ENVIRONMENTAL AND ENVELOPE STRESS RESPONSES IN THE AQUATIC ALPHA-PROTEOBACTERIUM CAULOBACTER CRESCENTUS

Our goal is to learn how the non-pathogenic alpha-proteobacterium Caulobacter crescentus (1, 2) responds to environmental stresses, particularly those that target its cellular membranes or promote increased antibiotic tolerance.Topic A: HipBA toxin-antitoxin systemsToxin-antitoxin (TA) systems are small operons that encode a toxin protein and a corresponding antitoxin, which can be a protein or an RNA (3). When free of inhibition, the toxin acts on various targets to inhibit cell growth or cause death. These ubiquitous modules have an addictive quality that stems from the differential stabilities of the toxin and antitoxin. While the toxin is stable and long-lived, the antitoxin is labile and subject to degradation. Therefore, the cell must continually produce the antitoxin to avoid dormancy or death.Although they seem detrimental, TA modules are involved in a variety processes that are beneficial to their bacterial hosts, including biofilm formation, phage resistance, stress responses, and persistence toward antibiotics (4-7). In contrast to antibiotic resistance, which is due to heritable genetic changes, persistence occurs when genetically susceptible bacteria enter a transient physiological state in which they are tolerant of otherwise lethal concentrations of drugs. Persister cells can then resume normal growth once the antibiotic is removed. TA systems are linked to persistence because toxin activation often induces dormancy, and most antibiotics target actively growing bacteria (8).The first TA system connected with persister formation was E. coli hipBA, named for high incidence of persistence (9, 10). When free of the antitoxin HipB, HipA functions as a serine/threonine kinase (STK), and among other targets, it phosphorylates the glutamyl tRNA synthetase GltX. Phosphorylated GltX is inactive, leading to a block in translation and entry into a dormancy via the stringent response (11, 12).Caulobacter crescentus is unusual in having three paralogous HipBA TA systems, whose functions we have begun to dissect. To summarize our preliminary data, all three hipBA modules encode bona fide TA systems, and kinase activity is necessary for the toxicity of each HipA protein. Deletion of hipBA1 or hipBA2 causes a reduction in the fraction of persisters formed during stationary phase, while ectopic expression of HipA1 or HipA2 increases the persister fraction. HipA1 and HipA2 phosphorylate GltX as well as two other tRNA synthetases, LysS and TrpS. Overexpression of GltX or TrpS, but not LysS, reduces the persister fraction in stationary phase, indicating that phosphorylation of GltX and/or TrpS is mechanistically linked to HipA-induced persistence. These results demonstrate that some, but not all, targets of HipA1 and HipA2 are involved in generating dormant persisters.In contrast, the hipBA3 mutant is exquisitely sensitive to oxidative and osmotic stresses during phosphate depletion. Phosphate depletion triggers the degradation of HipB3 by the ATP-dependent protease HslVU (13), which frees HipA3 to act on downstream targets. Activated HipA3 promotes the transcripton of dps, encoding a ferritin-like iron storage protein that detoxifies hydrogen peroxide (14). Although deletion of hipBA3 does not impair persister formation during late stationary phase, ectopic HipA3 expression increases the fraction of persister cells, much like HipA1 and HipA2. We will therefore identify substrates of HipA3 to determine how it stimulates dps transcription and promotes antibiotic persistence.Topic B: Lipid A synthesis and envelope stress responsesThe outer membrane (OM) of Gram-negative bacteria is a protective permeability barrier containing the glycolipid lipopolysaccharide. The O-antigen and core oligosaccharide components of LPS are frequently dispensable for growth, but the acylated lipid A molecule, anchored in the outer leaflet of the OM, is essential for the viability of nearly all Gram-negative bacterial species. Lipid A synthesis is performed by enzymes of the conserved Raetz pathway (15), which are present in the Caulobacter genome. However, Caulobacter lacks orthologs of signal transduction systems known to detect and respond to cell envelope stresses, such as unfolded periplasmic proteins or compromised barrier function (16).We previously identified a tyrosine phosphatase homolog, CtpA, important for Caulobacter cell envelope integrity. When CtpA is depleted, the cells die after developing large outer membrane blebs. Suppressor mutations conferring survival after ctpA deletion were identified in genes for O-antigen biosynthesis and in the transcription factor fur (ferric uptake regulator) (17). Surprisingly, the ctpA fur mutant contained 1000-fold less lipid A than wild-type Caulobacter. These results prompted us to attempt to delete lpxC, a conserved lipid A biosynthetic gene. A fur mutant was able to lose lpxC, and the resulting double mutant contained no detectable lipid A. Caulobacter crescentus is only the fourth Gram-negative species to survive the complete loss of lipid A (18-20), making it a key system for probing the functions of lipid A and for understanding how bacteria adapt to lipid A depletion.Since fur normally regulates transcription when bound to Fe2+ (21), we examined lipid A production by Caulobacter grown in iron-depleted medium. Both the fur deletion and iron limitation allowed Caulobacter to lose lipid A through chemical inhibition of the LpxC, but neither condition forces Caulobacter to stop synthesizing lipid A on its own. The fact that iron availability modulates the essentiality of lipid A in Caulobacter raises the possibility that lipid A may be conditionally essential in other bacteria, including plant and animal pathogens or symbionts.Although Caulobacter lacks known envelope stress responses, it possesses an essential two-component signal transduction system (cenKR) that affects cell envelope integrity (22). When the histidine kinase CenK or the DNA-binding response regulator CenR is depleted, they cells develop extensive membrane blebs and lose viability, very similar cells depleted of CtpA or LpxC. We therefore want to know how this signal transduction system regulates the synthesis or homeostasis of the cell envelope.Specific objectives forunderstanding the role of HipBA systems in Caulobacter stress responses:A1) Determine if Caulobacter mutants lacking hipBA3 have a decreased persister fraction in conditions of phosphate limitation, when HipA3 would normally be activated.A2) Identify Caulobacter proteins phosphorylated by HipA3 using quantitative phosphoproteomics.A3) Validate selected HipA3 substrates and determine which substrates are required for stress resistance and/or persister formation.A4) Reconstitute HipB3 proteolysis by HslUV in vitro to determine how it is triggered by phosphate limitation.Specific aims for understanding how Caulobacter survives in the absence of lipid A and adapts to envelope stress:B1) Determine if upregulation of degP and/or cpxP is necessary for the fur mutant to permit the loss of ctpA or lpxC. Caulobacter homologs of cpxP and degP are upregulated in the fur mutant (23), and in E. coli, these proteins combat envelope stress by degrading unfolded periplasmic proteins (24, 25).B2) Use an unbiased forward genetic approach to identify genes necessary for the fitness of the fur lpxC double mutant.B3) Characterize the transcriptional profile of the fur lpxC mutant using RNA-seq.B4) Characterize the CenR regulon using RNA-seq and ChIP-seq to identify genes downstream of the CenKR signaling system.B5) Isolate suppressor mutants capable of surviving in the absence of cenK and/or cenR. As with ctpA, the genes affected by suppressor mutations will provide clues to the essential function(s) of CenKR.B6) Determine if suppressor mutants lacking cenK or cenR (from B5) contain lipid A.