aCentre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney and Westmead Hospital, Westmead, New South Wales, Australia
bSchool of Life & Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia
bSchool of Life & Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia
cMicrobiology và Infectious Diseases, School of Medicine, Western Sydney University, Sydney, New South Wales, Australia
dAntibiotic Resistance & Smartphone Elements Group, Ingđê mê Institute for Applied Medical Retìm kiếm, Sydney, New South Wales, Australia
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FIG 3

Tn3 family transposons. The extents and orientations of various genes are shown by arrows, with thiông chồng arrows used to indicate antibiotic resistance genes (apart from those in gene cassettes). Terminal IR are indicated by black bars & putative sầu ancestral IR relics by gray bars. res sites are shown as blaông chồng boxes. (A) Tn3 family. Gene cassettes in Tn1331 are shown as narrower boxes. (B) Tn21 subfamily. For Tn21, the insertion site for class 1 In/Tn (see Fig. 4) and the 5-bp TSD are shown. Different integron structures và different cassettes may be present. IS4321 or IS5075 may be found inserted inkhổng lồ IRL and/or IRR, in the indicated orientations. (C) Tn4401. The approximate position of deletions that lead khổng lồ different promoter variants is indicated. Diagrams were drawn based on sequences from the following INSDC accession numbers: Tn2, AY123253; Tn1331, AF479774; Tn5393, AF262622; Tn1546, M97297; Tn21, AF071413; Tn1696, U12338; Tn6452; KY807920; Tn1721, X61367; và Tn4401, EU176011. The resistance genes shown confer resistance to the following antibiotics: blaTEM-1, broad-spectrum β-lactams; blaOXA-9, oxacillin; aadA1, streptomycin và spectinomycin; strAB, streptomycin; vanXAH, vancomycin/teicoplanin; mcr-5, colistin; tet(A), tetracycline; & blaKPC, carbapenems.

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blaTEM genes, including those encoding extended-spectrum β-lactamases (ESBL) or inhibitor-resistant (IRT) variants, have always been found within Tn1, Tn2, Tn3, or variants, hybrids, or fragments of these transposons. A degenerate relic of an IR just upstream of blaTEM suggests capture following adjacent insertion of an ancestral cryptic transposon (68). A derivative sầu of Tn3, named Tn1331, carries additional resistance genes in a region derived from a class 1 integron (Fig. 3A; see below) (72). Hybrid Tn1331-like elements with better matches to Tn1 or Tn2 in different segments are quite comtháng, including in association with blaKPC genes (73). Recombination between different copies of Tn2 in different locations may also contribute to spread of resistance genes that have sầu been inserted within this transposon by other thiết bị di động elements (74, 75).


Tn5393.Tn5393 carries the strAB (streptomycin resistance) ren pair in the position equivalent to that of blaTEM in Tn3. Complete copies of Tn5393 with different insertions have been identified (76), but fragments of Tn5393 appear to lớn be more common than the complete transposon in plinfobandarpkr.comids và genomic islands.


Tn1546.The transposons discussed above are all associated with antibiotic resistance in Gram-negative sầu bacteria, while the most notable thành viên of the Tn3 family in Gram-positive sầu bacterial species is Tn1546 (Fig. 3A). Similar to the tnpA and tnpR genes of Tn3, those of Tn1546 are transcribed in opposite directions & separated by the res site; it has 38-bp imperfect IR and creates 5-bp TSD on insertion (77). Tn1546 encodes resistance to lớn vancomycin via the vanA ren cluster, whose expression is regulated by the vanRS ren products. Variants of Tn1546 display significant heterogeneity, including deletions and/or one or more IS inserted inlớn the backbone structure (78). In some cases, these IS have also acquired additional resistance determinants, such as fosB3 (fosfomycin resistance) (79). Importantly, Tn1546 has been responsible for the spread of vancomycin resistance aao ước enterococcal populations around the world, largely facilitated by its association with conjugative sầu plinfobandarpkr.comids. Furthermore, Tn1546 has been delivered into methicillin-resistant S. aureus (MRSA) by plinfobandarpkr.comids on several occasions (see below).


