Perry R D, Pendrak M L, Schuetze P
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130-3932.
J Bacteriol. 1990 Oct;172(10):5929-37. doi: 10.1128/jb.172.10.5929-5937.1990.
The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.
鼠疫耶尔森菌吸收足够的外源性血红素或刚果红以形成色素沉着菌落的温度依赖性已被称为色素沉着表型（Pgm+）。自发突变为Pgm-表型会导致许多不同生理特征的丧失，包括在26℃下储存血红素和结合刚果红的能力。在本研究中，我们生成并分离了血红素储存阴性（Hms-）的转座子插入突变体，因此它们无法形成色素沉着菌落。这些突变是由于在染色体DNA的19.5千碱基（kb）SalI片段内单个迷你卡那霉素插入所致。对八个突变体的限制性位点分析确定了至少六个潜在不同的插入位点，跨越一个约10 kb的血红素储存（hms）基因座。从其中一个突变体KIM6-2012克隆了19.5 kb的SalI片段（包含约18 kb的鼠疫耶尔森菌DNA和迷你卡那霉素插入片段）。通过使用该克隆片段作为DNA探针，确定了自发突变为Pgm-表型的机制是大规模缺失事件。在来自九个不同鼠疫耶尔森菌菌株的自发Pgm-突变体中，缺失跨越至少18 kb的基因组DNA。与迷你卡那霉素插入片段相邻的DNA用于鉴定包含野生型hms基因座的克隆。含有该克隆的鼠疫耶尔森菌KIM的自发Pgm-突变体表现出Hms+表型。本研究中产生的hms::迷你卡那霉素突变和克隆的野生型hms基因座将极大地有助于确定鼠疫耶尔森菌中血红素储存的功能。
