Colanic Acid Research Articles

Colanic acid and lipopolysaccharide in Pectobacterium carotovorum Pcc21 serve as receptors for the bacteriophage phiPccP-2

Bacteriophages (phages) are viruses that specifically bind to and infect target bacteria. The phage phiPccP-2, belonging to the Myoviridae family, efficiently controls Pectobacterium spp. In the present study, we aimed to elucidate the mechanism of recognition of P. carotovorum Pcc21 by phiPccP-2. The EZ-Tn5 transposon mutant library of Pcc21 was used to screen for phage-resistant mutants. Among 4,072 mutants screened, 12 harbored disruptions in genes associated with the biosynthesis of either colanic acid (CA) or lipopolysaccharide (LPS) showed resistance to phiPccP-2. Complementation of 4 representative phage-resistant mutants with the corresponding genes fully restored the binding ability and lytic activity of PhiPccP-2. The amounts of CA or LPS structure in these mutants were significantly altered compared with those in the wild-type strain. Adsorption competition assays between CA and LPS extracted from Pcc21 and the natural receptors in Pcc21 showed that unbound phages were significantly increased, indicating that both CA and LPS are associated with the adsorption of the phiPccP-2 to Pcc21. In contrast, the adsorption of phiPccP-2 to extracted CA or LPS did not inactivate the lytic activity of phiPccP-2, indicating that the adsorption to the extracted CA or LPS is not sufficient for DNA injection. Treatment with polymyxin B, which disrupts LPS, interfered with phiPccP-2 adsorption to Pcc21. Furthermore, phage-resistant mutants showed reduced virulence in the host plant, suggesting a trade-off between phage resistance and bacterial virulence. Overall, our results indicate that both CA and LPS serve as receptors for the binding of phiPccP-2 to P. carotovorum Pcc21.

Multiplex Determination of K-Antigen and Colanic Acid Capsule Variants of Cronobacter sakazakii

Cronobacter sakazakii is associated with the ingestion of contaminated reconstituted powdered infant formula (PIF), resulting in necrotizing enterocolitis, sepsis and meningitis in neonatal infants. Potential virulence determinants include the variable capsular polysaccharides; K-antigen and colanic acid (CA). Strains encoding for the capsule variant K2:CA2 have been strongly associated with neonatal meningitis cases. This study aimed to develop and apply a multiplex PCR assay to determine C. sakazakii K-antigen and colanic acid types. Twenty-six strains of C. sakazakii which had previously been isolated from food and environmental sources were used. These cover 18 multilocus sequence types and four serotypes. Based on our research findings, we have identified two K-antigen types present. Specifically, the K1-antigen was observed in sequence types ST1, ST8, ST20, ST23, ST64, ST198, ST263, ST264 and ST406, while the K2-antigen was present in ST4, ST9, ST12, ST13, ST136, ST233, ST245 and ST405. Additionally, we detected colanic acid (CA) type 1 in sequence types ST1, ST8, ST9, ST20, ST245 and ST405, and colanic acid (CA) type 2 in ST4, ST12, ST13, ST23, and ST64. We compared the predicted K-antigen and colanic acid types with the entire genome sequences of the strains. The comparison showed complete agreement between the PCR amplification results and the genomic analysis of the K-antigen and colanic acid-encoding regions. This assay is a useful tool for rapid identification of C. sakazakii, K-antigen and colanic acid types, in routine diagnoses and foodborne investigations. In addition, it will contribute to our knowledge of virulence factors associated with life-threatening neonatal meningitis.

Optimized Protocol for Production and Extraction of Colanic Acid from E. coli Culture

Optimized Protocol for Production and Extraction of Colanic Acid from E. coli Culture

Chemical Induction of Longevity-Promoting Colanic Acid in the Host's Microbiota.

