More than Enzymes That Make or Break Cyclic Di-GMP—Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of
                    Escherichia coli

ABSTRACT The bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is “local” c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli . Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli . Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems. IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli , together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as “lonely players” without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of “local” c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.

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Enzymatically Active and Inactive Phosphodiesterases and Diguanylate Cyclases Are Involved in Regulation of Motility or Sessility in Escherichia coli CFT073

mBio ◽

10.1128/mbio.00307-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 39

Author(s):

Rachel R. Spurbeck ◽

Rebecca J. Tarrien ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Biofilm Formation ◽

Second Messenger ◽

Content Type ◽

E Coli ◽

Diguanylate Cyclases ◽

Genes Encoding ◽

K 12 ◽

Messenger Molecule

ABSTRACTIntracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. UropathogenicEscherichia coliregulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n= 11), DGC (n= 13), PilZ (n= 2), and both PDE and DGC (n= 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolateE. coliCFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenicE. coli.IMPORTANCEUropathogenicEscherichia coli(UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in anE. coliK-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain,E. coliCFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged fromE. coliK-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

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RNase I regulates Escherichia coli 2′,3′-cyclic nucleotide monophosphate levels and biofilm formation

Biochemical Journal ◽

10.1042/bcj20170906 ◽

2018 ◽

Vol 475 (8) ◽

pp. 1491-1506 ◽

Cited By ~ 8

Author(s):

Benjamin M. Fontaine ◽

Kevin S. Martin ◽

Jennifer M. Garcia-Rodriguez ◽

Claire Jung ◽

Laura Briggs ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Cyclic Nucleotide ◽

Biofilm Formation ◽

Physiological Role ◽

Rna Degradation ◽

Bacterial Biofilm ◽

Biological Functions ◽

Salvage Pathway ◽

E Coli

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2′,3′-cNMPs in Escherichia coli and demonstrates that 2′,3′-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2′,3′-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2′,3′-cNMPs.

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Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02431-20 ◽

2021 ◽

Author(s):

Jens Bo Andersen ◽

Kasper Nørskov Kragh ◽

Louise Dahl Hultqvist ◽

Morten Rybtke ◽

Martin Nilsson ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Single Cell ◽

Biofilm Formation ◽

Ectopic Expression ◽

Second Messenger ◽

Flow Cell ◽

Diguanylate Cyclases ◽

Biofilm Dispersal ◽

High Level

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. C-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We have previously presented evidence that ectopic expression in Pseudomonas aeruginosa of the Escherichia coli phosphodiesterase YhjH results in biofilm dispersal. More recently, however, evidence has been presented that induction of native c-di-GMP phosphodiesterases does not lead to dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for development of anti-biofilm drugs. However, here we demonstrate that induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed does result in dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and in a flow-cell biofilm system.

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Effect of Spermidine on Biofilm Formation in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00652-20 ◽

2021 ◽

Vol 203 (10) ◽

Author(s):

Kullathida Thongbhubate ◽

Yuko Nakafuji ◽

Rina Matsuoka ◽

Sonomi Kakegawa ◽

Hideyuki Suzuki

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Efflux Pump ◽

Conclusive Evidence ◽

Bacterial Biofilm ◽

Spermidine Synthase ◽

Periplasmic Binding Protein ◽

Content Type ◽

E Coli ◽

K 12

ABSTRACT Polyamines are essential for biofilm formation in Escherichia coli, but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene (speE) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ+ strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation. IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli. Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, ΔpotD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors.

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Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes

Applied and Environmental Microbiology ◽

10.1128/aem.01660-17 ◽

2017 ◽

Vol 83 (24) ◽

Cited By ~ 17

Author(s):

Juliane Schiebel ◽

Alexander Böhm ◽

Jörg Nitschke ◽

Michał Burdukiewicz ◽

Jörg Weinreich ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Polymeric Matrix ◽

Bacterial Biofilm ◽

Automated Image Analysis ◽

Host Immune System ◽

Content Type ◽

E Coli ◽

Initial Adhesion ◽

K 12

ABSTRACT Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes (“genotype”) and phenotypically analyzed the isolates for motility and curli and cellulose production (“phenotype”). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC. IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli. Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.