Tn21 Subfamily TransposonsIn members of the Tn21 subfamily of the Tn3 family, the tnpR & tnpA genes are in the same orientation và the res site is upstream of tnpR (80). This arrangement may give sầu a more stable transposition module than the organization in the transposons described above, as the tnpAtnpR genes are less likely to become separated by aberrant recombination in res (81). The 38-bp IR of Tn21 subfamily elements are the targets for the related IS4321 and IS5075 elements (IS110 family; encode a DDED transposase), which transpose via double-stranded circular intermediates and insert in one orientation at a specific position, presumably preventing further movement of the host transposon by transposition (82).


Tn21 and cthảm bại relatives.Tn21 (81) and related transposons (Fig. 3B) often carry a mercury resistance (mer) operon but are important in movement of antibiotic resistance genes, as they may also carry a class 1 integron (see below). Different members of this family have tnp regions that are ∼80% identical, và they carry different mer operons (e.g., Tn21 và Tn1696) (83) or other accessory genes (e.g., Tn1403) (84). Tn21 itself has an extra region between mer & the res site, và integrons with different structures và different cassette arrays are always found inserted at the same position in this extra sequence, flanked by the same TSD (81). In related transposons without this region, a class 1 integron may be inserted at different locations within the res site (see reference 21 for more details). Because such an interruption might affect resolution, this might help lớn explain why Tn21 is apparently more prevalent than other members of this subfamily (81).

A new mcr-type gen, mcr-5, was recently identified as part of a transposon designated Tn6452, identified in Salmonella, E. coli, and Cupriavidus gilardii (environmental Burkholderiaceae) (85, 86). The tnp region of Tn6452 is ∼80% identical to lớn that of Tn21, và Tn6452 is bounded by identical 38-bp IR và creates the expected 5-bp TSD.


Tn1721.Tn1721 consists of Tn1722 (tnpA, tnpR, & res) adjacent lớn a partial duplication of the IRR kết thúc of Tn1722 & the tet(A) tetracycline resistance determinant. The whole structure is flanked by 38-bp IR and has an extra internal copy of IRR (21). Tn1721 may have sầu been created by internal deletion of an ancestral composite element flanked by two copies of Tn1722 (68). As in the case of Tn5393, fragments of Tn1721/Tn1722 are more common than the complete element in plinfobandarpkr.comids and resistance islands in Gram-negative bacteria.


Tn4401Tn4401 (Fig. 3C), carrying blaKPC variants, also belongs khổng lồ the broader Tn3 family, but the comtháng mô tả tìm kiếm “Tn3 based” is not accurate, as the Tn3 và Tn4401 TnpA proteins are only about 39% similar/22% identical and the TnpR and nucleotide sequences are quite different. The organization is also different from that of Tn3, with blaKPC & the flanking ISKpn7 (upstream) & ISKpn6 (downstream) elements found between IRR of Tn4401 & the kết thúc of the tnpA ren. It appears that an ancestral transposon inserted upstream of blaKPC, with insertion of ISKpn6 disrupting the original IRR & forcing use of an alternative downstream sequence in subsequent transposition events (87).