Microbiota-derived metabolites have emerged as key regulators of longevity. The metabolic activity of the gut microbiota, influenced by dietary components and ingested chemical compounds, profoundly impacts host fitness. While the benefits of dietary prebiotics are well-known, chemically targeting the gut microbiota to enhance host fitness remains largely unexplored. Here, we report a novel chemical approach to induce a pro-longevity bacterial metabolite in the host gut. We discovered that specific Escherichia coli strains overproduce colanic acids (CAs) when exposed to a low dose of cephaloridine, leading to an increased lifespan in host Caenorhabditis elegans . In the mouse gut, oral administration of low-dose cephaloridine induces the transcription of the capsular biosynthesis operon responsible for CA biosynthesis in commensal E. coli , which overcomes the inhibition of CA biosynthesis above 30°C and enables its induction directly from the microbiota. Importantly, low-dose cephaloridine induces CA independently of its antibiotic properties through a previously unknown mechanism mediated by the membrane-bound histidine kinase ZraS. Our work lays the foundation for microbiota-based therapeutics through the chemical modulation of bacterial metabolism and reveals the promising potential of bacteria-targeting drugs in promoting host longevity.

Cellular Effects of Enterobacteriaceae Polysaccharide Colanic Acid.

Colanic acid (CA) is an exopolysaccharide found in Enterobacteriaceae. Recently, its ability to stimulate physical activity in mice and to prolong the lifespan of invertebrates has been described. In the current work, we use standard MTT assay, fluorescence microscopy, and flow cytometry to describe CA action on several cell lines of different origins. We observed slight antiproliferative activity against colorectal cancer (HCT-116), neuroblastoma (IMR-32), and myoblast (C2C12) cell lines at a concentration of 256 μg/mL, while other cell lines of non-cancerous origin (Vero, HPF) did not show any decrease in the MTT assay. In all cell lines, we observed a rearrangement of mitochondria localization using fluorescence microscopy. CA induces cell differentiation in the myoblast cell line (C2C12) at concentrations of 50-200 μg/mL. Briefly, we observed that the number of apoptotic cells increased and the metabolic activity in the MTT assay decreased, which was accompanied by changes in cell morphology, the quantity of ROS, and the potential of the mitochondrial membrane. Taken together, these results indicate that CA is specific in cytotoxicity to cell lines of different origins and can impact mitochondria and differentiation, consistent with its potential geroprotective function.

Engineering Escherichia coli to Efficiently Produce Colanic Acid with Low Molecular Mass and Viscosity.

Colanic acid (CA) is exopolysaccharide that presents growing potential in the food and healthcare industry as a versatile polymer. Previously, we have constructed the Escherichia coli strain WWM16 which can efficiently produce CA. In this study, WWM16 has been further engineered to produce a higher yield of CA with low molecular mass and viscosity. The gene mcbR encoding a transcriptional factor, and the genes opgD, opgG, and opgH related to the biosynthesis of osmoregulated periplasmic glucans were deleted in E. coli WWM16, and the resulting strain WWM166 produced 18.1 g/L CA. The expression level of wcaD encoding the polymerase in WWM166 was downregulated using CRISPRi. As a result, the strain WWM166/pWpD1 could produce 49.9 g/L CA with lower molecular mass. CA products were purified from both WWM166 and WWM166/pWpD1, and their molecular mass, viscosity, fluidity, hygroscopicity, and antioxidant activity were determined and compared. These findings demonstrate the potential application of CA with different molecular masses to prolong life and protect skin in the food and cosmetic industries.

Construction of a GTP regeneration system by regulating gene expression in the pathway

Guanosine 5′-triphosphate (GTP) provides precursors for the biological synthesis of various valuable compounds. However, the insufficient supply of GTP is often encountered when engineering microbes for the synthesis of certain products, which results in metabolic imbalance and unsatisfying product yield. To tackle this issue, a general strategy for improving GTP regeneration was developed in this study. The effects of dosage and combination strategy of genes related to GTP synthesis were examined to pinpoint the most critical genes influencing GTP production in Escherichia coli. A regeneration system of GTP was derived by the overexpression of genes guaA and guaB, the intracellular GTP concentration of which improved by 81%. To further verify the applicability of the engineered GTP regeneration system on strengthening the product synthesis, it was applied to the production of riboflavin and colanic acid that required GTP as a precursor. It turned out that the titer of riboflavin was increased by 95.8% and the titer of colanic acid was increased by 44%. The GTP regeneration system developed here can also be applied to other products that use GTP as a precursor or require GTP as a cofactor.

