Cues from the Membrane: Bacterial Glycerophospholipids

ABSTRACT

In this issue of the Journal of Bacteriology, V. W. Rowlett et al. unveil new Escherichia coli circuitry linking membrane glycerophospholipid (GPL) homeostasis to bacterial stress response and adaptation mechanisms (J Bacteriol 199:e00849-16, 2017, https://doi.org/10.1128/JB.00849-16). Glycerophospholipids comprise critical components of the dual-membrane envelope of Gram-negative bacteria and participate in many processes. The new evidence suggests that, in some instances, distinct E. coli GPL molecules function for distinct biochemistry and bacteria sense perturbations in membrane GPL concentrations to coordinate survival strategies. Understanding GPL sensing and remodeling mechanisms will be important moving forward, given the breadth of function for these molecules in bacteriology.

TEXT

Evolution provided nature with lipid amphiphiles to build barriers known as membrane bilayers. As a boundary and scaffold, the lipid bilayer comprises the principal surface of most cellular life. Humans have exploited the amphipathic properties of lipids for millennia and studied their biochemistry for nearly a century. Nevertheless, sparse knowledge exists of how particular lipids impact cell biology, especially in prokaryotes. In this issue of the Journal of Bacteriology, Rowlett et al. provide a biochemical and physiologic tour de force and analysis of Escherichia coli mutants lacking either the phosphatidylethanolamine (PE) or cardiolipin (CL) family of glycerophospholipids (GPL) (1).

Eugene P. Kennedy (1919 to 2011) and colleagues elucidated the GPL biosynthesis pathway between the mid-1950s and early 1970s by assaying rat liver mitochondria and E. coli extracts for catalysis (2–4). During the late 1970s and early 1980s, bacterial-mutant screens and gene-mapping approaches made it possible to identify the loci for these activities. Kennedy's pupils embraced the new approaches, especially Christian R. H. Raetz (1946 to 2011) and William Dowhan, an author of the work discussed here (1). Raetz thrust the field forward by designing groundbreaking mutant screens to identify lesions in genes like cdsA, pss, psd, and pgsA by colony autoradiography and in vivo activity screening (5–9). The approach worked elegantly, and by the 1990s, many E. coli open reading frames for Kennedy pathway enzymes had been mapped and mutated. Several genes for GPL biosynthesis were conditionally essential for bacterial viability, making their study complex. Rowlett et al. (1) revisit historic mutants, including a Pss-null mutant that lacks PE lipids and is viable only in the presence of nonphysiologic concentrations of MgCl₂ (20 to 50 mM) (10). The PE-deficient strain is compared to a more recently engineered CL-deficient E. coli clsABC triple mutant, in which what we now understand to be three distinct CL synthase enzymes are deleted (11).

Rowlett et al. identify strong phenotypes for PE- and CL-deficient E. coli strains, including dramatic alterations in morphology, envelope structure, O antigen chain length, carbon flux into surface molecules, outer membrane (OM) protein folding, energy production, membrane potential, metabolism, and biofilm formation (1). Removing PE has pleiotropic effects on E. coli physiology, and titrating pss expression in the mutant rescues phenotypes in a dose-dependent manner. Cardiolipin deficiency attenuates many of the same functions as PE depletion in the bacteria. Cardiolipin depletion also causes varied effects on bacterial synthesis and assembly of surface carbohydrates and biofilm formation. For example, the CL-deficient bacteria produce a longer O antigen structure than the wild type, while PE-depleted cells generate a more truncated molecule. For the latter, the O antigen chain length is restored to the wild type upon pss induction. Furthermore, the CL-deficient E. coli cells do not form biofilms, while bacteria in which PE is deleted are biofilm hyperproducers. Finally, bacteria in which CL is deleted that are exposed to hydrogen peroxide and basic pH grow better than the unexposed cells, a response not observed for the wild type. The data support the idea that E. coli cells employ CL molecules for specific functions.

Many of the enzymes and activities for GPL biosynthesis are conserved between E. coli and mammals. Cardiolipin is a major exception. The primary bacterial CL synthase for E. coli, ClsA, catalyzes a reaction whose substrates and mechanisms are distinct from those of the mammalian CL synthase in mitochondria (12). In fact, both ClsA and ClsB catalyze a condensation reaction between two phosphatidylglycerol (PG) substrates to form the CL product (11, 13). The third E. coli enzyme, ClsC, catalyzes a reaction involving PG and PE substrates (11). Therefore, multiple mechanisms and enzymes for CL synthesis exist, supporting the idea that the pathway is under strong positive selection for variation and redundancy. In further support of specialized roles for CL in Gram-negative bacteriology, the genes encoding pss, psd, and pgsA are each conditionally essential for bacterial viability, but clsA, encoding the major CL synthase, is not (10, 14–17). In fact, even the clsABC triple mutant assessed by Rowlett et al. shows no growth rate attenuation in rich broth medium compared to the growth of the wild type (1). Therefore, unlike most other major GPL families, CL is biochemically and genetically redundant for E. coli and performs specialized nonessential functions.

