Conjugative DNA transfer, antibiotic resistance and MDR bacteria

Antibiotics – a medical miracle Discovery of antibiotics changed the medical landscape Now possible to treat and cure bacterial infections that once killed millions Bacterial infection as cause of death plummeted Life expectancy increased by 8 years between 1944 and 1972 Much of this increase attributed to antibiotics

The antibiotic resistance problem Drug resistance happens quickly One study observed an increase from 0% to 28% drug resistant E. coli in less than 5 years Drug resistant bacteria are very wide spread occurring throughout the world Hospitals are centers for drug-resistant organisms MRSA There may be more deaths from MRSA infections in the US than from AIDS

CDC MDR TB rates worldwide

How antibiotics work

evolution.berkeley.edu How do drug resistant bugs arise?

evolution.berkeley.edu How do drug resistant bugs arise?

evolution.berkeley.edu How do drug resistant bugs arise?

evolution.berkeley.edu How do drug resistant bugs arise?

How did that 1 st drug resistant bug arise? A simple error in DNA replication that produced a mutation Occurs at low frequency Mutation is on the chromosome Mutation affects either ribosomal protein S12 or 16S rRNA to produce streptomycin resistance Does not explain MDR bugs or high rate of spread

A couple of pieces of information We know that drug resistance spreads at an alarming rate Far too fast to be the result of single mutations in the chromosome that arise independently We also know that bacteria become resistant to more than a single drug If this were the result of point mutations in the chromosome the rate would be even slower

Can you think of other mechanisms of drug resistance? A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms

Two questions 1– how are plasmids rapidly transferred in a bacterial population? 2 – how do plasmids encode resistance to multiple drugs? To understand the rapid increase in multiple drug resistant strains of bacteria there are two questions we must answer.

Bacterial conjugation Driven by conjugative plasmids; 1 st example called the fertility factor or F found in some but not all E. coli one of several different types of conjugative plasmid Mating only between cell with F (F + ) and cell without F (F – ) Transfer of information is one-way from donor to recipient Cells must be in close cell-cell contact for DNA transfer to occur

F Plasmid A 100 kbp plasmid (single copy) with ~ 100 genes – Replicates inside host cell using host machinery for replication – Partitions to daughter cells in a manner similar to chromosome

F Plasmid Contains genes encoding synthesis of pillin which is assembled into pili that allow cell contact F+ cells have pili and F- cells lack pilli F+ inhibited from making contact with other F+ cells

F Plasmid F + cells conjugate with F – cells – F + donates single-stranded copy of F to F – cell (rolling circle) – F + retains copy of plasmid, F - cell converted to F + by replication of ssDNA donated to the F- cell – Allows F plasmid to rapidly spread through a bacterial population

Bacterial Conjugation Bacterial conjugation is the primary mechanism used to spread antibiotic resistance among bacterial populations There will be several million infections involving antibiotic resistant bacteria this year This is now a very significant health problem

Pumping ssDNA

Inhibition of the Relaxase 1 nM 10 nM

Inhibition of DNA transfer

Transposable Genetic Elements A variety of colorful names have been used to describe these genetic elements Controlling elements Jumping genes Roving genes Mobile genetic elements Transposons Definition: Transposable genetic elements (transposons) are DNA segments that can insert themselves at one or more sites in a genome. They are ubiquitous among organisms and play an important role in genome evolution. Remarkably, almost 50% of our chromosomes consist of transposable elements We are still unsure of the normal genetic role, if any, of these elements

Composite versus simple Tns Composite Tns contain a variety of genes between two IS elements Transposase is encoded by one of the elements Individual IS elements cannot move Simple Tn contains short IRs at each end Encode their own transposase and other genes

R plasmids

Mechanisms of Transposition Transposition is catalyzed by an enzyme, transposase, encoded by the transposon The ends of the transposon are critical for transposition

Human genes contain transposons Human genome is typical in terms of abundance and distribution of mobile elements How do we survive? Elements inserted into introns Vast majority of elements cannot move There are instances of mutations caused by mobile elements

R plasmids

CDC TB rates worldwide