Stimulatory Effect on Mice by Bacterial Exopolysaccharide, Colanic Acid

Stimulatory Effect on Mice by Bacterial Exopolysaccharide, Colanic Acid

Silver nanoparticles facilitate phage-borne resistance gene transfer in planktonic and microplastic-attached bacteria

The spread of bacteriophage-borne antibiotic resistance genes (ARGs) poses a realistic threat to human health. Nanomaterials, as important emerging pollutants, have potential impacts on ARGs dissemination in aquatic environments. However, little is known about its role in transductive transfer of ARGs mediated by bacteriophage in the presence of microplastics. Therefore, this study comprehensively investigated the influence of silver nanoparticles (AgNPs) on the transfer of bacteriophage-encoded ARGs in planktonic Escherichia coli and microplastic-attached biofilm. AgNPs exposure facilitated the phage transduction in planktonic and microplastic-attached bacteria at ambient concentration of 0.1 mg/L. Biological binding mediated by phage-specific recognition, rather than physical aggregation conducted by hydrophilicity and ζ-potential, dominated the bacterial adhesion of AgNPs. The aggregated AgNPs in turn resulted in elevated oxidative stress and membrane destabilization, which promoted the bacteriophage infection to planktonic bacteria. AgNPs exposure could disrupt colanic acid biosynthesis and then reduce the thickness of biofilm on microplastics, contributing to the transfer of phage-encoded ARGs. Moreover, the roughness of microplastics also affected the performance of AgNPs on the transductive transfer of ARGs in biofilms. This study reveals the compound risks of nanomaterials and microplastics in phage-borne ARGs dissemination and highlights the complexity in various environmental scenarios.

Adhesion and corrosion effects of biofilms on steel surface mediated by hydrophilic exopolysaccharide colanic acid

The corrosion mechanism involving bacterial biofilms has long been a focus of the research on microbiologically influenced corrosion. In this study, biofilms formed by Pseudoalteromonas marine and its mutant demonstrated the ability to inhibit uniform corrosion. However, the excessive secretion of colanic acid by the mutant Δ01912 resulted in a significantly increased number of attached bacteria and the formation of unevenly hydrophilic biofilms, which aggravated localized pitting corrosion on the steel surface. This study provides evidence that unevenly hydrophilic biofilms can lead to local enrichment of chloride ions, thereby intensifying the pitting of metal materials.

LuxS-deficiency reduces persistence of Cronobacter to low-moisture but contributes to virulence after rehydration

Low‐moisture foods (LMF) have arisen an increasing concern as vehicles of foodborne pathogens. Cronobacter genus, a class A pathogen in powdered infant formula (PIF), is crucial to the safety of LMF. Researchers have concentrated more on the bacterial survival caused by key hazardous factors, yet they often ignore the alteration of virulence properties in the surviving strains following rehydration of LMF mediated by the key factors. Our previous transcriptional profiling showed that luxS might participate in desiccation response. Herein, we further investigated the role of Cronobacter LuxS under desiccation stress by combining with the phenotypic and gene analysis between the Cronobacter parent and luxS mutant strains. Desiccation stress destructing assays confirmed that luxS can significantly enhance the resistance of Cronobacter towards desiccation. Our results also showed that cell hydrophobicity, aggregation, motility, the content of polysaccharide, and AI-2 synthesis pathway involved in luxS-mediated desiccation response. The luxS mutant strain exhibited higher swimming and swarming motility, more content of capsular polysaccharide, and more rapid of aggregation, but lower hydrophobicity than that of the wild-type strain, whereas desiccation stress would result in a opposite effect on these cell surface properties in ΔluxS during rehydration. Additionally, the comparation of gene expression profiles indicated that low moisture would trigger Cronobacter luxS to promote transport osmoprotectants by regulating the expression of proX, proW, and treC, and suppress the expression of cpsG associated with polysaccharide colanic acid. Notably, this study also discovered for the first time that the luxS-deficiency dramatically attenuated adhesion and invasion to intestinal and brain cells, but ΔluxS subjected to desiccation could aggravate the cell virulence instead. Therefore, thinking the alteration of toxicity caused by low‐moisture, approach based on blocking the expression of the luxS gene to prevent Cronobacter in LMF needs to be adopted with caution.