Several variants of Tn4401 with different internal deletions have been distinguished by lowercase letters (88). The longest version, Tn4401b, has two experimentally confirmed promoters driving blaKPC expression: P2 (last 6 bp khổng lồ 24 bp downstream of the ISKpn7 IRR) & P1 (74 to lớn 46 bp upstream of the blaKPC start codon) (88). Deletions in Tn4401d (68 bp) and Tn4401a (99 bp; often incorrectly stated as 100 bp) remove regions between P1 and P2 that may size secondary structures but leave sầu both promoters intact (88), as does the 188-bp deletion in Tn4401h (89). The most common size, Tn4401a, gives the highest levels of resistance (88), while Tn4401h gives higher levels than those with Tn4401b (89). Tn4401c và Tn4401e have deletions of 216 bp (incorrectly reported as 215 bp) and 255 bp, respectively, ending at the same place (27 bp upstream of the blaKPC start codon) & leaving P2 only, which was found khổng lồ result in reduced blaKPC expression (88). Three other blaKPC contexts have either no deletion in this region (Tn4401f <90>) or the 216-bp (Tn4401g <91>) or 255-bp (another Tn4401h variant <92>) deletion, but regions upstream of blaKPC bởi not match the complete Tn4401 sequence, và these may better be considered “non-Tn4401 elements” (NTEKPC) that contain only part of Tn4401 (93).


Tn7.The characteristics of Tn7 have sầu been reviewed several times (see references 94 & 95 & references therein). Tn7 carries the tnsABCDE genes (Fig. 4A) & uses a “cut-and-paste” transposition mechanism. TnsB & TnsA together khung a heteromeric transposase that excises Tn7 from its original site. IR of ∼28 bp are present at each extremity of Tn7, but there are also four 22-bp TnsB binding sites within 90 bp of the IRL end and three within 150 bp of the IRR kết thúc (95). TnsC is an adaptor for target capture that communicates between TnsA/B và either TnsD, directing insertion to a single chromosomal attTn7 site just downstream of the conserved glmS gen of Gram-negative sầu bacteria, or TnsE, to lớn target the lagging strands of replicating conjugative plinfobandarpkr.comids. This allows both vertical và horizontal transmission (95). Transposition generates 5-bp TSD, và Tn7 carries a class 2 integron (see below).


FIG 4

Tn7-like transposons. Most features are shown as described in the legkết thúc lớn Fig. 3. (A) Tn7. The asterisk indicates the position of a comtháng stop codon in intI2. The diagram was drawn based on the sequence from INSDC accession number AP002527. (B) Evolution of class 1 In/Tn. The diagrams show capture of intI1/attI1/Pc và ren cassettes, with qacE in the last position, by a Tn5053-lượt thích transposon. Subsequent deletion of parts of the final qacE cassette and tni region and insertion of sul1 create the 3′-CS, giving a typical “clinical” class 1 In/Tn which is not self-transposable, e.g., In2. Diagrams are based on information in reference 21 và sequences from INSDC accession numbers U67194 & AF071413. Different extents of tni và different IS may be present beyond the 3′-CS (see Fig. 5 in reference 21 for further details). (C) Tn552. Gene names shown in parentheses indicate relationships lớn those in Tn7/Tn5053 elements. The diagram was drawn based on the sequence from INSDC accession number X52734. (D) Transposons making up resistance islands in A. baumannii. The top diagram represents Tn6022; differences in minor variants Tn6021 (a short region with only 90% identity matches Tn6172) và Tn6022Δ are shown. The main part of the Tn6174 diagram corresponds to lớn the hypothetical, ancestral Tn6173, which is also related khổng lồ Tn6022 (percentage identities in different regions shown above) but has the ars/feo region replacing uspA and sup. Tn6174 itself has the two insertions shown above sầu the diagram. Tn6172 was generated from Tn6174 by addition of Tn5393 (Fig. 3) & an internal deletion. In AbGRI1-0, a region flanked by Tn6022 & Tn6172 is inserted inlớn the chromosomal comM ren. The backbone Tn6019 of AbaR3-like islands is related lớn Tn6022 (percent identities in different regions are shown) but contains an additional segment, shown above the relevant diagram. Various regions containing different antibiotic resistance genes are found between the two copies of Tn6018 (designated RR). Diagrams are based on previously published information (109, 110) and on sequences from the following INSDC accession numbers: Tn6022, CP012952; Tn6021 and Tn6164, CP012005; Tn6022Δ, JN247441; Tn6172, KU744946; and Tn6019, FJ172370. (E) GIsul2 (15.460 kb <188> rather than the initially reported 15.456 kb <61>, apparently due khổng lồ errors in the S. flexneri sequence). ars, arenite/arsenate resistance gene; TA, toxin-antitoxin system; alpA, regulation gene. The diagram is based on information in reference 188 & the sequence from INSDC accession number KX709966. The resistance genes shown confer resistance to the following antibiotics: aadA1, streptomycin & spectinomycin; sat2, streptothricin; dfrA1dfrB3, trimethoprim; qacE, quaternary ammonium compounds; sul1 & sul2, sulfonamides; blaZ, penicillins; và strAB, streptomycin.