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Tight Modulation of Escherichia coli Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors

Applied and Environmental Microbiology ◽

10.1128/aem.02625-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3391-3403 ◽

Cited By ~ 46

Author(s):

Sandra Da Re ◽

Benjamin Le Quéré ◽

Jean-Marc Ghigo ◽

Christophe Beloin

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Medical Settings ◽

E Coli ◽

Antigen 43 ◽

Genetically Manipulated ◽

The Impact ◽

Potential Use ◽

Bioremediation Processes

ABSTRACT Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.

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Escherichia coli O157:H7 Strains Isolated from High-Event Period Beef Contamination Have Strong Biofilm-Forming Ability and Low Sanitizer Susceptibility, Which Are Associated with High pO157 Plasmid Copy Number

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-113 ◽

2016 ◽

Vol 79 (11) ◽

pp. 1875-1883 ◽

Cited By ~ 8

Author(s):

RONG WANG ◽

BRANDON E. LUEDTKE ◽

JOSEPH M. BOSILEVAC ◽

JOHN W. SCHMIDT ◽

NORASAK KALCHAYANAND ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Biofilm Formation ◽

Copy Number ◽

Plasmid Copy Number ◽

Bacterial Biofilm ◽

Escherichia Coli O157 ◽

Plasmid Copy ◽

E Coli ◽

High Event

ABSTRACT In the meat industry, a high-event period (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by Escherichia coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute to HEPs. We conducted the present study to further characterize E. coli O157:H7 strains isolated during HEPs for their potential to cause contamination and to investigate the genetic basis for their strong biofilm-forming ability and high sanitizer resistance. Our results show that, compared with the E. coli O157:H7 diversity control panel strains, the HEP strains had a significantly higher biofilm-forming ability on contact surfaces and a lower susceptibility to common sanitizers. No difference in the presence of disinfectant-resistant genes or the prevalence of antibiotic resistance was observed between the HEP and control strains. However, the HEP strains retained significantly higher copy numbers of the pO157 plasmid. A positive correlation was observed among a strain's high plasmid copy number, strong biofilm-forming ability, low sanitizer susceptibility, and high survival and recovery capability after sanitization, suggesting that these specific phenotypes could be either directly correlated to gene expression on the pO157 plasmid or indirectly regulated via chromosomal gene expression influenced by the presence of the plasmid. Our data highlight the potential risk of biofilm formation and sanitizer resistance in HEP contamination by E. coli O157:H7, and our results call for increased attention to proper and effective sanitization practices in meat processing facilities.

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Control of Growth and Persistence of Listeria monocytogenes and β-Lactam-Resistant Escherichia coli by Thymol in Food Processing Settings

Molecules ◽

10.3390/molecules25020383 ◽

2020 ◽

Vol 25 (2) ◽

pp. 383 ◽

Cited By ~ 1

Author(s):

Maria Grazia Cusimano ◽

Vita Di Stefano ◽

Maria La Giglia ◽

Vincenzo Di Marco Lo Presti ◽

Domenico Schillaci ◽

...

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Food Processing ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Free Living ◽

Biofilm Growth ◽

Food Industries ◽

E Coli ◽

Reference Strains

The main objective of this study was to evaluate the efficacy of thymol in controlling environmental contamination in food processing facilities. The effect of thymol was tested as an agent to prevent planktonic and bacterial biofilm growth of twenty-five Listeria monocytogenes isolates from a variety of foods and five Escherichia coli isolates from a farm. The E. coli isolates were positive for extended spectrum β-lactamase (ESBL) genes. All isolates and reference strains were susceptible to thymol at Minimum inhibitory concentration (MIC) values ranging from 250 to 800 μg/mL. An interesting activity of interference with biofilm formation of L. monocytogenes and E. coli was found for thymol at sub-MIC concentrations of 200, 100, 75, and 50 μg/mL. Anti-biofilm activity ranging from 59.71% to 66.90% against pre-formed 24-h-old L. monocytogenes biofilms at concentrations of 500 or 800 µg/mL, corresponding to 2× MIC, was determined against free-living forms of six isolates chosen as the best or moderate biofilm producers among the tested strains. The property of thymol to attack L. monocytogenes biofilm formation was also observed at a concentration of 100 µg/mL, corresponding to 1/4 MIC, by using a stainless-steel model to simulate the surfaces in food industries. This study gives information on the use of thymol in food processing setting.