Bacterial networks in Atlantic salmon with Piscirickettsiosis

An unbalanced composition of gut microbiota in fish is hypothesized to play a role in promoting bacterial infections, but the synergistic or antagonistic interactions between bacterial groups in relation to fish health are not well understood. We report that pathogenic species in the Piscirickettsia, Aeromonas, Renibacterium and Tenacibaculum genera were all detected in the digesta and gut mucosa of healthy Atlantic salmon without clinical signs of disease. Although Piscirickettsia salmonis (and other pathogens) occurred in greater frequencies of fish with clinical Salmonid Rickettsial Septicemia (SRS), the relative abundance was about the same as that observed in healthy fish. Remarkably, the SRS-positive fish presented with a generalized mid-gut dysbiosis and positive growth associations between Piscirickettsiaceae and members of other taxonomic families containing known pathogens. The reconstruction of metabolic phenotypes based on the bacterial networks detected in the gut and mucosa indicated the synthesis of Gram-negative virulence factors such as colanic acid and O-antigen were over-represented in SRS positive fish. This evidence indicates that cooperative interactions between organisms of different taxonomic families within localized bacterial networks might promote an opportunity for P. salmonis to cause clinical SRS in the farm environment.

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Regulation on Pathway Metabolic Fluxes to Enhance Colanic Acid Production in Escherichia coli.

Colanic acid (CA) is a natural polysaccharide macromolecule with rich and unique biological properties and is a promising candidate for use in food and cosmetics. To date, the efficient biosynthesis of CA and the influence of product accumulation on the strains used have yet to be precisely investigated. Herein, bottlenecks in the CA metabolic pathway were untangled by finely regulating the expression of manA, cpsG, fcl, and rcsA. Engineered strains produced CA at >1 g/L in shake flasks without dependence on cold temperatures, and it was verified in a 1 L bioreactor with a titer up to 18.64 g/L within 24 h. The accumulation of CA caused a decrease in the saturated fatty acid content (represented by C16:0 and C18:0) in the cell membrane. This study demonstrated pathway engineering for efficient CA production in cell factories and provided insights into the barriers and solutions faced in the biosynthesis of natural products.

Potential of bacteriophage PCT27 to reduce the use of agrochemicals to control Pectobacterium carotovorum subsp. Carotovorum in Chinese cabbage (Brassica pekinensis)

As antimicrobial resistance (AMR) continues to spread among bacteria and poses a serious threat to global health, the indiscriminate use of antibiotics in various fields, including agriculture, should be avoided. Here, we isolated a novel bacteriophage (phage), PCT27, specific for Pectobacterium carotovorum subsp. carotovorum (Pcc), a phytopathogen responsible for soft rot of crops, and investigated its potential as an adjunct to reduce the use of agrochemicals in Pcc control. Phage PCT27, which belongs to the family Autographiviridae, inhibited in vitro growth of Pcc within 1 h of infection by recognizing colanic acid as a host receptor. More than 90% of the virions rapidly adsorbed to the host cells within 3 min, supporting the efficient control of Pcc by PCT27. PCT27 supplementation reduced the minimum inhibitory concentration of streptomycin by 50% for Pcc control in vitro. Accordingly, PCT27 successfully complemented an antibiotic-based agrochemical in preventing Pcc infection in Chinese cabbage leaves and seedlings by reducing the dose of the agrochemical by a quarter. As a biocontrol agent for Pcc that complements agrochemicals, PCT27 can not only prevent economic losses due to crop decay but would also contribute to addressing the AMR crisis for sustainable agriculture and environmental safety.