Tn402-lượt thích transposons.Tn402 (also called Tn5090) và other members of the Tn5053 family may carry a class 1 integron (see below) or a mer operon. They are bounded by 25-bp IR, create 5-bp TSD, và carry the tniABQR genes (Fig. 4B). TniA and TniB are related lớn TnsB and TnsC of Tn7, respectively (96), but transposition occurs via formation of a cointegrate, which also requires TniQ (also called TniD) và resolution by the TniR (TniC) resolvase acting at the adjacent res site (97). These transposons target the res site of Tn21 subfamily transposons but also resolution sites found on plinfobandarpkr.comids (98). Different Tn402-lượt thích tni regions, including hybrids, have been identified in association with class 1 integrons (99).


Tn552.Strains of S. aureus resistant khổng lồ penicillin emerged shortly after its therapeutic introduction, & Tn552-like elements are believed to be the origin of all β-lactamase genes in staphylococci (100). Tn552 itself carries genes encoding proteins related lớn TnsB (orf490) & TnsC (orf271) of Tn7 (94) as well as binL, encoding a serine recombinase, separated from the blaI, blaR1 (both encoding regulators <101>), and blaZ (β-lactamase) genes by a res site (Fig. 4C) (102). It is bounded by 116-bp IR & creates 6- or 7-bp TSD on transposition. Tn552-like transposons are sometimes found in the chromosome but are often carried by multiresistance plinfobandarpkr.comids and, like Tn5053-lượt thích elements, are usually inserted within the res site of the plinfobandarpkr.comid"s resolution system (103–106). In many cases, genetic rearrangements are evident within or in the vicinity of these elements, presumably mediated by interactions between the transposon & plinfobandarpkr.comid resolution systems and repeated transposition events inkhổng lồ them (15).


A. baumannii resistance islands.Antibiotic resistance islands (AbaR & AbGRI1) found in global clones (GC) of A. baumannii are described in this section, as they are based on transposons related lớn Tn7 & Tn402 (95, 107, 108). Like Tn7 và Tn402, these transposons may target a specific site(s), as they are generally inserted into lớn the chromosomal comM gen (encoding a protein of unknown function with an ATPase domain <109>), flanked by the same 5-bp TSD (ACCGC), but also on plinfobandarpkr.comids (110). These transposons are bounded by 25-bp IR và carry tniCAB, encoding Tn7 TnsA-, TnsB-, và TnsC-like proteins, as well as tniDE (orf2 và -3) and various downstream genes (Fig. 4D). Different resistance genes are inserted at different places in these transposon backbones.

Variants of the same basic transposon structure have been named Tn6022, Tn6022Δ (2.85-kb deletion), and Tn6021 (differences in part of tniCA) (Fig. 4D), while other variants are more complex. Tn6172 appears to have sầu evolved from a hypothetical transposon, Tn6173, by addition of other elements to give sầu Tn6174, followed by incorporation of Tn5393 & a subsequent large internal deletion (110). AbGRI1-0 consists of Tn6022 và Tn6172 flanking a region containing orf5 khổng lồ orf11int (encoding a tyrosine recombinase) & may be derived from a plinfobandarpkr.comid-borne region. AbGRI1 variants may be derived from AbGRI1-0 by addition of resistance genes or deletions due khổng lồ recombination between homologous transposon segments (110). In some A. baumannii isolates, a single Tn6022-like transposon is inserted into comM, e.g., AbaR4, which consists of Tn6022 with Tn2006 (Table 1) inserted.

