TRANSPOSITIONAL MUTAGENESIS AND MUTACINS IN S MUTANS

龋齿是一种传染性和传播性疾病，而变种 链球菌被认为是主要的病原体。龋齿 仍然影响着很大一部分人口，包括在美国 而且是全世界的。一些变形链球菌菌株能产生一种 一种名为突变菌素的抗菌物质，它能杀死 相同的或密切相关的物种。突变蛋白的生产就是其中之一 变形链球菌的特征，这可能有助于其定植能力 并在口腔中持续存在。出于这个原因，突变体可能是 被认为是致命性因素。因为它们独特的抑制作用 属性，关于突变体的知识可以应用于最终的控制 变形链球菌感染的发病机制。不幸的是， 人们对它的监管或在什么情况下的监管知之甚少。 制造。因为这些物质是小批量生产的，而且只有在 某些确定的条件，过去尝试用生化方法分离突变菌素 都没有成功。我们已经成功地提纯了突变蛋白 多肽并测定其氨基酸组成和N-末端 序列。结合转座突变，结构基因 因为突变体已经被明确定位在从 变形链球菌UA96的染色体。 因此，这一提议需要继续进行基因表征 利用各种已被证实的遗传技术对突变体基因座进行研究。这个 这里的目的是在分子水平上了解突变体是如何 生产和加工的。它的体积很小，加上它的核糖体 人工合成允许广泛的基因操作。其中一种方法是 称为定点突变，它允许单个碱基对改变 结构基因，它反过来改变了氨基酸组成 粘附素多肽。多肽组成的变化可能会导致 突变素性质的变化导致光谱增强 活动或物理特性。因为穆塔金非常像 一组重要的多肽抗生素，称为l抗生素，它们是 由其他革兰氏阳性细菌产生，发展的潜力 突变蛋白成为一种治疗剂有着很大的希望。另外，因为 突变素基因座可能聚为一个单一单位，它应该是 有可能将整个基因座转移到其他细菌中，如非 致龋性效应菌株，例如血链球菌或无毒变形链球菌 紧张。这构成了替代疗法的基础。的表达 突变蛋白可以帮助工程菌株定植到 一个现有的生态环境或防止入侵的S. 变种进入口腔。 有几个与健康有关的长期利益的研究 牧羊人的制作。首先，可以开发一种手段来提高大型 规模化生产用于治疗应用的防腐剂。第二，知识 对这种基因的控制或调节将使我们深入了解 这种重要有机体的毒力因素。最后，也是重要的一点， 突变蛋白具有广泛的抗菌活性，包括一种 对人类致病菌化脓性链球菌和致病菌有明显的杀菌作用 肺炎链球菌。在抗药性日益增强的环境中 人类病原体，另类抗菌剂，如突变体，可能会有生命- 作为一种新的抗生素，节省了价值。

Dental caries is an infectious and transmissible disease and the mutans streptococci have been implicated as major causative agents. Dental caries still affects a large segment of the population, both in the United States and world wide. Some strains of Streptococcus mutans produce an antibacterial substance called mutacin which kill other bacteria of the same or closely related species. The production of mutacin is one characteristic of S. mutans which may contribute to its ability to colonize and be sustained in the oral cavity. For this reason, mutacin may be considered a virulence factor. Because of their unique inhibitory properties, knowledge about mutacin may be applied to the eventual control of the pathogenesis of mutans streptococcal infections. Unfortunately, very little is known about its regulation or under what conditions it is made. Because these substances are made in small quantities and only under certain defined conditions, past attempts to isolate mutacin biochemically have been unsuccessful. We have successfully purified the mutacin polypeptide and determined its amino acid composition and N-terminal sequence. Combined with transpositional mutagenesis, the structural gene for mutacin has been definitively located on a cloned segment of DNA from the chromosome of S. mutans UA96. Accordingly, this proposal entails the continued genetic characterization of the mutacin locus using a variety of proven genetic techniques. The objective here is to understand, at the molecular level, how mutacin is produced and processed. Its small size coupled with its ribosomal synthesis permits a wide range of genetic manipulation. One such method is called site-directed mutagenesis which allows single base-pair changes in the structural gene which, in turn, changes the amino acid composition of the mutacin polypeptide. A change in peptide composition could cause changes in the properties of mutacin leading to enhanced spectrum of activity or physical properties. Because mutacin closely resembles the important group of peptide antibiotics called lantibiotics which are produced by other gram-positive bacteria, the potential for developing mutacin into a therapeutic agent holds great promise. In addition, because the mutacin gene locus is likely clustered as a single unit, it should be possible to transfer the entire gene locus into other bacteria such as non- cariogenic effector strains, e.g., S. sanguis or an avirulent S. mutans strain. This constitutes the basis of replacement therapy. Expression of mutacin could either assist the colonization of the engineered strain into an existing ecological milieu or prevent the colonization of invading S. mutans into the oral cavity. There are several long-term health-related interests for the study of mutacin production. First, a means could be developed to enhance the large scale production of mutacin for therapeutic application. Second, knowledge of the control or regulation of this gene will give us insight into one of the virulence factors of this important organism. Lastly and importantly, mutacin exhibits a wide range of antibacterial activity, including a pronounced bactericidal action against the human pathogens S. pyogenes and S. pneumoniae. In a growing climate of increased drug resistance among human pathogens, alternative antimicrobials such as mutacin may have life- saving value as a new antibiotic.