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The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

ABSTRACT: The bacterial extracellular matrix encases cells and protects them from host-related and environmental insults. The Escherichia coli master biofilm regulator CsgD is required for the production of the matrix components curli and cellulose. CsgD activates the diguanylate cyclase AdrA, which in turn stimulates cellulose production through cyclic di-GMP (c-di-GMP). Here, we identified and characterized a CsgD- and AdrA-independent cellulose production pathway that was maximally active when cultures were grown under reducing conditions or when the disulfide bonding system (DSB) was compromised. The CsgD-independent cellulose activation pathway was dependent on a second diguanylate cyclase, called YfiN. c-di-GMP production by YfiN was repressed by the periplasmic protein YfiR, and deletion of yfiR promoted CsgD-independent cellulose production. Conversely, when YfiR was overexpressed, cellulose production was decreased. Finally, we found that YfiR was oxidized by DsbA and that intraprotein YfiR disulfide bonds stabilized YfiR in the periplasm. Altogether, we showed that reducing conditions and mutations in the DSB system caused hyperactivation of YfiN and subsequent CsgD-independent cellulose production.

SUBMITTER: Hufnagel DA

PROVIDER: S-EPMC4248800 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

Hufnagel David A DA DePas William H WH Chapman Matthew R MR

Journal of bacteriology 20140811 21

The bacterial extracellular matrix encases cells and protects them from host-related and environmental insults. The Escherichia coli master biofilm regulator CsgD is required for the production of the matrix components curli and cellulose. CsgD activates the diguanylate cyclase AdrA, which in turn stimulates cellulose production through cyclic di-GMP (c-di-GMP). Here, we identified and characterized a CsgD- and AdrA-independent cellulose production pathway that was maximally active when cultures ...[more]

PMID: 25112475

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Project description:The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

Project description:BackgroundRetinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.ResultsAmong the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.ConclusionsIn this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.

Dataset Information

The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

Publications

The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

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