Introduction: In the last lecture, we discussed bacterial DNA: chromosomal, plasmid and phage. Today, I will introduce you to mobile genetic elements called tranposable elements or transposons. Transposons are important in the evolution of multiple antibiotic resistance. Further, transposon mutagenesis has been an invaluable tool in the genetic analysis of pathogenic bacteria.

IV. Transposons

A. General properties

• Ubiquitous in nature; found on chromosome, plasmids and phage
• Transposition is Rec-independent
• frequency: 10^-4-10^-7
• encode transposase
• inverted terminal repeats at their ends
• Minimal target sequence specificity, bordering on random
• Like plasmids, often confer antibiotic resistance.
• Regulated: Transpostion occurs at low frequency. Controlled by transposase and its repressor.
• Generate polar mutations

B. Types: Based on mode of transposition

• Conservative: Copy number doesn't increase upon transpositon:
• Replicative: Tn is copied upon transposition resulting in two copies
• Conjugative: Transposon encodes Tra functions, excises and transfers to another host.

C. Transposons as tools to study pathogenesis

1. Transposon mutagenesis. The elegant genetic analysis possible in a few bacteria such as E. coli and B. subtilis are not possible in most pathogens. Indeed, even natural isolates of E. coli are often refractile to genetic analysis such as transformation and transduction. As a consequence, methods have been developed which facilitate the introduction of transposable elements into a wide variety of both gram negative and gram positive bacteria. Transposable elements can be introduced into the genome of bacteria where they insert somewhat randomly thus causing insertion mutations. Since the average bacterial species has approximately 3000 genes, one can saturate the chromosome with ease. Furthermore, the transposon provides a molecular tag which can be subsequently used to identify the mutated gene and clone it. In combination with genomics, transposons are a powerful approach to identify genes.

2. Features of transposon mutagenesis:

• generate random (or semi-random) insertion mutations
• tag mutated gene allowing isolation and characterization of that and surrounding genes.
• generate polar muations.

3. Introduction of transposons: There are many different methods to introduce transposons into bacteria. The choice will depend on the nature of the target bacterium. Here are a few methods based on the use of suicide vectors. In each case, a replicon containing the transposon is propogated in E. coli then transferred into the target bacterium where the Tn is selected.

• A temperature-sensitive replicon:
• Bump plasmid by introduction of an incompatible replicon
• Transfer plasmid lacking essential replication protein supplied in trans in donor cell
• phage that lacks replication in host.

E. Before, I discuss the use of transposons, let me clarify the difference between a transcriptional and translational fusion and discuss the concept of a reporter:

• Reporter: A reporter is used in biological systems to quantitate gene or protein expression. The reporter usually is an enzyme that has properties making it easy to measure:
• Examples include lacZ encoding beta-galactosidase; phoA encoding alkaline phosphatase; GFP or green fluorescent protein.
• Transcriptional fusions rely on the promoter of a target gene, but contain their own ribosome binding site and start codon. Lac-fusions express beta-galactosidase under the transcriptional control of another gene.
• Translational fusions are in-frame fusions betweent two proteins, so that the promoter, ribosome binding site, start codon, and some or all of the coding sequence are fused in-frame with another protein.

F. TnlacZ: mediates transcriptional fusions:

G. TnphoA: One example of a wonderful system for identifying mutants using translational fusions is called TnPhoA. This transposon has at one end the gene encoding alkaline phosphatase but without its signal sequence. Alkaline phosphatase is inactive in E. coli unless it is exported outside the cytoplasmic membrane (it is normally in the periplasm). When TnPhoA inserts into a gene in the appropriate orientation and in the correct reading frame, a translational fusion will result. If the gene into which TnPhoA inserts encodes a secreted protein, then the fusion will be exported. The presence of a secreted fusion can be monitored by including in the agar medium a substate for alkaline phosphatase which results in a blue color upon hydrolysis. Thus, one can screen colonies on agar medium for the rare blue colony. Approximately 5-10% of bacterial proteins have signal sequences, which means about 250 secreted proteins. Also, most virulence factors are secreted proteins (toxins, pili, etc). Taken together, this approach allows an investigator to isolate insertion mutations within many of the bacterial secreted proteins in a very short time. If one knows how virulence factors are regulated in that species, one can look for colonies which are conditionally blue.

VI. Pathogenicity Islands

The term pathogenicity island has been used to refer to large chromosomal regions in pathogenic bacteria that encode virulence genes. It appears that pathogenicity islands can confer complex virulence phenotypes and were acquired by bacteria from unrelated organisms, leading to interesting hypotheses about how bacterial pathogens evolved. It is likely that mechanisms that generate pathogenicity islands continue to operate and may contribute to the emergence of bacterial pathogens with new virulence properties.

Analysis of bacterial populations often reveals that virulence genes, or antibiotic resistance genes, are or were mobile. Hints may include the presence of a transposase, integrase, etc.