Info

individual signature tagged mutants

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input pool recovered pool

Hybridize to replicate filters

recovered pool

Hybridize to replicate filters

Identify attenuated mutants

Administer to animal model

Administer to animal model

Isolate in vivo and in vitro bacterial mRNA and use as template for cDNA synthesis

Hybriidiizse to xDNA

Identify mRNAs only present in vivo

antibiotic resistance gene antibiotic resistance gene in vitro reporter gen (lacZ)

in vivo reporter gen (purA, cat, hly, tnpR)

random xDNA fragments in vitro reporter gen (lacZ)

in vivo reporter gen (purA, cat, hly, tnpR)

random xDNA fragments

Administer plasmid library to animal model

Recover in vivo active sublibrary

Identify in vitro inactive clones as in vivo induced

Figure 2 Schematic representation of the basic principles of STM (A), SCOTS (B) and (R-)IVET (C). Abbreviations: IVET, in vivo expression technology; SCOTS, selective capture of transcribed sequences; STM, signature tagged mutagenesis.

strategies have mainly been applied for the identification of genes from pathogens which are important during infection of their animal host. Signature tagged mutagenesis (STM) utilizes a negative selection strategy in which an animal host is infected with a pool of sequence-tagged insertion mutants. Mutated genes represented in the initial inoculum but not recovered from the host are essential for growth in the host (27,28). A major advantage of STM is that this type of screen provides direct proof for the importance of the mutated genes in the relevant niche. Unfortunately, only limited numbers of mutants can be screened per animal model. Therefore, large scale animal experiments are required for genome-wide mutant screens and for this reason STM screens are labor-intensive. In addition, mutants that are slow-growing, contain mutations in genes encoding redundant functions, or that can be complemented in a mixed population remain undetected or are at least underrepresented (29). Moreover, mutants for genes that are essential in the laboratory can never be obtained and, therefore, their importance for persistence in vivo cannot be investigated using this technique. Nevertheless, the STM strategy has been applied successfully to identify genes important in Gl-tract colonization by at least six enteric pathogens, including Klebsiella pneumoniae, Vibrio cholerae, and Escherichia coli (27). Lipopolysaccharides have been recognized as an important factor in Gl-tract persistence and colonization of several Gram-negative bacteria, as they have emerged as a common theme in the STM-based studies. In addition, the importance of the global regulator of anaerobic metabolism Fnr was highlighted by several STM screens, which is not surprising considering the low oxygen tension in the colon. Moreover, the alternate sigma factor RpoN was found in several of the STM screens and is likely to associate with RNA polymerase to promote the transcription of genes that are specifically required in the Gl-tract niche. Finally, STM studies revealed the importance of specific adhesins, including the type IV pili of Vibrio cholerae and Citrobacter rodentium (27).

A second strategy that has been applied for the identification of in vivo transcribed genes is selective capture of transcribed sequences (SCOTS). cDNA is prepared from total RNA isolated from infected cells, or tissue samples. cDNA mixtures obtained are then enriched for sequences that are transcribed preferentially during growth in the host, using hybridizations to biotinylated bacterial genomic DNA in the presence of cDNA similarly prepared from bacteria grown in vitro. This strategy is very effective for the identification of highly abundant genes in situ which are also expressed to a lower level in the laboratory. In contrast to the STM strategy, genes that are essential in the laboratory can be investigated for their importance in GI-tract colonization. Nevertheless, major disadvantages of SCOTS are the instability of bacterial mRNA for the construction of cDNA libraries, the low abundance of mRNA from transiently or lowly expressed genes, and the technical difficulty in isolation of sufficient high-quality mRNA from small populations of bacteria in vivo (29). SCOTS has only been applied in a limited number of studies and the majority of these screens was performed to identify bacterial genes expressed within macrophages (30-33). More recently, the first SCOTS strategy utilizing an animal model to identify genes important during infection was performed (34). This approach resulted in the identification of Escherichia coli genes of which the expression is either relatively abundant or induced in vivo. Similar to the STM approaches described above, this SCOTS approach revealed the induction of expression of genes involved in pilus formation and lipopolysaccharide (LPS) biosynthesis. Other genes identified included iron-responsive and plasmid- and phage-encoded genes (34).

The third strategy that has been used to identify genes that are specifically induced or required during infection is in vivo expression technology (IVET). Similar to SCOTS, the IVET strategy is capable of identifying genes that are non-essential or redundant, while in an STM approach genes are only identified that are essential in vivo. An important difference between IVET and SCOTS lies in the fact that SCOTS is capable of identifying genes that are active in the laboratory, but, nevertheless, are induced in the host, while IVET only identifies in vivo induced genes that are very lowly or not expressed in the laboratory. The IVET approach relies on the generation of transcriptional fusions of genomic sequences to a reporter gene encoding an enzymatic activity. Nowadays, four variations of IVET utilizing different reporter genes have evolved as discussed in the section below.

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