The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid

The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional 1H and 13C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the β-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS.

De novo 2′‐fucosyllactose bioproduction through modular engineering of a novel Escherichia coli K12‐derived strain

AbstractThe most abundant human milk oligosaccharide 2′‐fucosyllactose (2′‐FL) is a valuable component that has gained significant attention from the food industry. To biosynthesize 2′‐FL, various Escherichia coli K12 derivatives have been genetically modified. To further enhance the application performance of E. coli K12, a novel E. coli K12 derivative BL27 was used as a chassis cell in this study, and modular pathway enhancement was performed to achieve de novo synthesis of 2′‐FL. The futC gene encoding α‐1,2‐fucosyltransferase was introduced, and the wcaJ gene was knocked out to prevent the conversion of GDP‐ l‐fucose to colanic acid. Next, the effects of overexpressing transcriptional regulators rcsA and rcsB and knocking out transcriptional regulators mcbR and waaF were evaluated to optimize the colanic acid pathway. The expression level, solubility, and activity of FutC were improved through genomic integration, TrxA‐tag fusion, and double mutation in F40S/Q237S. Fermentation conditions were optimized to achieve maximum 2′‐FL titers of 3.86 and 23.56 g/L in shake‐flask and fed‐batch cultivation, respectively. Over 85% of the products were successfully excreted into extracellular and almost no byproduct 2′,3‐difucosyllactose was generated. This study has explored a new microbial platform and modification strategies for the synthesis of 2′‐FL and provides opportunities for its commercial production.

Regulating Cardiolipin Biosynthesis for Efficient Production of Colanic Acid in Escherichia coli.

Colanic acid has broad application prospects in the food and healthcare market due to its excellent physical properties and biological activities. In this study, we discovered that colonic acid production in Escherichia coli could be enhanced by regulating cardiolipin biosynthesis. Single deletion of clsA, clsB, or clsC related to cardiolipin biosynthesis in E. coli MG1655 only slightly increased colonic acid production, but double or triple deletion of these three genes in E. coli MG1655 increased colonic acid production up to 2.48-fold. Previously, we have discovered that truncating lipopolysaccharide by deletion of the waaLUZYROBSPGQ gene cluster and enhancing RcsA by deletion of genes lon and hns can increase colonic acid production in E. coli. Therefore, these genes together with clsA, clsB, or/and clsC were deleted in E. coli, and all the resulting mutants showed increased colonic acid production. The best colonic acid production was observed in the mutant WWM16, which is 126-fold higher than in the control MG1655. To further improve colonic acid production, the genes rcsA and rcsD1-466 were overexpressed in WWM16, and the resulting recombinant E. coli WWM16/pWADT could produce 44.9 g/L colonic acid, which is the highest titer reported to date.

Progressive Sub-MIC Exposure of Klebsiella pneumoniae 43816 to Cephalothin Induces the Evolution of Beta-Lactam Resistance without Acquisition of Beta-Lactamase Genes.

Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in soils and water supplies in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 μg/mL to 7.5 μg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 μg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increased. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS may contribute to the resistant phenotype. These data demonstrate that very low sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.

Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli.

The fundamental biology and application of bacterial exopolysaccharides is gaining increasing attention. However, current synthetic biology efforts to produce the major component of Escherichia sp. slime, colanic acid, and functional derivatives thereof have been limited. Herein, we report the overproduction of colanic acid (up to 1.32 g/L) from d-glucose in an engineered strain of Escherichia coli JM109. Furthermore, we report that chemically synthesized l-fucose analogues containing an azide motif can be metabolically incorporated into the slime layer via a heterologous fucose salvage pathway from Bacteroides sp. and used in a click reaction to attach an organic cargo to the cell surface. This molecular-engineered biopolymer has potential as a new tool for use in chemical, biological, and materials research.