The diversity of inducible and constitutively expressed erm(C) genes and association to different replicon types in staphylococci plasmids

Abstract

The aim of this study was to analyze the diversity of the macrolide resistance gene, erm(C) in relation to structural alterations affecting the gene expression. In addition, the association of erm(C) to mobile genetic elements (MGEs) in staphylococci mainly from Danish pigs was investigated. In total, 78 erythromycin-resistant isolates were screened for erm(C) by PCR. The erm(C) genes incl. the upstream regulatory region were sequenced and the expression types were characterized phenotypically (agar diffusion test) and genotypically (sequence analysis). Phylogenetic analysis of erm(C) was compared with structural alterations affecting the gene expression. Plasmids carrying erm(C) from seven selected isolates were fully or partially sequenced. Thirty-seven isolates were shown to be erm(C) positive and erm(C) from pigs were all constitutively expressed, mainly caused by different sized deletions (118, 111, 107, 70, 66, 16 and 3 bp) in the regulatory region. Duplication (63 bp) and substitutions were also found to cause a constitutive phenotype. Only one horse isolate had an inducible expression type. Phylogenetic analysis showed that structural alterations have happened in different erm(C) allele groups and not only in one group. Furthermore erm(C) was found mainly on plasmids (~2.4-8 kb) and gene sequence types correlated with plasmid replication (rep) gene types. One erm(C) type was linked to an IS257 element able to circularize. In conclusion, structural alterations giving rise to constitutive expression of erm(C) have happened several times in the evolution of erm(C). Interestingly, the diversity of erm(C) appears to be linked to the plasmid type or MGE carrying the gene.

Figure 1. Schematic representation of the inducible erm(C) gene and structural alterations in the upstream regulatory region, which lead to constitutive expression of erm(C). The erm(C) mRNA begins at +1 and besides the erm(C) gene (735 bp) it contains an upstream leader ORF (60 bp). Ribosomal binding sites, Shine Dalgarno sequence (SD) precedes both genes. In the absence of an inducer, the inverted repeat sequences (IR), IR1-IR4 form a double hairpin structure, rendering the erm(C) start codon non-accessible to the ribosome. Predicted pairing of IR sequences is indicated with arrows (threshold for predicted pairing was ΔG < -41.84 kJ/mol, -10 kcal/mol) and hypothetical pairing are indicated with dotted arrows. Structural alterations that have not previously been reported are highlighted with an asterix (*). (A) The upstream region of erm(C) from the S. aureus isolate (7504026–1) shown to have an inducible phenotype aligned with two GenBank sequences known to have an inducible phenotype. (B). The region upstream erm(C) containing deletions resulting in a constitutive phenotype. The alignment shows the different size deletions found in sequences from this study that had a constitutive phenotype. (C). Substitutions (underlined in read) in the upstream region of erm(C) from four S. hyicus isolates were predicted to result in the pairing of IR2 with IR3, which explains the constitutive phenotype (see text). (D) The upstream region of erm(C) in the S. aureus isolate (65–5) predicted to have a constitutive expression of erm(C) due to a 63 bp duplication containing a truncated erm(C)´ including an additional SD2´/IR4´.

Figure 2. Phylogenetic gene tree of erm(C) compared with structural alterations in the upstream regulatory region of erm(C) conferring constitutive expression. Sequences from this study (marked in bold letters) were mainly isolated from pigs (Table 1), whereas the GenBank sequences were mainly from human isolates. Structural alterations causing constitutive expression of erm(C) are marked with a color code, and the type of alteration is noted with a number which indicates the size (in bp) of deletion or duplication causing the constitutive phenotype. Bootstrap values are indicated at branch points (out of 1,000 generated NJ trees). The relatively low bootstrap values at some branch points within group 4 can be explained by single nucleotide differences between sequences in this group.