Exploitation of new bacteriophages for generic strain engineering methods and functional genomic analysis of diverse bacteria

在快速基因克隆和高通量DNA测序方法出现之前，对大多数细菌的遗传学，生物化学和生理学只有非常有限的知识。在这些技术发展之前，对细菌的最复杂的研究是用E。大肠杆菌K12菌株，其具有大量的背景信息，基于通过体内和体外遗传操作的组合进行本发明遗传分析和菌株构建的能力。近年来，确定基因组序列的能力以令人难以置信的速度发展，因此现在可以在一天内确定新细菌的完整DNA序列（尽管基因注释需要相当长的时间）。研究基因表达的方法学（例如通过Q-RT-PCR分析）的其他技术进步显著增强了研究细菌中特定基因表达调控的能力，对于这些细菌没有或只有最少的遗传分析方法可用。然而，在“新”细菌中全面和有意义地利用总基因组序列信息的瓶颈通常是能够产生定义的特定突变体并在转录组学、蛋白质组学和代谢组学中对这些突变体进行生理学上严格的研究。为了实现这一目标，研究人员通常不得不尝试从经过充分研究的细菌中转移现有的遗传和分子生物学方法进行诱变和互补（通常是基于质粒的）-结果差异极大。在缺乏严格定义的突变体和干净的互补策略的情况下，比较组学研究的准确性充其量是值得怀疑的，最坏的情况是根本不存在。因此，具有用于在菌株之间转移定义的突变基因和用于细菌中的菌株工程化的简便且稳健的方法将是非常有用的，对于所述细菌，完整的基因组序列是已知的，但是对于所述细菌，除了生物信息学预测之外，几乎没有其他信息。在本项目中，我们将分离和开发一些细菌病毒-广义转导病毒（GTP）-用于一系列已被基因组测序的细菌宿主，但目前几乎没有可用的基因工程方法。这些GTP将是有用的细菌菌株的建设，需要强大的比较野生型和突变株的功能基因组学研究计划。此外，我们将开始工程改造我们在细菌小鼠病原体中发现的新噬菌体（phiNP）。这种噬菌体是温和的，并且将其基因组以单拷贝整合到细菌染色体中，位于靠近参与蛋白质合成和核糖体再循环的有效控制的细菌基因（tmRNA或ssrA）末端的精确位置。当噬菌体基因组整合到细菌基因序列中时，它产生靶序列复制，使得tmRNA靶基因在功能上重建，因此在细菌宿主中不存在作为获得病毒DNA的结果的明显缺陷。细菌tmRNA基因在细菌中分布非常广泛，甚至在高等细胞的质体基因组中也有发现。因此，这种细菌病毒可以被操纵以制备衍生物，其将提供用于将突变和正常基因、诱变转座子和其他遗传元件转移到含有保守靶序列的细菌宿主的染色体中的工具。因此，通过基因工程方法，我们打算获得基于phiNP的工具，其在诱变、克隆和互补分析中具有广泛的宿主范围适用性。GTP和phiNP衍生技术的结合将扩大可以进行强大功能基因组学的细菌宿主的数量，这将使更多的研究人员能够进行多样化和严格控制的“组学研究”。

Until the advent of rapid gene cloning and high throughput DNA sequencing methods, there was only very limited knowledge on the genetics, biochemistry and physiology of most bacteria. Prior to the development of such technologies, the most sophisticated studies on bacteria were done with E. coli K12 strains for which there was tremendous background information based on the ability to do inventive genetic analysis and strain constructions by a combination of in vivo and in vitro genetic manipulation. In recent years the ability to determine genome sequences has advanced at an incredible pace and so it is now possible to determine the complete DNA sequence of a new bacterium within a day (although gene annotation takes considerably longer). Additional technical advances in the methodologies for studying gene expression (e.g. by Q-RT-PCR analysis) have enhanced significantly the ability to investigate regulation of expression of specific genes in bacteria for which there are no, or minimal, genetic analysis methods available. However, a bottleneck in the full and meaningful exploitation of total genomic sequence information in 'new' bacteria is often the ability to make defined, specific mutants and to genetically complement such mutants for physiologically rigorous studies in transcriptomics, proteomics and metabolomics. To achieve this the researcher usually has to try to transfer existing genetic and molecular biology methods for mutagenesis and complementation (usually plasmid-based) from well-studied bacteria - with extremely variable outcomes. In the absence of rigorously defined mutants and clean complementation strategies, the veracity of comparative 'omic studies is questionable, at best, and non-existent at worst. Consequently it would be very useful to have facile and robust methods for transferring defined mutant genes between strains and for strain engineering in bacteria for which the full genomic sequences are known, but for which there is little other information - except bioinformatic prediction. In this project we will isolate and develop some bacterial viruses - generalised transducing phages (GTPs) - for a range of bacterial hosts which have been genomically sequenced, but for which there is little in the way of genetic engineering methodology currently available. These GTPs will be useful for bacterial strain constructions that are required for robust comparisons of wild type and mutant strains in functional genomics research programmes. In addition, we will start the engineering of a new bacteriophage (phiNP) that we discovered in a bacterial mouse pathogen. This phage is temperate and integrates its genome into the bacterial chromosome in single copy at a precise location towards the end of a bacterial gene involved in the efficient control of protein synthesis and ribosome recycling (tmRNA or ssrA). When the phage genome integrates into the bacterial gene sequence it creates a target sequence duplication such that the tmRNA target gene is functionally reconstituted and thus there is no obvious defect in the bacterial host as a consequence of acquiring the virus DNA. The bacterial tmRNA gene is very widely distributed in bacteria and is even found in plastid genomes of higher cells. Consequently, this bacterial virus could be manipulated to make derivatives that will provide tools for transferring mutant and normal genes, mutagenic transposons and other genetic elements into the chromosomes of bacterial hosts containing the conserved target sequence. Therefore, by gene engineering methods, we intend to derive phiNP-based tools with a broad host range applicability in mutagenesis, cloning and complementation analysis. The combination of GTPs and phiNP-derived technologies will broaden the number of bacterial hosts for which powerful functional genomics can be performed and this should enfranchise more researchers for diverse - and rigorously controlled - 'omic studies.