The backbone of regions referred to as AbaR3-lượt thích is Tn6019, which is related khổng lồ Tn6022 but has a different, longer region downstream of tniE (Fig. 4D) (109). A composite transposon-type structure consisting of two directly oriented copies of Tn6018 flanking different resistance regions is inserted in this backbone. Components of these resistance regions are apparently derived from a plinfobandarpkr.comid related lớn R1215 from Serratia marcescens, which is not stably maintained in A. baumannii (111).

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GENE CASSETTES AND INTEGRONS

A ren cassette is a small điện thoại element (∼0.5 to lớn 1 kb) consisting of a single ren (occasionally two), typically lacking a promoter, và an attC recombination site. Gene cassettes can exist in a không lấy phí circular form but are nonreplicative sầu và are usually found inserted into an integron (Fig. 1), characterized by an intI gen, an attI recombination site, & a promoter (Pc). intI encodes an atypical site-specific tyrosine recombinase, which has an extra domain compared khổng lồ other members of this family (112), that catalyzes recombination between the attI site of the integron and the attC site of a cassette. This inserts the cassette inkhổng lồ the integron in the orientation that allows expression of the cassette-borne ren from the Pc promoter. Multiple cassettes may be inserted into lớn the same integron lớn create a cassette array (often incorrectly referred khổng lồ as a “cassette”) that may confer multiresistance (Fig. 1). Different classes of integron have sầu been defined based on the sequence of IntI (called IntI1, IntI2, IntI3, etc., with cognate attI1, attI2, & attI3 sites), with class 1 being the first reported and most comtháng in antibiotic-resistant clinical isolates. Integrons and gene cassettes have been reviewed many times (e.g., see references 112–114).


Cassette Integration and ExpressionattC sites associated with different cassettes differ in sequence, but all include two pairs of conserved 7- or 8-bp core sites at their outer ends (R″-L″ & R′-L′ <112> or 1L-2L và 2R-1R <114>). These are separated by a region of variable length that usually shows inverted repeatedness. Although the sequence similarity between different attC sites is low, single-stranded versions each size a conserved secondary structure, with two or three unpaired, protruding extrahelical bases. These are recognized by the IntI recombinase and are important in directing recombination lớn the bottom strvà, ensuring that insertion occurs in one orientation only (112).

The most efficient IntI-mediated reaction is recombination between the double-stranded attI site & the single-stranded, folded attC site to lớn insert a cassette into lớn the first position in an array. IntI-mediated excision of cassettes typically occurs between two single-stranded, folded attC sites. IntI1 activity is regulated by LexA (SOS response master regulator) binding to a site overlapping the −10 box of the intI1 promoter, repressing expression to minimize unnecessary cassette shuffling. If the SOS response is triggered, repression is lifted, giving increased integrase activity when adaptation is required (112). Formation of single-stranded DNA during conjugation also favors both attC folding & recombination, as well as triggering of the SOS response, so that incoming cassettes are more likely to be integrated (112). intI2 expression is not regulated by the SOS response (115).

In class 1 integrons, the Pc promoter lies within the int1 ren, và minor sequence variations give an inverse relationship between Pc strength & IntI1 activity (116). In some class 1 integrons, insertion of three G"s between potential −35 và −10 sites gives optimal 17-bp spacing, activating an additional promoter (P2) (116). attI2 of class 2 integrons contains two active Pc promoters, also with variants of different strengths (115). Expression of cassette genes is reduced with increasing distance from Pc và P2. Rather than being due to effects of attC secondary structure on transcription, as first proposed (117), this appears to be due to lớn effects on translation (112). This means that cassettes can be carried at less cost at the “back” of an array but still have the potential khổng lồ be shuffled lớn the “front” of the array. Some cassette genes laông xã a ribosome binding site (RBS), and ORF-11 (118) and the recently identified ORF-17 (119) in attI1 may contribute to expression if the cassette is the first in the array.


