(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator [1]

['Samuel A. Adeleye', 'Waksman Institute Of Microbiology', 'Department Of Genetics', 'Rutgers University', 'Piscataway New Jersey', 'United States Of America', 'Srujana S. Yadavalli']

Date: 2024-05 

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions–Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE’s secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of “one gene, one enzyme, one function”, which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

In stressful environments, proteins in many organisms can take on extra roles. When Escherichia coli bacteria are exposed to antimicrobial compounds, the cell activates the PhoQ/PhoP signaling system, increasing the production of an enzyme called QueE. QueE is usually involved in the formation of queuosine (Q) tRNA modification. When cells make abundant QueE, it interacts with a vital division protein, FtsZ, disrupting division and causing elongation − a "moonlighting" function. Detailed study of QueE reveals specific regions involved in Q biosynthesis or cell division. QueE in organisms closely related to E. coli also has dual roles, while distant relatives are unifunctional. Comparative analysis identifies a unique E. coli QueE region regulating cell division. This study shows QueE’s versatility in linking and impacting distinct cellular processes such as RNA metabolism, protein translation, cell division, and stress response.

In this study, using QueE as a model, we investigate the molecular determinants that allow a protein to perform two distinct functions. We analyze QueE’s dual roles in tRNA modification and cell division by utilizing a specialized northern blotting technique and microscopy, respectively, as readouts. By examining single alanine mutants and variants of E. coli QueE (EcQueE), we establish that the catalytic activity required for QueE’s biosynthetic role is dispensable for its function as a cell division regulator. As a corollary, we identify several individual amino acid residues and a specific region in EcQueE, which are necessary to cause filamentation but not for Q biosynthesis. Analysis of QueE homologs from different bacteria shows that the moonlighting functions of QueE are conserved among other enterobacteria, suggesting this mode of cell division regulation is widespread.

(a) Schematic representation of the dual roles of QueE upon strong activation of the PhoQ/PhoP two-component system. QueE is an enzyme required for the biosynthesis of queuosine (Q) tRNA modification. E. coli cells under strong PhoQP-activating stress conditions such as exposure to sub-MIC levels of specific antimicrobial peptides, upregulate QueE, which then modulates cell division in addition to Q biosynthesis. (b) Methods used to study the two activities of QueE–APB (N-acryloyl-3-aminophenylboronic acid) gel and northern blotting to detect Q-tRNAs and microscopy to observe filamentation. (c) APB gel-northern blot detecting tRNA Tyr in total RNA samples of E. coli wild-type (WT, MG1655) and ΔqueE (SAM31) cells encoding either an empty vector (pEB52) or wild-type (WT) QueE (pRL03). G-tRNA Tyr = unmodified tRNA Tyr , Q-tRNA Tyr = Q-modified tRNA Tyr . (d) Representative phase-contrast micrographs of E. coli wild-type (WT, MG1655) and ΔqueE (SAM31) cells expressing either an empty vector (pEB52) or WT QueE (pRL03), scale bar = 5 μm. For (c) and (d), cells were grown in supplemented MinA minimal medium for 2 hours (OD 600 = 0.2–0.3) and induced with IPTG (“+”) for 3 hours.