To understand the stress responses elicited by PE and CL deficiency, the authors developed gene-reporter assays, Western blots, and second-messenger measurements to detect the abundance and activity of key regulators. Many of the promoters chosen for the reporter constructs are regulated by more than one transcription factor, making the results challenging to interpret quantitatively. Fortunately, the authors probe further and directly measure the protein and nucleotide abundance. The analysis provides a snapshot of the stress response elicited by the loss of either PE or CL. They blot for the housekeeping sigma factor, RpoD, the alternative sigma factors, RpoE, RpoH, and RpoS, a periplasmic protease, DegP, a chaperone, DnaK, and a heat shock-related stress protein, GroL. The data suggest that PE- and CL-deficient cells activate a variety of cytoplasmic and envelope-related stress response mechanisms. Both PE- and CL-deficient bacteria activate the stringent response pathway, since the levels of the nucleotide alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as ppGpp, increase in the mutants compared to their levels in the wild-type bacteria. Subtle differences in the levels of key regulators exist between the PE and CL deletion mutant bacteria. In general, PE depletion caused dramatic increases in all the stress-related regulatory molecules analyzed and a strong decrease in the housekeeping molecule RpoD. In contrast, CL deficiency caused a less dramatic increase in the regulators, especially RpoE and RpoS, while the levels of RpoD were not affected. Thus, E. coli might harbor distinct sensory mechanisms to detect perturbations in either PE or CL abundance. Cardiolipin deletion increased the level of ppGpp but did not cause a concomitant decrease in RpoD, suggesting that the response to CL perturbation is complex. A principal role for the ppGpp second messenger is to decrease rpoD promoter activity, such that RNA polymerase is made available to the alternative sigma factors. Therefore, bacterial adaptation to CL depletion might involve the simultaneous expression of homeostatic and stress response systems. The results are consistent with E. coli sensing membrane lipid perturbation by regulatory mechanisms.

Evidence in the literature supports the idea that bacterial stress sensors might respond to GPL concentrations directly. For example, the CpxA inner membrane (IM) sensor kinase responds to a variety of signals originating within the E. coli envelope, and PE and PG deficiencies induce CpxA activity (18–21). Phosphatidylethanolamine is synthesized from serine, and the Pss enzyme associates with the ribosome (22). The primary ppGpp synthetase in E. coli, RelA, also interacts with the ribosome, where it detects uncharged tRNAs that accumulate in response to amino acid starvation (23). Perhaps signaling occurs between Pss and RelA at the ribosome to control ppGpp increases during fluctuations in amino acid substrate availability. Finally, the OM sensor lipoprotein, RcsF, detects OM stress, including alterations in lipid content (24). It is now recognized that RcsF can be threaded through the lumen of OM beta-barrel proteins to access the OM outer leaflet and detect lipid perturbations (25, 26). Therefore, E. coli regulators harbor mechanisms to sense and respond to alterations in membrane lipid content.

From the late 1970s to the early 1980s, Raetz and others continued to probe the mutants that were defective for GPL biosynthesis. A particular temperature-sensitive mutant has a mutation in pgsA, the enzyme that catalyzes the first committed step in the synthesis of PG and CL, which caused the accumulation of two unknown glycolipid species within the E. coli membrane, lipids X and Y (27). These molecules were identified as synthetic precursors to the lipid A disaccharolipid, which comprises the majority of the OM outer leaflet and forms the hydrophobic base of the LPS superstructure. The data strongly indicated a critical intersection between acidic GPL and lipid A biosynthesis. However, the field was propelled toward identifying the genes encoding the lipid A biosynthesis enzymes. Soon it was determined that lipid A could be regulated by Gram-negative bacteria to increase resistance to antibiotics and decrease pathogen recognition by innate immune receptors. Until recently, there was minimal evidence to suggest that Gram-negative bacteria regulate the GPL composition of the envelope in response to changes in their environment. The exception again was CL, whose content increases upon entry into stationary phase (28, 29). Recent studies in Salmonella enterica serovar Typhimurium demonstrate that the PhoPQ virulence regulators control OM acidic-GPL content to increase OM barrier function and intracellular survival during pathogenesis in mice (30). The chemical modifications regulated include increased CL trafficking to the OM and increased activity of the OM palmitoyltransferase/phospholipase A1 enzyme, PagP (30, 31). In fact, PhoPQ activation promotes PagP palmitoylation of both lipid A and PG substrates within the OM outer leaflet (31).

Gram-negative bacteria likely harbor a variety of mechanisms to alter membrane GPL content. However, it remains challenging to study GPL molecules, in part owing to the biochemical complexity of the cell envelope, which consists of several distinct layers of varied chemical composition.

Within the dual-membrane cell envelope of E. coli, GPL structures interact with other lipid amphiphiles, such as neutral glycerolipids, lipopolysaccharides, membrane-derived glycolipids, and capsular exopolysaccharides. Unfortunately for experimentalists, bacteria rely upon GPL to synthesize, bind, and assemble most of the other envelope constituents. This has made gene knockout studies difficult to interpret. Consequently, progress in understanding the signals and regulatory conditions that cue bacteria to alter membrane GPL composition has been slow, and sparse knowledge exists for how alterations in GPL composition and structure affect physiology and adaptation. Only recently have we begun to understand the mechanisms by which GPL perturbance might cue bacterial sensors of stress. The work by Rowlett et al. (1) and others continues to probe this biology, and many questions remain.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.