Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics

Abstract

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Figure 1. Morphology of L. johnsonii wild type and spontaneous mutant strains.

Wild type rough (FI9785, a, b, e) and smooth (FI10386, c, d, f) colony variants showing colony phenotype on agar (a, c), organisation within colonies (b, d, magnification ×40) and with SEM (e, f, bar = 1 µm).

Figure 2. Identification of a protein change associated with the smooth colony phenotype.

2-D gel electrophoresis of proteins extracted from wild type rough (FI9785, R) and smooth colony variant (FI10386, S) strains of L. johnsonii. Left, whole gel image of proteins extracted from wild type showing location of enlarged section in top right; bottom right, equivalent section from gel of smooth variant. The spots corresponding to EpsC are circled.

Figure 3. Location of epsC in a predicted exopolysaccharide biosynthesis cluster and features of its amino acid sequence.

a) Putative eps operon of L. johnsonii FI9785 showing predicted gene functions. Striped, transcriptional regulation; dark grey, chain length determination, polymerisation and regulation; light grey, priming glycosyltransferase; white, glycosyltransferases; black, polymerisation and export. b) Alignment of the EpsC protein with CpsD from Streptococcus pneumoniae (Q54520); #, amino acids involved in the nucleotide binding site cd02035, *, D88 mutated to N88 in smooth variant FI10386, regions with similarity to the Walker A motif (GKT/S) and the Walker B motif (hhhD) are underlined.

Figure 4. Effect of the expression of the wild type epsC gene in the smooth colony strain.

Morphology of smooth colony variants containing the L. johnsonii expression construct with no insert (a) or containing the mutant epsC^D88N (b) or the wild type epsC (c).

Figure 5. Total sugar content of L. johnsonii strains.

Results are the mean of triplicate measurements +/− standard deviation, figures above bars represent the p value calculated using an independent t-test with unequal variance, comparing each strain to the wild type.

Figure 6. Effect of the deletion of epsE on L. johnsonii morphology.

a) Aggregation of L. johnsonii ΔepsE FI10844 (right) compared to L. johnsonii FI9785 (left) after 24 h growth at 37°C. b) SEM of FI10844 cells, bar = 1 µm.

Figure 7. Adhesion of L. johnsonii strains to HT29 monolayers.

Results are the mean of triplicate experiments with three replicates per experiment +/− standard deviation, figures above bars represent the p value calculated using an independent t-test with unequal variance, comparing each strain to the wild type.