Incorporating LsrK AI‐2 quorum quenching capability in a functionalized biopolymer capsule

Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation, and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer‐2, AI‐2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI‐2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI‐2 in culture fluids. Correspondingly, AI‐2 mediated QS activity is diminished. All components of this system are “biofabricated”—they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the “artificial cell” capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI‐2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans.

     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.     

Quorum-sensing system in Acidithiobacillus ferrooxidans involved in its resistance to Cu²⁺

Aims: The purpose of this study was to search for the relationship between quorum sensing (QS) and Cu²⁺ resistance in Acidithiobacillus ferrooxidans. Methods and Results: Resistance to Cu²⁺ of A. ferrooxidans significantly decreased with the treatment dose of a synthetic QS blocker (5Z)‐4‐bromo‐5‐(bromomethylene)‐2(5H)‐furanone (FUR). Relative differences in expression of the QS genes afeI, afeR and Cu²⁺ resistance‐associated genes afe0329, afe0454 were examined in the presence of Cu²⁺ and/or FUR compound. The expression of QS genes afeI and afeR increased significantly with 50 mmol l^?1 Cu²⁺ in the culture, while for samples treated with both 50 mmol l^?1 Cu²⁺ and 0·01 μg ml^?1 FUR compound, they showed little changes compared with control, and the expression of afe0329 and afe0454 genes increased slightly either. These results showed that QS system was positively related to the mechanism of Cu²⁺ resistance. Conclusions: QS system in A. ferrooxidans involved in its resistance to Cu^2+. Significance and Impact of the Study: The mechanisms of Cu²⁺ resistance in A. ferrooxidans could be revealed on a population level rather than on a single‐cell level. Our work also provides useful data for further selection of A. ferrooxidans strains with suitable Cu²⁺ resistance that could probably increase the bioleaching efficiency.     

Robust and sensitive control of a quorum‐sensing circuit by two interlocked feedback loops

The quorum‐sensing (QS) response of Vibrio fischeri involves a rapid switch between low and high induction states of the lux operon over a narrow concentration range of the autoinducer (AI) 3‐oxo‐hexanoyl‐L ‐homoserine lactone. In this system, LuxR is an AI‐dependent positive regulator of the lux operon, which encodes the AI synthase. This creates a positive feedback loop common in many bacterial species that exhibit QS‐controlled gene expression. Applying a combination of modeling and experimental analyses, we provide evidence for a LuxR autoregulatory feedback loop that allows LuxR to increase its concentration in the cell during the switch to full lux activation. Using synthetic lux gene fragments, with or without the AI synthase gene, we show that the buildup of LuxR provides more sensitivity to increasing AI, and promotes the induction process. Elevated LuxR levels buffer against spurious variations in AI levels ensuring a robust response that endows the system with enhanced hysteresis. LuxR autoregulation also allows for two distinct responses within the same cell population.     

Autonomous bacterial localization and gene expression based on nearby cell receptor density

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site‐specific synthesis of bacterial quorum sensing autoinducer‐2 (AI‐2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI‐2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area‐based switch in synthetic biology that will find use beyond the proposed cancer model here.     

Ratiometric fluorescent scaffold giving discrete response towards iodide ion: a combined experimental and DFT study

A novel ratiometric fluorescent receptor (Z)‐2‐(4‐[diethylamino]‐2‐hydroxybenzylideneamino) pyridine‐3‐carbaldehyde (3) bearing one phenolic OH and one aldehyde group as recognition sites was synthesized and characterized. The anion recognition behaviour of receptor 3 was evaluated by various spectroscopic (UV‐visible, fluorescence and ¹H nuclear magnetic resonance) methods and was validated by computational studies. The receptor showed fast response time, excellent selectivity and reproducibility towards iodide ion detection among the other surveyed anions, with a binding constant of 6.12 × 10⁴ M^?1 and a detection limit of 0.24 μM, thus confirming its potential applicability as a fluorescent sensor for iodide Copyright © 2014 John Wiley & Sons, Ltd.     

