Gene Cassette Encoding a 3-N-Aminoglycoside Acetyltransferase in a Chromosomal Integron

The large number and variety of antibiotic resistance genes found in gene cassettes associated with class 1 integrons in multiply antibiotic-resistant organisms have always indicated the existence of a large pool of gene cassettes from which they are drawn (10). The discovery of integrons, sometimes harboring very large arrays of gene cassettes, in the chromosomes of many different bacteria has confirmed the existence of this pool, but to date very few cassettes containing resistance genes have been found among these cassettes. Known examples are the catB9 (12) and blaP7 (blaP9) (7, 8) cassettes found in the cassette arrays of different Vibrio cholerae strains.

Though 3-N-aminoglycoside acetyltransferases [AAC(3) enzymes] have been classified into several groups based on differences in the spectrum of aminoglycosides they modify, they fall into only two groups based on the relationships between the proteins (6). The five characterized aacC genes found in gene cassettes (see Table 1) encode proteins of 154 to 158 amino acids that are related to one another and form family A (6). They belong to the aac(3)-I phenotypic group and confer resistance to gentamicin, sisomicin, and fortimicin but not to tobramycin, amikacin, or kanamycin (1, 3, 6, 11, 13, 15).

TABLE 1.

aacC-A genes and gene cassettes

Gene namea	Previous name(s)	Length (bp)		Integron type	GenBank accession no.	Reference(s)
		Cassette	59-be
aacC-A1	aacC1 (aac(3)-Ia)	577	109	Class 1	X15852	15
aacC-A2	aac(3)-Ib	>498	>33	Class 1	L06157	13
aacC-A3	aac(3)-Ic	590	112	Class 1	AJ511268	11
aacC-A4	aacC4	576	108	Class 1	AF318077	4, 9
aacC-A5	aac(3)-Idb	564	78	Class 1	AY463797	1, 6
aacC-A6		577	112	Class 1	AY884051
aacC-A7		558	75	Genomic	CP000282d
aacC-A8		NAc			Nonee
aacC-A9		NAc			Nonef
aacC-A10		NAc			CP000155

^aNumbers have been assigned in the order of release of the GenBank record (5). Only A1 to A5 have been demonstrated to confer aminoglycoside resistance.

^bThe designation -Id for this gene (GenBank accession numbers AB114632, AY458224, and AY563051) is incorrect (1-3).

^cNA, not applicable (because this gene is not part of an identifiable cassette).

^dGenome of Saccharophagus (formerly Microbulbifer) degradans 2-40. Cassette is at 2,333,600 to 2,334,159 bp.

^eTwo copies (98% identical) are found in the draft genome of Paracoccus denitrificans PD1222, U.S. Department of Energy Joint Genome Institute (the sequence can be obtained at http://genome.jgi-psf.org/draft_microbes/parde/parde.home.html or via GenBank accession no. AAIT00000000).

^fFound in the draft genome of Sphingopyxis alaskensis RB2256, U.S. Department of Energy Joint Genome Institute (the sequence can be obtained at http://genome.jgi-psf.org/draft_microbes/sphal/sphal.home.html or via GenBank accession no. AAIP00000000).

Our recent searches have revealed further potential aacC-A-type genes (Table 1). One is in a cassette found in a class 1 integron, but three of them are in draft sequences of bacterial genomes. The aacC-A7 gene in the complete Saccharophagus degradans (formerly Microbulbifer degradans) genome also lies within an identifiable gene cassette. The putative AacC-A7 [AAC(3)-Ig] acetyltransferase encoded by this cassette is 51 to 60% identical to other cassette-encoded AacC-A enzymes and most closely related to AacC-A5 [AAC(3)-Ie]. Alignment of the established and putative AacC-A sequences (Fig. 1) reveals 45 completely conserved amino acids, or 62 if only AacC-A1 to AacC-A7 are considered. It therefore seems reasonable to assume that aacC-A7 also confers resistance to aminoglycoside antibiotics.

FIG. 1.

Alignment of proteins in the AacC-A [AAC(3)-I] family. Amino acids completely conserved in all sequences are shown as white on black and are indicated by uppercase letters below the sequence. Amino acids conserved in 9 of the 10 sequences are indicated by lowercase letters below. The sequences of AacC-A proteins were obtained from the GenBank accession numbers listed in Table 1.

This represents only the third identification of a potential antibiotic resistance gene associated with a gene cassette that is part of the genome of a microorganism that harbors an integron. Though each aacC-A cassette has an identifiable 59-be (sometimes called an attC site) made up of two simple sites and a central region as is characteristic for 59-be (14), in most cases they are not close relatives of one another and some are not closely related to ones found in any other known cassette (6, 11). The 59-be of the Saccharophagus aacC-A7 cassette is not closely related to a 59-be in any of the many cassettes in that organism's genome. Its closest relative is in a cassette from Nitrosococcus oceani. Thus, if, as is generally assumed, cassettes that arose in a particular organism have very similar 59-be, it is unlikely that the aacC-A7 cassette was originally formed in S. degradans.