GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli

Abstract

Curli are extracellular surface fibers that are produced by many members of the Enterobacteriaceae and contribute to biofilm formation. The environmental cues that promote biofilm formation are poorly understood. We found that deletion of the N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase gene, nagA, resulted in decreased transcription from the curli-specific promoters csgBA and csgDEFG and a corresponding decrease in curli production in Escherichia coli. nagA is in an operon that contains nagB, nagC, nagD, and nagE, whose products are required for utilization of GlcNAc as a carbon source. NagC is a repressor of the nagBACD and nagE genes in the absence of intracellular GlcNAc-6P. We found that nagC mutants were also defective in curli production. Growth of a wild-type strain on media containing additional GlcNAc reduced curli gene transcription to a level similar to the level observed when nagA was deleted. The defect in curli production in nagA or nagC mutants was alleviated by deletion of the GlcNAc transporter gene, nagE. Curli-producing DeltanagA suppressor mutants whose cells were unable to take up GlcNAc were isolated. These results suggest that elevated levels of intracellular GlcNAc-6P signal cells to down-regulate curli gene expression.

FIG. 1.

Schematic diagram the of the nag genes. nagBACD are expressed from a single transcript, and nagE is divergently transcribed. The intergenic region is 330 bp long. NagC represses transcription from the nagBACD and nagE promoters in the absence of GlcNAc-6P.

FIG. 2.

ΔnagA and ΔnagC strains are defective in curli production. (A) CR-YESCA plate containing Wt strains (Wt_c, C600; Wt_m, MC4100) and ΔnagA strains (ΔnagA_c, MMB103; ΔnagA_m, MMB5) transformed with pTrc99A or pNagA after 48 h of growth at 26°C. (B) CR-YESCA plate containing Wt (C600), ΔnagBACD ΔnagE (MMB87) (Δnag), ΔnagA (MMB103), ΔnagB (MMB120), ΔnagC (MMB171), ΔnagD (MMB161), ΔnagE (MMB152), ΔnagEA (SKM2), and ΔnagEC (MMB185) strains after 48 h of growth at 26°C. (C and D) Electron micrographs of Wt (MC4100) (C) and ΔnagA (MMB5) (D) strains. Scale bars = 400 nm.

FIG. 3.

Steady-state levels of CsgA and CsgG are reduced when intracellular levels of GlcNAc-6P are high. (A and B) Western blots of whole-cell lysates from different strains in a C600 background after 48 h of growth at 26°C on YESCA plates developed with anti-CsgA antibodies (A) or with anti-CsgG antibodies (B). (C) Whole-cell Western blot of Wt strain MMB200 transformed with pRJ800 after 48 h of growth at 26°C on YESCA plates or YESCA (pH 8.1) plates with or without 10 mM GlcNAc developed with anti-CsgA antibodies. The samples in even-numbered lanes in panels A and C were treated with formic acid (FA) prior to electrophoresis.

FIG. 4.

CR⁺ ΔnagA suppressors develop after extended growth on YESCA plates. After 96 h of growth on CR-YESCA plates, CR⁺ ΔnagA suppressors arose. These suppressors were mixed with CR⁻ ΔnagA isolates, and the two populations were isolated. (A) CR-YESCA plate containing the Wt (C600), original ΔnagA (MMB103), CR⁺ ΔnagA suppressor, and CR⁻ ΔnagA (isolate mixed with the CR⁺ ΔnagA suppressor) strains after growth for 48 h at 26°C. (B and C) The strains were streaked onto W minimal medium plates with glucose (B) or GlcNAc (C) as the sole carbon source and incubated at 37°C for 48 h.

FIG. 5.

CR⁺ ΔnagA suppressors map to nagE or nagC. (A) Intergenic region between nagBACD and nagE and part of the nagE open reading frame. The start codons for nagB and nagE are indicated by boldface type and arrows. The arrowheads indicate places where IS1 or IS5 elements are inserted into the nagE promoter or gene in the CR⁺ ΔnagA suppressors. In one suppressor the underlined sequence is duplicated, and in another suppressor the sequence indicated by gray type is deleted; both result in premature stop codons in nagE. (B) CR-YESCA plate containing Wt (C600), ΔnagC (MMB171), and ΔnagA (MMB103) strains transformed with pTrc99A, pNagC, or pNagC4 after 48 h of growth at 26°C. (C) Plate containing MacConkey agar with 1% GlcNAc and the Wt (C600) and ΔnagC (MMB171) strains transformed with pTrc99A, pNagC, or pNagC4 after overnight growth at 37°C.