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Monday Article #52: Gram positive vs. negative bacteria: phosphorylation for survival

A discussion of survival mechanisms which respond to a change in osmolarity and increase in temperature involving phosphorylation in Escherichia coli (Gram negative) and Bacillus subtilis (positive) bacteria


Bacterial stress responses, observed in all living organisms, have long been an area of interest of the global scientific community. Some of the stress conditions experienced by prokaryotes include an increase in temperature, change in pH or osmolarity. The driving force of evolution has ensured that prokaryotes develop specific survival mechanisms to deal with induced cellular stress (White et al., 1995). Phosphorylation is a key biochemical reaction which has a key role in prokaryotic protective mechanisms in which most of them are ATP-dependent. Phosphorylation is a post-translational modification which involves the addition of phosphates to proteins. Well-known examples would be the phosphorylation of bacterial protein kinases in two component signalling systems and phosphorylation of the GroEL-ES chaperonin complex (Stock et al., 1989). This essay will focus on the role of the aforementioned molecular mechanisms in response to an increase in temperature and osmotic stress. We will then proceed to a discussion on the mechanistic similarities and differences in Escherichia coli (Gram negative) and Bacillus subtilis (positive) bacteria.

The role of two component signalling systems in Escherichia coli osmoregulation.

Osmotic stress is sensed in both high and low osmolarity conditions, and it is because of this stress response that E.coli is able to thrive in both extreme environments (Kempf and Bremer, 1998). E.coli have evolved to deal with osmotic stresses firstly through sensing the environmental change via the two component cell signalling system (Breland et al., 2017).

The two component signal transduction system is composed of a histidine (His) kinase sensor (EnvZ) and a cognate response regulator protein (OmpR) whose function is to mediate cellular response through expression of the OmpF and OmpC genes, which encodes for outer membrane protein (Omp) synthesis, which depends on the cellular osmolarity. The signal transduction process involves autophosphorylation of the EnvZ osmosensor followed by a phosphotransfer to the OmpR protein. EnvZ possesses two enzyme activities; a kinase activity for phosphorylation and a phosphatase activity for dephosphorylation of OmpR (Stock et al.,1989) (Figure 1).

The signal is then received at the N-terminal input domain of EnvZ and subsequent His residue phosphorylation occurs at the C-terminal transmitter domain. This causes the binding of EnvZ to OmpR, and phosphotransfer takes place via phosphorylation of the aspartate (Asp) residue on the receiver domain of OmpR. The signal will continue to pass through OmpR until it is transmitted to target genes OmpC or OmpF through the output domain. Determination of OmpC or OmpF transcription depends on the initial signal sensed by EnvZ. Finally, phosphatases will dephosphorylate OmpR once the function is fulfilled. This also resets the EnvZ-OmpR system to a non-stimulated state (White et al.,1995, Kenney and Anand, 2020).

OmpF and OmpC are transcribed during low and high osmolarity conditions respectively. These conditions also affect kinase and phosphatase activity of the EnvZ osmosensor and OmpR-P concentration (Kenney and Anand, 2020).

Figure 1: A schematic outlining the EnvZ-OmpR two-component system. Phosphorylation

plays a key role in regulation of this signalling system. Source: Stock et al.,1989.

GroEL-ES chaperonin system and proteases play a part in E.coli heat shock response

Besides osmotic stress, one of the other widely studied stress conditions experienced by E. coli include thermal stress (induced at temperatures between 30- 42°C) (Arsène et al.,2000).

Housekeeping σ70 and σE factors activates the promoter, initiating transcription of the RNA polymerase (RNAP) and subsequently the encoding gene for heat shock, rpoH. This activates the σ32 regulon (class 1 heat shock genes, HSG) in E.coli leading to production of heat shock proteins (HSPs) such as DnaK and the ATP-dependent GroEL-ES chaperonin system (Roncarati and Scarlato, 2017).

The DnaJ-DnaK chaperone proteins, in association with nucleotide exchange factor (NEF) GrpE transports misfolded proteins to the barrel-shaped GroEL-ES chaperonin complex The GroEL structure will then be bound to exposed hydrophobic regions of the misfolded protein. Protein refolding will proceed upon phosphorylation whereby ATP binding induces a conformational change and the subsequent recruitment of GroES to form the cap of the protein complex. Phosphorylation of the complex is required again, but on the opposite side in order for release of both GroES and the refolded protein into the cytoplasm (Lennarz and Lane, 2013) (Figure 2).

However, if GroEL-ES fails to refold the protein, FtsH, a HSP in the form of a protease, is tasked with degradation of the protein. FtsH, being ATP-dependent, would require phosphorylation for polypeptide substrate reorganisation for transportation to the proteasome to be metabolised. The broken down amino acids produced will be utilised by E.coli for production of new proteins (Missiakas et al.,1996; Wawrzynów et al., 1996).