Our previous work found that an enzyme–QueE–involved in the biosynthetic pathway for queuosine (Q) tRNA modification also plays a role in stress response [ 10 ]. Q is a hypermodified guanosine that is found ubiquitously at the wobble position of the anticodon loop of specific tRNAs–tRNA His , tRNA Tyr , tRNA Asp , and tRNA Asn [ 11 – 14 ]. Q-tRNA modification is crucial to maintain translation fidelity and efficiency [ 15 ]. It has also been implicated in redox, virulence, development, and cancers [ 14 , 16 , 17 ]. Despite its universal distribution and importance, only bacteria are capable of de novo synthesis of Q from guanosine triphosphate (GTP), and eukaryotes salvage precursors of Q from diet or gut bacteria [ 11 , 14 ]. In the biosynthesis of Q, three enzymes QueD, QueE, and QueC, are required to produce a vital intermediate PreQ 0 [ 18 ]. Specifically, QueE (also called 7-carboxy-7-deazaguanine or CDG synthase) catalyzes the conversion of the substrate CPH 4 (6-carboxy-5,6,7,8-tetrahydropterin) to CDG (7-carboxy-7-deazaguanine) [ 19 , 20 ]. The role of QueE in the biosynthesis of Q has been well characterized, and crystal structures of QueE homologs from Bacillus subtilis, Burkholderia multivorans, and Escherichia coli have been solved, providing insights into the catalytic mechanism [ 19 – 22 ]. More recently, a second function for QueE has been described during the stress response of E. coli cells exposed to sub-MIC levels of cationic-antimicrobial peptides (AMP) [ 10 ]. During this response, the PhoQ/PhoP two-component signaling system, which plays an important role in sensing antimicrobial peptides and several other signals [ 23 – 25 ] is strongly activated, leading to an increase in QueE expression ( Fig 1A ). When QueE is upregulated in the cell, it binds at the site of cell division and blocks septation. Consequently, E. coli cells grow as long heterogeneous filaments ranging from a few microns to hundreds of microns in length. It has been shown that QueE localizes to the septal Z-ring, a vital structure in bacterial cell division [ 26 , 27 ], inhibiting septation post-Z ring formation in an SOS-independent manner [ 10 ]. This QueE-mediated filamentation phenotype is also observed under other conditions that activate the PhoQ/PhoP system robustly, such as when cells lacking MgrB (a negative feedback inhibitor of PhoQ [ 28 , 29 ]) are grown under magnesium limitation. Although historically filamentation was considered a sign of death, it can also be a crucial adaptive response to stress [ 30 – 33 ].

Moonlighting proteins are multifunctional molecules that challenge the one-gene-one-function paradigm [ 1 , 2 ]. They are prevalent across all kingdoms of life, from humans to bacteria. In bacteria, they have been shown to link vital metabolic processes to physiological stress responses such as regulation of cell size [ 3 ], adhesion [ 4 ], bacterial virulence, and pathogenicity [ 5 , 6 ]. Moonlighting proteins associated with cell division are particularly intriguing because they connect this fundamental process to many additional networks that can profoundly influence cellular physiology. Specific examples include glucosyltransferases–OpgH in Escherichia coli and UgtP in Bacillus subtilis, which modulate cell division based on nutrient availability [ 3 , 7 , 8 ]. In E. coli, DnaA, known for its role in initiating DNA replication, also acts as a transcription factor regulating gene expression [ 7 ]. In B. subtilis, protein DivIVA has been implicated in chromosome segregation and spore formation apart from its role in cell division [ 9 ]. Studying moonlighting proteins provides a fascinating path to understanding protein functionality, interactions, and complexity within a cell. While our understanding of moonlighting proteins in bacteria has advanced significantly in recent years, several knowledge gaps and challenges remain in this field of research. One of the primary knowledge gaps is the precise molecular mechanisms underlying moonlighting. While some moonlighting functions have been identified and characterized, we often lack a comprehensive understanding of how a single protein can perform multiple functions.

Results

Increased expression of QueE leads to its secondary activity in E. coli cell division To study the molecular determinants in QueE contributing to its dual functions, we utilize two distinct readouts (Fig 1B). Firstly, to test QueE’s function in the formation of Q modification, we adapted published methods based on a specialized gel containing N-acryloyl-3-aminophenylboronic acid (APB) and northern blotting to detect queuosinylated tRNAs (Q-tRNAs) [34,35]. In this technique (APB gel and northern blotting), modified Q-tRNAs containing cis-diol reactive groups migrate slower than unmodified guanosinylated-tRNA (G-tRNAs) [35]. Secondly, to examine QueE’s ability to cause filamentation, we use phase contrast microscopy and monitor cell morphology. Using ΔqueE cells harboring queE on an IPTG-inducible plasmid, we analyzed Q-tRNA formation and filamentation in the presence or absence of the inducer. Wild-type (WT) cells carrying an empty plasmid were included as a control. As a positive control, WT cells show the formation of Q-tRNATyr (Fig 1C). As expected, ΔqueE cells carrying an empty vector show a band corresponding to unmodified guanosinylated-tRNATyr (G-tRNATyr) but not queuosinylated-tRNATyr (Q-tRNATyr), confirming that QueE is required to produce Q. The complementation of ΔqueE cells with WT QueE restores the formation of intact Q-tRNATyr regardless of induction, indicating that the overexpression of the enzyme is not necessary for the complementation of Q-tRNA synthesis activity. Regarding cell morphology, cells lacking QueE do not filament. ΔqueE cells expressing basal levels of QueE in the absence of inducer also do not filament, however, ΔqueE cells induced to express WT QueE show filamentation (Fig 1D,[10]), implying that increased expression of QueE is needed for cell division inhibition.