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Trigger phosphodiesterases as a novel class of c-di-GMP effector proteins

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0498 ◽

2016 ◽

Vol 371 (1707) ◽

pp. 20150498 ◽

Cited By ~ 36

Author(s):

Regine Hengge

Keyword(s):

Escherichia Coli ◽

Cell Cycle ◽

Biofilm Formation ◽

Promoter Region ◽

Cell Cycle Progression ◽

Effector Proteins ◽

Bacterial Biofilm ◽

Cycle Progression ◽

Allosteric Control ◽

Diguanylate Cyclases

The bacterial second messenger c-di-GMP controls bacterial biofilm formation, motility, cell cycle progression, development and virulence. It is synthesized by diguanylate cyclases (with GGDEF domains), degraded by specific phosphodiesterases (PDEs, with EAL of HD-GYP domains) and sensed by a wide variety of c-di-GMP-binding effectors that control diverse targets. c-di-GMP-binding effectors can be riboswitches as well as proteins with highly diverse structures and functions. The latter include ‘degenerate’ GGDEF/EAL domain proteins that are enzymatically inactive but still able to bind c-di-GMP. Surprisingly, two enzymatically active ‘trigger PDEs’, the Escherichia coli proteins PdeR and PdeL, have recently been added to this list of c-di-GMP-sensing effectors. Mechanistically, trigger PDEs are multifunctional. They directly and specifically interact with a macromolecular target (e.g. with a transcription factor or directly with a promoter region), whose activity they control by their binding and degradation of c-di-GMP—their PDE activity thus represents the c-di-GMP sensor or effector function. In this process, c-di-GMP serves as a regulatory ligand, but in contrast to classical allosteric control, this ligand is also degraded. The resulting kinetics and circuitry of control are ideally suited for trigger PDEs to serve as key components in regulatory switches. This article is part of the themed issue ‘The new bacteriology’.

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Label-free quantitative proteomic analysis of the inhibition effect of Lactobacillus rhamnosus GG on Escherichia coli biofilm formation in co-culture

Proteome Science ◽

10.1186/s12953-021-00172-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Huiyi Song ◽

Ni Lou ◽

Jianjun Liu ◽

Hong Xiang ◽

Dong Shang

Keyword(s):

Escherichia Coli ◽

Proteomic Analysis ◽

Biofilm Formation ◽

Lactobacillus Rhamnosus ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Label Free ◽

Quantitative Proteomic Analysis ◽

E Coli ◽

Protein Ubiquitination

Abstract Background Escherichia coli (E. coli) is the principal pathogen that causes biofilm formation. Biofilms are associated with infectious diseases and antibiotic resistance. This study employed proteomic analysis to identify differentially expressed proteins after coculture of E. coli with Lactobacillus rhamnosus GG (LGG) microcapsules. Methods To explore the relevant protein abundance changes after E. coli and LGG coculture, label-free quantitative proteomic analysis and qRT-PCR were applied to E. coli and LGG microcapsule groups before and after coculture, respectively. Results The proteomic analysis characterised a total of 1655 proteins in E. coli K12MG1655 and 1431 proteins in the LGG. After coculture treatment, there were 262 differentially expressed proteins in E. coli and 291 in LGG. Gene ontology analysis showed that the differentially expressed proteins were mainly related to cellular metabolism, the stress response, transcription and the cell membrane. A protein interaction network and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that the differentiated proteins were mainly involved in the protein ubiquitination pathway and mitochondrial dysfunction. Conclusions These findings indicated that LGG microcapsules may inhibit E. coli biofilm formation by disrupting metabolic processes, particularly in relation to energy metabolism and stimulus responses, both of which are critical for the growth of LGG. Together, these findings increase our understanding of the interactions between bacteria under coculture conditions.

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