Figure 2: A cartoon showcasing the GroEL-GroES chaperonin system. Phosphorylation is

key for this system to function. Source: Lennarz and Lane, 2013.

Osmotic stress in Bacillus subtilis is handled by OpuA expression and ABC transporters

The OpuA gene is expressed when B. subtilis is faced with osmotic stress. The EnvZ-OmpR regulon is not conserved in B. subtilis. OpuA gene encodes for ATP-binding cassettes (ABC) transporters, which can either be influx and efflux transporters and are mediated by the σB regulon. An ABC transporter is a multisubunit protein consisting of two transmembrane domains and two ATP-binding domains. The OpuA ABC transporter uptakes glycine betaine (GB) as an osmoprotectant. Interestingly, OpuA tends to be expressed dominantly although other transporters like OpuB and OpuC also exist (Kempf and Bremer, 1995; Hoffmann et al., 2013) (Figure 3).

The transporter first begins in its closed state, unbounded to GB or ATP. It then converts to an open conformation whereby the GB substrate enters the transmembrane domain space and binds to the transmembrane domains, closing it. This results in a conformational change and an increase in affinity which allows binding of two ATPs to the ATP-binding domains. This causes another change in conformation in the transmembrane domain which releases GB to the other side of the periplasm. ATP hydrolysis is then required to reset the OpuA transporters (Locher, 2009).

Figure 3: OpuA ABC transporter pathway. The transmembrane domain is located within the periplasm and the ATP-binding domain is located inside the periplasm as indicated by the blue arrows. Modified figure; adapted from: Hoffmann et al., 2013.

Coordination of the heat shock response in B.subtilis.

Unlike E.coli, the GroEL-ES chaperonin system is not conserved in Gram positive Bacillus subtilis. This is because that B. subtilis utilises the σB regulon (class 2 HSG) instead of the σ32 regulon (class 1) in E.coli (Haldenwang and Losick, 1979). The σB regulon is also induced by oxidative and starvation stress in addition to raised temperatures. Since that is the case, only general stress proteins are coded by the σB regulon (Hecker et al., 1996). The regulon tends to encode for HSG at extreme temperatures of 48 - 50°C (Narberhaus 1999; Servant and Mazodier 2001).

The σB regulon is suppressed by an anti-sigma factor encoded by the rsbW gene. This would result in preventing σ factor binding to RNAP. The RsbW protein also possesses serine kinase activity which is involved in phosphorylation of the RsbV anti-sigma factor, rendering the protein inactive.

B. subtilis utilises a unique “2-partner module switching” mechanism dictated by phosphorylation. It is important to note that this mechanism is only understood partially (Alper et al 1994). Each one of these modules contains 3 partner proteins whose binding is mediated by phosphorylation of one of the partner proteins. When exposed to heat stress, the RsbV anti-sigma factor is dephosphorylated by phosphatases RsbP or RsbU. RsbV then attacks the σB-RsbW complex. This stimulates “partner switching” of RsbW from σB to RsbV, with σB being released in the process. σB regulon is then free to transcribe general stress proteins for ATP-dependent protein refolding (Schumann, 2003) (Figure 4).

Just like E.coli, the FtsH protease will cleave unwanted proteins if general stress proteins are unable to refold the misfolded protein. The same ATP-dependent process occurs and the protein would be redirected to the proteasome for degradation (Duerling et al., 1997).

Figure 4: The “2-partner module switching”. All of these proteins work together to modulate σB as shown. σB is responsible for transcription initiation of general stress genes in Bacillus subtilis. Source: Schumann, 2003.

Summary of key distinctions

As discussed, there are striking differences in the systems used in both E.coli and B.subtilis in responding to osmotic and heat stress. From the cases presented here and with prior knowledge on Gram positive and negative bacteria, we can speculate that E.coli uses the EnvZ-OmpR regulon when dealing with osmotic stress because porins are able to be formed on the outer membrane of E.coli and not in B. subtilis. We believe most of these differences are due to the morphology of both Gram bacteria types.


To summarise, we have discussed a series of molecular mechanisms involving phosphorylation and dephosphorylation in response to osmotic and thermal stress. Despite the obvious differences, it is evident that phosphorylation is pivotal in order for these systems to be fully functional, therefore ensuring the survival of both Gram bacteria species. When dealing with osmotic stress, there is the EnvZ-OmpR regulon and expression of the OpuA gene. However, when responding to thermal stress, the σ32 and σB regulons modulate HSG. The protease FtsH seems to be highly conserved in Gram bacteria. It is important for microbiologists to study the differences in Gram bacteria stress responses in order to apply this knowledge in developing drugs which will act against multi-drug resistance (MDR) posed by pathogenic forms of these bacteria. Furthermore, we are also able to apply our knowledge on these bacterial systems when comparing with eukaryotic systems in response to these stresses as molecular mechanisms tend to be conserved to an extent.


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This article was prepared by Eldrian Tho