Class 1 IntegronsTn402 (Fig. 4B) seems to lớn have resulted from capture of the intI1/attI1/Pc combination, found on the chromosomes of betaproteobacteria in association with a qacE cassette (resistance lớn antiseptics), by a Tn5053 family transposon (120). In the more comtháng “clinical” or “sul1-type” class 1 integrons, part of the tni region has been replaced by the 3′ conserved segment (3′-CS) (Fig. 4B). The longest versions of the 3′-CS include the qacEΔ1 gene, derived from the qacE cassette, và sul1 (encoding resistance to lớn the early sulfonamide antibiotics), but only part of this region may be present. The term “class 1 In/Tn” has been suggested khổng lồ encompass structures with intI1/attI1/Pc và either a full or truncated tni region (21). The 25-bp IR of class 1 In/Tn are known as IRi (at the integrase end) & IRt (at the tni end), and the region from IRi to lớn the end of the attI1 site is called the 5′ conserved segment (5′-CS). While some class 1 In/Tn have sầu lost tni transposition functions, there is evidence that they can be moved, presumably by compatible Tni proteins available in the same cell (121). Class 1 In/Tn may also move sầu with an upstream ISPa17 element, which has IR related to lớn IRi & IRt (122).

The first few class 1 In/Tn identified were given In numbers, In0 (no cassettes) to In6, intended lớn specify all components, including the cassettes, the length of the 3′-CS and tni region, and any additional elements, such as IS. “In2-like” (with IS1326 plus IS1353 inserted) & “In4-like” (with a shorter 3′-CS và inverted IRt ends of tni separated by IS6100) integrons seem khổng lồ be the most comtháng (114). INTEGRALL (123; http://integrall.bio.ua.pt/) now keeps a registry of In numbers, but these really correspond only khổng lồ different cassette arrays. So-called “complex” class 1 integrons, usually with partial duplications of the 3′-CS, are created by insertion of circles containing ISCR1 và an associated resistance gene(s) by recombination into lớn the 3′-CS or an existing ISCR1 element (Fig. 2G). The boundary with position 1,313 of the 3′-CS is used to define the ter kết thúc of ISCR1, although this may not be the original end (21).


Other Integron ClassesClass 2 integrons, associated with Tn7 (Fig. 4A) và variants, often have a nonfunctional IntI2 ren due lớn an internal stop codon &, probably as a consequence, house a limited variety of cassettes (124). Class 3 integrons are more similar khổng lồ class 1 integrons & also appear to lớn be associated with Tn402-like transposons (125). Only a few examples have sầu been identified, mostly carrying cassettes that encode β-lactamases. Class 4 was previously used khổng lồ refer to lớn an integron found in the Vibrio cholerae chromosome. This và other “sedentary chromosomal integrons” (SCI; formerly called CI) may contain very large arrays of cassettes (>170 in V. cholerae), which all tkết thúc khổng lồ have sầu very similar attC sites. Although cassettes containing resistance genes trang điểm a minority of those in SCI, they appear lớn be the source of cassettes found in “mobile” integrons (112). “Mobile” integron types, now designated class 4 và class 5 integrons (112), appear to lớn be rare & have sầu not been identified in the species of interest here.


