Biology subjects

Underhill, S. A. M.

Publications and source records attributed to Underhill, S. A. M..

4 recordsLinked to original sources

bioRxiv2022

Redundancy in citrate and cis-aconitate transport in Pseudomonas aeruginosa

Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the TCA cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Loss of tctDE has been associated with sensitization to aminoglycosides, possibly linking tricarboxylate transport to enhanced antimicrobial resistance. In this work, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate porin; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to its inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. Author SummaryPseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. In some cases, there may be links between antibiotic uptake and the transport of metabolic molecules, making cross-membrane transport medically important. In this work we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.

microbiology↗

bioRxiv2019

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence signaling peptide (CSP) induces bimodal expression of comX, with only a fraction of cells in the population becoming transformable. Curiously, bimodal comX activation in response to CSP is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but in defined media lacking small peptides CSP induces no response in comX. In addition, growth on certain sugars other than glucose increases the proportion of the population that activates comX in response to CSP, relative to growth on glucose. By investigating the connection between media and bimodal comX expression, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, which is the origin of comX bimodality. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the intracellular XIP/ComS signal. Deletion of pepO eliminates bimodality in comX, leading to a unimodal comX response to CSP in defined and complex media. We also find that CSP upregulates comR in a carbohydrate source-dependent fashion, providing an additional stimulus to the ComRS feedback system. Our data provide mechanistic insight into how CSP regulates the bistable competence circuit and explain the puzzle of growth medium-dependence in S. mutans competence regulation.

biophysics↗

bioRxiv2018

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

AbstractEntry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX.\n\nAuthor SummaryHeterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

microbiology↗

bioRxiv2017

Intercellular communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses and ecological competitiveness. One example is activation of natural genetic competence by ComR and the comX-inducing peptide (XIP) in Streptococcus mutans. Although the competence pathway can be activated by addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signal molecule has not been rigorously tested. Co-culture model systems were developed that included a \"sender\" strain that overexpressed the XIP precursor (ComS) and a \"responder\" strain harboring a GFP reporter fusion to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using i) planktonic culture systems, ii) cells suspended in an agarose matrix or iii) cells growing in biofilms. XIP was shown to be freely diffusible and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with endogenously-produced peptide(s) in populations in various environments and physiologic states.\n\nIMPORTANCEThe comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids and addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence and evolution of a ubiquitous human pathogen and related organisms.

microbiology↗