Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917

Abstract

Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917.

FIG. 1.

Assessment of the genome size of E. coli strain Nissle 1917 by I-CeuI restriction, followed by PFGE. Genomic DNA of E. coli strains MG1655, Nissle 1917, CFT073, and 536 was digested with the indicated restriction enzyme(s). Restriction fragments were separated by PFGE. Lane 1, lambda ladder PFGE marker; lanes 2, 6, and 10, E. coli strain MG1655; lanes 3, 7, and 11, E. coli strain Nissle 1917; lanes 4, 8, and 12, E. coli strain CFT073; lanes 5, 9, and 13, E. coli strain 536; lane 14, low-range PFGE marker.

FIG. 2.

Comprehensive genomic map of E. coli strain Nissle 1917 based on the chromosome of E. coli strain MG1655. GEIs I_{Nissle 1917} to IV_{Nissle 1917} and smaller genomic islets coding for fitness factors have been indicated according to their chromosomal insertion site next to tRNA-encoding genes. The positions of the tRNA-encoding genes, which seem to be possible chromosomal insertion sites for horizontally transferred DNA, are indicated as well as those of chromosomal restriction sites of CeuI. Grey marks indicate tRNA genes with sequence contexts identical to that of K-12 strain MG1655. Black marks indicate tRNA genes with sequence contexts identical to that of UPEC O6 strain CFT073. Dotted marks indicate tRNA genes with an as-yet-unknown downstream region. fim, type 1 fimbrial determinant; mch/mcm, microcin M- and H47-encoding determinants; foc, F1C fimbrial determinant; iro, salmochelin-encoding determinant; ybt, yersiniabactin-encoding determinant; iuc, aerobactin-encoding determinant; sat, Sat protease-encoding determinant; iha, Iha adhesin-encoding determinant; sap, Sap-like autotransporter-encoding determinant; kps, capsule determinant; chu, Chu hemin uptake determinant; wa*/wb*, gene clusters required for LPS biosynthesis.

FIG. 3.

Assessment of the genome content of strain Nissle 1917 by DNA arrays. (A) Genome comparison of nonpathogenic E. coli strain Nissle 1917 and E. coli K-12 strain MG1655 by Panorama E. coli gene arrays. The individual chromosomes are displayed linearly and in equal length. Missing and/or undetectable ORFs are marked by vertical black lines in the individual chromosomes. The positions of the undetectable ORFs refer to the E. coli MG1655 chromosome. The positions of tRNA genes frequently used as chromosomal insertion sites of horizontally acquired DNA elements, those of 10 prophages of strain MG1655, and the chromosomal origin and terminus of replication are marked within the map of E. coli strain MG1655. (B) Detection of various genes of the flexible gene pool of E. coli and Shigella in nonpathogenic E. coli strain Nissle 1917 by the E. coli pathoarray. The genes are grouped by typical E. coli pathotypes. Missing and/or undetectable ORFs are marked by vertical black lines.

FIG. 4.

Genetic structure of E. coli Nissle 1917-specific GEIs (GEI I_{Nissle 1917} to GEI IV_{Nissle 1917}). Important GEI regions or fitness-conferring determinants are highlighted with different colors or patterns. The argW downstream region, identified as a part of GEI III_{Nissle 1917}, is underlined. The localization of tRNA-encoding genes is indicated as well as that of the DNA regions included in the PCR-screening approach for the detection of E. coli strain Nissle 1917-specific sequences.

FIG. 5.

Comparison of the genetic organization of the left-hand end of GEI II_{Nissle 1917} and the pheV-associated PAI of E. coli strain CFT073, demonstrating the loss of the alpha-hemolysin-encoding determinant (hly) and large parts of the P-fimbrial operon (pap) in strain Nissle 1917. Homologous regions between the two islands are highlighted by identical colors. The color code is identical to that shown on Fig. 4.