Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion

Abstract

We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3")-Ia cassettes. The first cassette in integron 2 contains the ant(2")-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3")-Ii-aac(6')-IId, which has been characterized for its bifunctional ANT(3")-I and AAC(6')-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla(OXA-10) cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla(OXA-10) cassette is incomplete and that the 3' conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3")-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.

FIG. 1.

Mapping of integrons of S. marcescens SCH909. (a) General structures of integrons 1, 2, and 3 found in S. marcescens SCH909. The arrows show the direction of transcription. (b) PCR amplification from SCH909. The PCR products were separated by electrophoresis through 1.0% agarose. Lanes 1 and 14, 1-kb DNA ladder; lanes 2 to 13, SCH909 with the following primers: lane 2, Sulpro3 and 3′-CS; lane 3, Sulpro3 and dfrA1; lane 4, Sulpro3 and ant(3")-Ia; lane 5, Sulpro3 and ant(3")-Ii; lane 6, Sulpro3 and aac(6′)-Ib; lane 7, Sulpro3 and ant(2")-Ia; lane 8, Sulpro3 and aac(3)-Ia; lane 9, aac(3)-Iacooh and 3′-CS; lane 10, ant(3")-Iacooh and 3′-CS; lane 11, aac(3)-Iacooh and ant(3")-Ia; lane12, ant(2")-Iacooh and aac(6′)-Ib; and lane 13, tnpR and BLATEM. Primer Sulpro3 is a rightward primer in the 5′ conserved segment. Primers with gene names are leftward primers near the beginning of their respective cassettes. Primers with “cooh” are rightward primers near the end of their respective cassettes. Primer 3′-CS is a leftward primer in the 3′ conserved sequence. These primers are used to determine gene order in integrons (14).

FIG. 1.

Mapping of integrons of S. marcescens SCH909. (a) General structures of integrons 1, 2, and 3 found in S. marcescens SCH909. The arrows show the direction of transcription. (b) PCR amplification from SCH909. The PCR products were separated by electrophoresis through 1.0% agarose. Lanes 1 and 14, 1-kb DNA ladder; lanes 2 to 13, SCH909 with the following primers: lane 2, Sulpro3 and 3′-CS; lane 3, Sulpro3 and dfrA1; lane 4, Sulpro3 and ant(3")-Ia; lane 5, Sulpro3 and ant(3")-Ii; lane 6, Sulpro3 and aac(6′)-Ib; lane 7, Sulpro3 and ant(2")-Ia; lane 8, Sulpro3 and aac(3)-Ia; lane 9, aac(3)-Iacooh and 3′-CS; lane 10, ant(3")-Iacooh and 3′-CS; lane 11, aac(3)-Iacooh and ant(3")-Ia; lane12, ant(2")-Iacooh and aac(6′)-Ib; and lane 13, tnpR and BLATEM. Primer Sulpro3 is a rightward primer in the 5′ conserved segment. Primers with gene names are leftward primers near the beginning of their respective cassettes. Primers with “cooh” are rightward primers near the end of their respective cassettes. Primer 3′-CS is a leftward primer in the 3′ conserved sequence. These primers are used to determine gene order in integrons (14).

FIG. 2.

Protein alignment of the putative maturase, Smtr, in SCH909 with eubacterial group II intron-encoded proteins from an Acinetobacter sp. (Acin.sp.) (J. H. Yum et al., unpublished; GenBank accession number AF369871; ORF II), N. europaea (N. euro) (JGI, unpublished), P. alcaligenes (PaOrX6) (36), Streptococcus pneumoniae (S. pneu) (5), and Lactococcus lactis (LlltrA) (18). Consensus amino acids (four or more identical in the six sequences) are in bold letters. Domains conserved among intron-encoded proteins are denoted by the lines above the alignment. RT1 through RT7, RT-like domains (19); z, domain of undetermined function in non-long terminal repeat retroelements; x, maturase-specific domain (19). Numbers in parentheses indicate numbers of amino acids not shown.

FIG. 3.

General features of Smtr and its insertion site. (a) Secondary structure model of domains V and VI of the group II intron, Smtr, within integron 2 from SCH909. The boundary of the intron and the ant(2")-Ia attC site is indicated by a vertical line. The bulging adenine residue involved in lariat formation is indicated by an asterisk. (b) Nucleotide sequence alignment of the ant(2")-Ia stop codon and its attC site (59-base element) (4) with the ant(2")-Ia gene and Smtr-intron boundary from SCH909). ant(2")-Ia stop codons are indicated by asterisks. Exon sequences are shown in bold letters. (c) Nucleotide sequence alignment of the group II introns of SCH909 and N. europaea. The latter is adjacent to an attC site on one end; there is no recognizable ORF on the other end.

FIG. 3.

General features of Smtr and its insertion site. (a) Secondary structure model of domains V and VI of the group II intron, Smtr, within integron 2 from SCH909. The boundary of the intron and the ant(2")-Ia attC site is indicated by a vertical line. The bulging adenine residue involved in lariat formation is indicated by an asterisk. (b) Nucleotide sequence alignment of the ant(2")-Ia stop codon and its attC site (59-base element) (4) with the ant(2")-Ia gene and Smtr-intron boundary from SCH909). ant(2")-Ia stop codons are indicated by asterisks. Exon sequences are shown in bold letters. (c) Nucleotide sequence alignment of the group II introns of SCH909 and N. europaea. The latter is adjacent to an attC site on one end; there is no recognizable ORF on the other end.