The Scr and Csc pathways for sucrose utilization co-exist in E. coli, but only the Scr pathway is widespread in other Enterobacteriaceae

Abstract

Most Escherichia coli isolates from humans do not utilize D-sucrose as a substrate for fermentation or growth. Previous work has shown that the Csc pathway allows some E. coli to utilize sucrose for slow growth, and this pathway has been engineered in E. coli W strains to enhance use of sucrose as a feedstock for industrial applications. An alternative sucrose utilization pathway, Scr, was first identified in Klebsiella pneumoniae and has been reported in some E. coli and Salmonella enterica isolates. We show here that the Scr pathway is native to an important subset of E. coli phylogroup B2 lineages that lack the Csc pathway but grow rapidly on sucrose. Laboratory E. coli strains derived from MG1655 (phylogroup A, ST10) are unable to utilize sucrose and lack the scr and csc genes, but a recombinant plasmid-borne scr locus enables rapid growth on and fermentation of sucrose. Genome analyses of Enterobacteriaceae indicate that the scr locus is widespread in other Enterobacteriaceae; including Enterobacter and Klebsiella species, and some Citrobacter and Proteus species. In contrast, the Csc pathway is limited mostly to E. coli, some Shigella species (in which csc loci are rendered non-functional by various mutations), and Citrobacter freundii. The more efficient Scr pathway likely has greater potential than the Csc pathway for bioindustrial applications of E. coli and other Enterobacteriaceae using sucrose as a feedstock.

Keywords: Csc; Escherichia coli; Scr; evolutionary genomics; metabolism; sucrose.

Figure 1

Function and genetic organization of the Scr and Csc pathways for sucrose utilization. Inferred structure and function of the Scr and Csc pathways (reviewed in Reid and Abratt, 2005). Organization of the E. coli genetic loci encoding the Scr and Csc pathways, and the regulators controlling them (cscR and scrR, respectively). The transcriptional organizations of the loci are shown in solid arrows above the genes. Approximate locations of IS3 insertions (scrY) and recombination event (scrB) discussed in Results are indicated below the scr locus. Figure created using BioRender.

Figure 2

Assays for sucrose utilization in liquid and agar media. Culture growth in M9 minimal salts broth with either glucose or sucrose as sole carbon/energy source was monitored in a spectrophotometer at 600 nm, whom on the left of each panel. M9 agar plates with either glucose (M9G) or sucrose (M9S) were photographed 24 h after streaking.

Figure 3

Utilization of sucrose conferred by recombinant plasmids with scr genes. Life side of the figure diagrams inserts in recombinant plasmids (named on the right side) containing the entire scr operon, or fragments thereof. Plasmids were introduced into E. coli SCU-113 (B1, ST5974, scr⁻ csc⁻). In the table, “+” indicates that fast fermentation/growth was observed, “+/−” indicates that slower fermentation and growth was observed, and “−” indicates that no fermentation/growth was observed.

Figure 4

Rare spontaneous conversion of Suc⁻ SCU-175 (scrY::Is3) to Suc⁺ phenotype. The left panel shows that SCU-175 showed no growth on M9S plates after 48 h, with the exception of rare spontaneous colonies. When those colonies were restreaked on M9S plates (right panel, bottom half of plate) they were Suc⁺, in contrast to the parental strain streaked on the same plate as a control (right panel, top half).

Figure 5

Conservation of the scrY promoter and regulatory region. DNA sequences upstream of the scrY start codon were aligned using the Geneious alignment algorithm. An identical base at a position is indicated by “.”, and gaps by “−”. Annotations above the aligned sequences are based on Cowan et al. (1991) and Jahreis and Lengeler (1993).

Figure 6

Representative structures of E. coli, Shigella, and C. freundii csc loci. Shigella species and strain names are shown on the left side. Note that in the C. freundii locus, the cscA and cscR genes are in the same relative locations, but their orientations are each flipped.

Figure 7

Conservation of the cscA promoter and regulatory region. DNA sequences upstream of the cscA start codon were aligned using the Geneious alignment algorithm. Identity with the consensus sequence is indicated by “.”, and gaps by “−”. Annotations above the aligned sequences are based on Jahreis et al. (2002).