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，人们对大多数细菌的遗传学、生物化学和生理学的了解非常有限。在这些技术发展之前，最复杂的细菌研究是用大肠杆菌K12菌株进行的，由于能够通过体内和体外遗传操作的结合进行创造性的遗传分析和菌株构建，因此有大量的背景信息。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，因此现在有可能在一天内确定一种新细菌的完整DNA序列（尽管基因注释需要相当长的时间）。研究基因表达方法的其他技术进步（例如Q-RT-PCR分析）大大提高了研究细菌中特定基因表达调控的能力，而目前没有或很少有遗传分析方法可用。然而，在“新”细菌中充分和有意义地利用总基因组序列信息的瓶颈通常是制造明确的、特定的突变体的能力，以及在转录组学、蛋白质组学和代谢组学中对这些突变体进行遗传补充的能力。为了实现这一点，研究人员通常必须尝试将现有的遗传和分子生物学方法用于诱变和从经过充分研究的细菌中进行互补（通常是基于质粒的）——结果极不相同。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往好了说是值得怀疑的，往坏了说是不存在的。因此，如果有一种简便而可靠的方法来在菌株之间转移已定义的突变基因，以及在已知完整基因组序列但除了生物信息学预测之外几乎没有其他信息的细菌中进行菌株工程，将是非常有用的。在这个项目中，我们将分离和开发一些细菌病毒-通用转导噬菌体(GTPs) -用于一系列细菌宿主，这些细菌宿主已经进行了基因组测序，但目前几乎没有可用的基因工程方法。这些GTPs将有助于在功能基因组学研究项目中进行野生型和突变型菌株的可靠比较所需的菌株构建。此外，我们将开始对我们在细菌性小鼠病原体中发现的一种新的噬菌体（phiNP）进行工程设计。这种噬菌体是温和的，并将其基因组整合到细菌染色体的单个拷贝中，位于细菌基因末端的精确位置，该基因参与有效控制蛋白质合成和核糖体循环（tmRNA或ssrA）。当噬菌体基因组整合到细菌基因序列中时，它会产生一个目标序列重复，从而使tmRNA目标基因在功能上被重组，因此在获得病毒DNA的细菌宿主中没有明显的缺陷。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来制造衍生物，这些衍生物将为将突变和正常基因、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提供工具。因此，我们打算通过基因工程的方法，在诱变、克隆和互补分析中获得基于phphp的广泛宿主适用性的工具。gtp和phinp衍生技术的结合将扩大细菌宿主的数量，这些细菌宿主可以进行强大的功能基因组学研究，这应该使更多的研究人员能够进行多样化的、严格控制的基因组学研究。

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.