Phenotypic persistence and external shielding ultraviolet radiation inactivation kinetic model

Aim: To develop an inactivation kinetic model to describe ultraviolet (UV) dose‐response behaviour for micro‐organisms that exhibit tailing using two commonly referenced causes for tailing: physical shielding of micro‐organisms and phenotypic persistence. Methods and Results: Dose‐response data for Escherichia coli, Mycobacterium terrae and Bacillus subtilis spores exposed to UV radiation were fit to the phenotypic persistence and external shielding (PPES) model. The fraction of persistent micro‐organisms in the original population (N_persistent/N_total) that exhibited reduced sensitivity to UV radiation was estimated by the PPES model as approx. 10^?7, 10^?5 and 10^?4 for E. coli, B. subtilis spores and Myco. terrae, respectively. Particle shielding effects were evaluated for Myco. terrae and resulted in additional reduction in UV sensitivity. Conclusions: Tailing occurred in laboratory experiments even when clumping and shielding were eliminated as major factors in UV resistance, suggesting that phenotypic persistence in addition to shielding may be important to consider when evaluating dose‐response curves for disinfection applications. Significance and Impact of the Study: The PPES model provides a mechanistically plausible tool for estimating the dose–response behaviour for micro‐organisms that exhibit tailing in dispersed and aggregated settings. Accurate dose‐response behaviour (including the tailing region) is critical to the analysis and validation of all UV disinfection systems.     

Difference in the distribution and speciation of cellular nickel between nickel‐tolerant and non‐tolerant Nicotiana tabacum L. cv. BY‐2 cells

To evaluate Ni dynamics at the subcellular level, the distribution and speciation of Ni were determined in wild‐type (WT) and Ni‐tolerant (NIT) tobacco BY‐2 cell lines. When exposed to low but toxic levels of Ni, NIT cells were found to contain 2.5‐fold more Ni (14% of whole‐cell Ni values) in their cell walls than WT cells (6% of whole‐cell Ni values). In addition to higher levels of Ni in the apoplast, a higher proportion (94%) of symplastic Ni was localized in the vacuoles of NIT cells than in the vacuoles of WT cells (81%). The concentration of cytosolic Ni in the NIT cells was significantly lower (18 nmol g^?1 FW) than that in the WT cells (85 nmol g^?1 FW). In silico simulation showed that 95% of vacuolar Ni was in the form of Ni‐citrate complexes, and that free Ni²⁺ was virtually absent in the NIT cells. On the other hand, the amount of free metal ions was markedly increased in WT cells because free citrate was depleted by chelation of Ni. A protoplast viability assay using BCECF‐AM further demonstrated that the main mechanism that confers strong Ni tolerance was present in the symplast as opposed to the cell wall.     

A highly selective fluorescent probe for pyrophosphate detection in aqueous solutions

A novel and simple fluorescence enhancement method is introduced for selective pyrophosphate (PPi) sensing in an aqueous solution. The method is based on a 1:1 metal complex formation between tris(8‐hydroxyquinoline‐5‐sulphonate) thulium(III) [Tm(QS)₃] and PPi ion. The linear response covers a concentration range of 1.6 × 10^?7–1.0 × 10^?5 mol/L PPi and the detection limit is 2.3 × 10^?8 mol/L. The association constant of Tm(QS)₃–PPi complex was calculated as 2.6 × 10⁵ mol/L. Tm(QS)₃ shows a selective and sensitive fluorescence enhancement toward PPi ion in comparion with I₃^?, NO₃^?, CN^?, CO₃^2?, Br^?, Cl^?, F^?, H₂PO₄^? and SO₄^2?, which is attributed to higher stability of the inorganic complex between pyrophosphate ion and Tm(QS)₃. Copyright © 2011 John Wiley & Sons, Ltd.     

Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

Effect of ultra‐strong static magnetic field on bacteria: Application of Fourier‐transform infrared spectroscopy combined with cluster analysis and deconvolution

A new method based on Fourier‐transform infrared (FTIR) spectroscopy combined with cluster analysis and deconvolution was established to investigate the biological effect of an ultra‐strong static magnetic field (SMF) of 10.0 T on Escherichia coli and Staphylococcus aureus. FTIR spectroscopy was applied to characterize the spectroscopic fingerprints of these bacterial cells with or without the treatment of the SMF. After the calculation, the results of cluster analysis indicated that the SMF had significant effects on E. coli compared with S. aureus, and the effects were reflected by the changes of spectral region of 1500–1200 cm^?1. The deconvolution results of this major indication region showed that the composition and conformation of nucleic acid, protein, and fatty acid of E. coli were altered under the magnetic conditions. Bioelectromagnetics 30:500–507, 2009. © 2009 Wiley‐Liss, Inc.     

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

E. coli (P450pyrTM‐GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g^?1 cdw) for the biohydroxylation of N‐benzylpyrrolidine 1 and a GDH activity of 106 U g^?1 protein. The E. coli cells were used as efficient biocatalysts for highly regio‐ and stereoselective hydroxylation of alicyclic substrates at non‐activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N‐benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio‐ and stereoselectivity, giving (S)‐N‐benzyl‐3‐hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM‐GDH) was found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpyrrolidin‐2‐one 3 , improving the regioselectivity from 90% of the wild‐type P450pyr to 100% and giving (S)‐N‐benzyl‐4‐hydroxylpyrrolidin‐2‐one 4 in 99% ee as the sole product. A high activity of 15.5 U g^?1 cdw was achieved and (S)‐ 4 was obtained in 19.4 mM. E. coli (P450pyrTM‐GDH) was also found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpiperidin‐2‐one 5 , increasing the ee of the product (S)‐N‐benzyl‐4‐hydroxy‐piperidin‐2‐one 6 to 94% from 33% of the wild‐type P450pyr. A high activity of 15.8 U g^?1 cdw was obtained and (S)‐ 6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM‐GDH) represents the most productive system known thus far for P450‐catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM‐GDH) provide with simple and useful syntheses of (S)‐ 2 , (S)‐ 4 , and (S)‐ 6 that are valuable pharmaceutical intermediates and difficult to prepare. Biotechnol. Bioeng. 2013; 110: 363–373. © 2012 Wiley Periodicals, Inc.     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Structure‐kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin

The emergence of Gram‐negative “superbugs” exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram‐negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure‐translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC‐ E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC‐expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k_on ≈ 2 × 10⁴ M^?1 s^?1). Zwitterionic compounds with highly polar groups attached to the penem‐2 ring, including panipenem, imipenem and doripenem exhibit faster k_on (>10⁴ M^?1 s^?1), while meropenem and biapenem with fewer exposed polar groups exhibit slower k_on (～5 × 10³ M^?1 s^?1). Tebipenem pivoxil and razupenem exhibit ～13‐fold slower k_on (～1.5 × 10³ M^?1 s^?1) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure‐kinetic relationships of carbapenem translocation are governed by H‐bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups). Proteins 2014; 82:2998–3012. © 2014 Wiley Periodicals, Inc.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Reversible non‐genetic phenotypic heterogeneity in bacterial quorum sensing

Bacteria co‐ordinate their social behaviour in a density‐dependent manner by production of diffusible signal molecules by a process known as quorum sensing (QS). It is generally assumed that in homogenous environments and at high cell density, QS synchronizes cells in the population to perform collective social tasks in unison which maximize the benefit at the inclusive fitness of individuals. However, evolutionary theory predicts that maintaining phenotypic heterogeneity in performing social tasks is advantageous as it can serve as a bet‐hedging survival strategy. Using Pseudomonas syringae and Xanthomonas campestris as model organisms, which use two diverse classes of QS signals, we show that two distinct subpopulations of QS‐responsive and non‐responsive cells exist in the QS‐activated population. Addition of excess exogenous QS signal does not significantly alter the distribution of QS‐responsive and non‐responsive cells in the population. We further show that progeny of cells derived from these subpopulations also exhibited heterogeneous distribution patterns similar to their respective parental strains. Overall, these results support the model that bacteria maintain QS‐responsive and non‐responsive subpopulations at high cell densities in a bet‐hedging strategy to simultaneously perform functions that are both positively and negatively regulated by QS to improve their fitness in fluctuating environments.