneous nonmucoid strains with the algT18 and algT29 alleles have a four- to fivefold reduction in the accumulation of algT transcripts compared with the wild-type mucoid strain. Likewise, a plasmid-borne algT-cat transcriptional fusion was about 3-fold less active in the algT18 and algT29 backgrounds compared with the mucoid wild-type strain, and it was 20-fold less active in an algT::Tn501 background. These data indicate that algT is autoregulated. The spontaneous algT missense alleles also caused about fivefold-reduced expression of the adjacent negative regulator, algN (also known as mucB). Transcripts of algN were essentially absent in the algT::Tn501 strain. Thus, algT regulates the algTN cluster, and the two genes may be cotranscribed. A primer extension analysis showed that algT transcription starts 54 bp upstream of the start of translation. Although the algT promoter showed little similarity to promoters recognized by the vegetative sigma factor, it was similar to the algR promoter. This finding suggests that AlgT may function as a sigma factor to activate its own promoter and those of other alginate genes. The primer extension analysis also showed that algT transcripts were readily detectable in the typical nonmucoid strain PAO1, which was in contrast to a weak signal seen in the algT18 mutant of FRD. A plasmid-borne algT gene in PAO1 resulted in both the mucoid phenotype and high levels of algT transcripts, further supporting the hypothesis that AlgT controls its own gene expression and expression of genes of the alginate regulon.