This newly established ESR is described as a complementary mechanism for managing envelope stress, entirely independent to the other ESR pathways previously described (McBroom and Kuehn, 2007).
Vesicular release occurs throughout the growth of Gram-negative bacteria and involving the blebbing out and subsequent fission of the OM (reviewed by (McBroom and Kuehn, 2005, Mayrand and Grenier, 1989). This allows controlled regulation of envelope components through the removal of unwanted material, as well as providing intercellular messaging and transport vehicles. A role for vesicles in host-pathogen interactions has been indicated as they provide an alternative means for the release of toxins to host cells and surrounding environments (McBroom and Kuehn, 2005, Kuehn and Kesty, 2005). McBroom and Kuehn (2007) have suggested that during envelope stress, such releases allow removal of toxic protein species and other damaged material. Vesiculation would maintain envelope homeostasis and promote cellular survival in an advantageous manner through this “selective disposal” of misfolded polypeptides. This was supported by the observation of a positive correlation between increases in vesiculation and bacterial survival, with this ‘bulk-flow mechanism’ sufficiently relieving envelope stresses in the absence of previously described ESRs (McBroom and Kuehn, 2007).
Due to the independent nature of OM vesicle production, there appears not to be a regulatory link between this stress response and those previously described. How this novel ESR is induced and the means by which cells transmit such signals to the OM vesicle machinery is still unknown and requires further investigation. The regulatory mechanisms controlling vesicle contents also requires more attention as this selective process is essential for the correct application of vesiculation as an ESR.
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1.11 BaeSR
The BaeSR stress response was first discovered during a search for new 2CST systems, and was later characterised as the third ESR (Raffa and Raivio, 2002). However, compared to the σE and Cpx ESRs, little work has
been conducted into BaeSR. This ESR is yet to be characterised in depth, most likely due to the fact that deletion of baeS or baeR produce few noticeably phenotypes (Appia-Ayme et al., 2011).
Overexpression of the RR, BaeR, increases bacterial resistance to beta- lactam antibiotics, novobiocin (an aminocoumarin antibiotic) and bile salts in
E. coli (Raffa and Raivio, 2002). Functional overlap with the Cpx-pathway
has been observed as double deletion mutants of the RR in both two- component systems (baeR and cpxR) present an increased sensitivity to envelope stress than the single mutants alone. An overlap is also observed in the genes regulated by the Cpx and BaeSR pathways, of which spy is a prominent example. However, the core BaeSR regulon in E. coli contains three genes, amongst others, encoding members of the resistance nodulation-cell division (RND) family of multidrug transporters; mdtA, acrB and acrD (Bury-Mone et al., 2009). Sodium tungstate (Na2WO4) is a natural
substrate of MdtABC influx pumps (Leblanc et al., 2011). In S. Typhimurium
baeR is up-regulated in the presence of tungstate and baeR deletion
mutants have an increased sensitivity to tungstate compared to the isogenic parent strain (Appia-Ayme et al., 2011). Strains carrying mutations in
mdtABCD and/or acrAB or acrD lose their ability to grow on tungstate,
whereas single mutations of the RND efflux pump systems do not show sensitivity. CpxR also regulates transcription of mdtA and acrD, but a cpxR deletion does not result in tungstate sensitivity. BaeR is therefore the primary regulatory of these genes in response to tungstate waste removal and is critically required for S. Typhimurium resistance to Na2WO4 (Appia-Ayme et
al., 2011). Functional overlap between these RND transporters exists for waste disposal of tungstate from the cell, suggesting a role for BaeSR in
Salmonella survival from the stresses inflicted from water and soil where
Expression of BaeSR is also growth phase linked, like σE, as induction of
baeSR increases during stationary phase growth when grown in LB (alkaline
conditions) (Appia-Ayme et al., 2011, Miticka et al., 2003). Other inducers of BaeR activity include: iron, copper, zinc and high concentrations of indole, a compound found in the intestines and faeces due to bacterial decomposition of tryptophan.
Indole at µM concentrations is utilised by bacteria as a signalling molecule in the intestines of humans and other mammals (Botsford and Demoss, 1972), a process encouraged by the induction of tnaA (an indole producing tryptophanase) by the alkaline pH and low nutrient concentrations present within the intestines (Han et al., 2011). The survival rate of a S. Typhimurium
baeR mutant is no different to the isogenic parent strain in the presence of
mM indole concentrations (Appia-Ayme et al., 2011). However, in a baeR deletion strain of E. coli, 1 mM indole significantly reduces cell viability (Raffa and Raivio, 2002). E. coli is an indole producer whereas Salmonella is not and it has been hypothesised that differences in survival of these mutant strains is a result of dissimilarities in the indole producing and sensing pathways of these two bacterial species (Appia-Ayme et al., 2011).
BaeSR is required for Salmonella enterica serovar Dublin (S. Dublin) colonisation and subsequent systemic salmonellosis in orally infected cattle (Pullinger et al., 2010). Appia-Ayme and colleagues (2011) ascertained that in a murine typhoid model, BaeSR is not required for S. Typhimurium infection, with no significant attenuation of a baeR mutant observed, compared to the isogenic parent strain SL1344, after oral infection.
The BaeSR system is also associated with zinc stress tolerance in E. coli and S. Typhimurium (Nishino et al., 2007, Wang and Fierke, 2013). This function of the Bae ESR with be discussed in Chapter 5 and will therefore not be mentioned here.
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1.12 ZraPSR
The ZraPSR system is discussed in detail in Chapter 5. Below is a brief introduction to this newly characterised ESR.
The majority of investigations conducted on the ZraSR 2CST system are associated with zinc homeostasis in E. coli. Zinc plays an essential role in cell metabolism. However, at high concentrations, accumulation of this metal is cytotoxic and strict homeostatic mechanisms are needed to prevent this. Numerous E. coli genes involved in such processes, including zraR, have been identified; regulated by zinc inducible promoters (Outten et al., 1999). In response to elevated Zn2+ concentrations, both the RR, ZraR, and the HK, ZraS activate zraP, which is divergently transcribed from zraSR (Franke et al., 2001). Within the zraP-zraSR intergenic region, a σ54 dependent promoter is located where it is believed ZraR, as a bEBP, binds. Noll et al. (1998) showed that in the absence of transcriptional activators, this promoter is completely switched off, whereas the weak constitutive promoter of zraSR allows for basal levels of expression of both the RR and SK (Ravikumar et al., 2011, Noll et al., 1998, Leonhartsberger et al., 2001).
Although originally described as a zinc responsive 2CST system, Appia- Ayme et al. (2011) provided the first evidence of the involvement of ZraPSR in envelope stress. A regulatory link, directly or indirectly, between ZraPSR and the BaeSR system was observed; overexpression of BaeR results in significant transcriptional repression of zraP. Other links between ZraP and BaeR have been indicated in the presence of tungstate (Appia-Ayme et al., 2011) and this compound was shown to induce all ESR pathways, but in a
baeR mutant background, zraP and zraSR were both highly up-regulated at
the transcriptional level. These results concur with previous observations in
E. coli that showed repression of baeR upon zinc addition, proposed to be a