QueE regulates cell length in a dose-dependent manner Expression of queE is upregulated during filamentation of E. coli cells in response to sub-lethal concentrations of a cationic antimicrobial peptide, C18G [10]. These elongated cells vary from ~2 to hundreds of microns in length, with an average size of ~20 μm. Interestingly, queE expression from an IPTG-inducible promoter on a plasmid in wild-type (WT) cells also causes filamentation. To systematically examine the effect of increasing QueE expression on cell length, we performed an IPTG titration using the inducible plasmid encoding E. coli QueE (EcQueE) in MG1655 ΔlacZYA cells. As the IPTG concentration is increased from 0 to 500 μM, we observe a significant increase in the mean cell length, ranging from approximately 3 to 25 μm, respectively (Fig 2). The average cell length obtained for uninduced cells carrying queE on a plasmid is similar to that of the cells containing an empty vector. This indicates that any leaky expression of queE encoded by the plasmid has a negligible impact on the cell length. The average cell lengths of 18–25 μm obtained for 250–500 μM of IPTG are comparable to those observed for WT cells grown in the presence of sub-MIC level of cationic antimicrobial peptide or ΔmgrB cells starved for Mg2+ [10], suggesting that overexpression of QueE on a plasmid is a good proxy for upregulation of this enzyme under strong PhoQP-activating stress conditions. It is to be noted that the average cell lengths at higher levels of induction are likely underestimated due to the technical limitations in fitting some of the long filaments into the imaging window during microscopy. Overall, our data show a gradual and linear increase in the extent of filamentation with induction. To confirm if higher induction indeed results in increased levels of QueE protein in cells, we cloned and expressed a His 6 -tagged EcQueE on an IPTG-inducible plasmid. This construct behaves similarly to our untagged QueE in causing filamentation upon induction and allows us to monitor His 6 -EcQueE levels at different inducer concentrations (Fig (i)a and (i)b in S1 Text). Consistent with our expectation, higher IPTG concentrations correlate with higher amounts of His 6 -EcQueE in cells as visualized by western blotting (Fig (i)c and (i)d in S1 Text), and average cell lengths correlate with QueE abundance (Fig (i)f in S1 Text). As an added control, we show that the His 6 -EcQueE maintains the biosynthetic activity to produce Q-tRNAs (Fig (i)e in S1 Text). Together, our data indicate that QueE levels need to be about 3-fold or higher than basal (uninduced) expression level to cause cell elongation by greater than 2-fold. Induction with IPTG at 250–500 μM (average cell lengths ranging from 18–25 μm) leads to an estimated 7 to 8-fold increase in QueE levels. In filaments produced under PhoQP activation conditions (WT cells grown in the presence of sub-MIC level of cationic antimicrobial peptide or ΔmgrB cells starved for Mg2+ [10],), the cell lengths obtained are comparable to plasmid overexpression, where the QueE levels would be upregulated by ~7 fold. Collectively, these results show that QueE levels modulate cell division frequency in a dose-dependent manner. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Effect of dose-dependent expression of EcQueE on septation. (a) Measurement of cell lengths of E. coli MG1655 ΔlacZYA (TIM183) cells expressing QueE. Strains containing a plasmid encoding E. coli QueE (pRL03) or empty vector (pEB52) were grown in supplemented MinA minimal medium for 2 hours (OD 600 = 0.2–0.3) and induced with IPTG for 3 hours at the indicated concentration. Gray circles represent individual cells, mean cell length values are indicated in red, and the horizontal gray bars represent the median. Data are obtained from four independent experiments and the number of cells analyzed for each inducer concentration is indicated by (n). Statistical analysis was done using t-test displaying significance at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and “ns” = P >0.05 (b) Representative phase-contrast micrographs of ΔlacZYA cells expressing QueE (pRL03) at the indicated inducer concentration, scale bar = 5 μm. https://doi.org/10.1371/journal.pgen.1011287.g002

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011287

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/