Requirements of Escherichia coli to survive stress induced by the secretin, pIV : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand
Pathogenic Gram-negative bacteria utilise complex multiprotein and functionally
unrelated trans-envelope machineries to secrete toxins and other virulence factors.
Such machineries are referred to as secretion systems. These contain large,
membrane-inserted homologous channels called a secretin. These secretion systems
include the type II and III secretion systems (T2SS and T3SS), type IV pili assembly
system (T4PS), and the filamentous phage assembly-secretion system (FFSS). Secretins
are homomultimers with radial symmetry blocked by an inner gate or septum and
have a pore size of up to 10 nm.
As determined by previous studies on the FFSS secretin, pIV, and the T3SS secretin,
InvG, there is a cost associated with the insertion of large membrane channels.
Membrane integrity is disrupted, leaving the bacterial cell highly susceptible to
antibiotics and environmental stressors. As a result, Gram-negative bacteria have
developed stress response pathways which upregulate genes to mitigate this secretininduced
stress. These are the Phage Shock Protein response (Psp), Conjugative plasmid
expression (Cpx), Regulation of capsular synthesis (Rcs), and SoxRS Superoxide
response (Sox). Not all individual genes within these stress response pathways are
necessarily required for the survival of Escherichia coli expressing secretin.
Stress can be induced in E. coli by expression of leaky pIV mutants as they are open,
not gated, under physiological conditions and imitate the actively secreting channel. A
synthetic lethality assay was performed to determine the importance of the key
regulators from four stress response pathways (PspF, CpxR, RcsA, RcsB, SoxR, and
SoxS) on cell viability in the presence of the leaky secretin mutant, pIV-E292K. Here it
was determined that the Psp, Rcs and, (to a lesser extent), Cpx regulons, confer a
protective effect on E. coli K-12 experiencing stress induced by pIV-E292K. Expression
of pIV-E292K mutant also induced an Rcs-dependent capsular polysaccharide
phenotype indicating upregulation of Rcs in response to leaky pIV production. These
three responses are potential drug targets in the fight against antibiotic-resistant
infections. Inhibition of the stress response may prevent mitigation of membrane
stress, thereby killing the channel-expressing bacteria.