Gene Cassettes & Antibiotic ResistanceA wide variety of ren cassettes containing resistance genes (named after the ren carried) have been identified (114; see http://ứng dụng.spokade.com/rac/feature/danh sách for updated lists). The most clinically relevant are those carrying genes encoding β-lactamases or aminoglycoside-modifying enzymes. The former include metallo-β-lactamases (MBL; class B), with the VIM và IMP types being the most comtháng. Cassette-borne genes also encode class A GES enzymes, which are either ESBL or carbapenemases (with a mutation at amino acid 170), và class D OXA-10-like (which include ESBL variants) & OXA-1-like enzymes. Variants of the comtháng aacA4/aac(6′)-Ib cassette may confer resistance lớn tobramycin plus gentamicin and/or amikacin or low-level resistance to lớn fluoroquinolones due to lớn different point mutations. Different fusions that compensate for the lachồng of an RBS in this cassette also create AacA4 proteins with different N-terminal ends (114). Certain cassette arrays (e.g., ∣dfrA17aadA5∣ và ∣dfrA12gcuFaadA2∣, giving resistance khổng lồ trimethoprim <dfr> & khổng lồ streptomycin và spectinomycin <aadA>; gcu indicates a gen cassette of unknown function) are very comtháng in class 1 integrons (114).

Gene cassettes may be interrupted at a specific position in the attC site by an IS1111-attC element related khổng lồ IS4321/IS5075 (see above) or by a group II intron (114). These small, Mobile, site-specific elements encode a catalytic RNA (ribozyme) & a reverse transcriptase (126). A role for these introns in creation of gene cassettes has been suggested (127), but there are also arguments against this (112). Sometimes the partial attC site that follows an IS1111-attC element or an intron does not belong to lớn the preceding cassette, suggesting IS- or intron-mediated deletion (114, 128), which may be a way of streamlining arrays. Group II introns, named using a combination of a species abbreviation và a number (129; http://webapps2.ucalgary.ca/~groupii), are also found inserted into lớn conjugative plinfobandarpkr.comids (75), ICE, và pathogeniđô thị islands (16).


Gene Cassettes và Integrons in Gram-Positive BacteriaGene cassettes and/or integrons have been reported for a few Gram-positive bacterial species, including Corynebacterium glutamicum (on a plinfobandarpkr.comid transferable khổng lồ E. coli) (130), Staphylococcus (e.g., see reference 131), & Enterococcus (e.g., on a transferable plinfobandarpkr.comid <132>). However, many studies report only detection of intI1 by PCR, with sequencing of fragments in some cases. Searches with the class 1 integron 5′-CS or 3′-CS against sequences from Staphylococcus & Enterococcus species in GenBank (including the whole-genome shotgun contigs database ) identified very few examples, most of which were fragments & none of which provided evidence of linkage lớn the chromosome or plinfobandarpkr.comids. Thus, there is presently no conclusive evidence demonstrating the existence of integrons in these genera.


MITEs AND TIMEs

MITEs are nonautonomous (i.e., incapable of self-transposition) derivatives of bacterial IS or transposons that retain the IR but which have sầu lost central parts, including the transposase gene(s) (134). Pairs of MITEs, including Tn3-derived inverted-repeat miniature elements (TIMEs) (135), appear khổng lồ have sầu been involved in mobilization of resistance genes. For example, a composite transposon-lượt thích structure flanked by two copies of a 288-bp TIME (referred to as an integron mobilization unit ) was shown to transpose the intervening integron fragment when a Tn3 family transposase was provided (136). Two copies of the same 439-bp MITE were also identified flanking integron fragments carrying different cassette arrays in different Acinetobacter isolates (133, 137). MITEs and TIMEs may provide an explanation for movement of resistance genes if full-length IS or transposons cannot be found, but they can be difficult lớn identify.


RESISTANCE PLinfobandarpkr.comIDS

Plinfobandarpkr.comids are important vehicles for the carriage of other MGE & acquired antimicrobial resistance genes associated with these elements in both Gram-negative sầu and Gram-positive sầu genera, and they vary in kích thước from less than a kilobase to lớn several megabases (138). Their extrachromosomal existence stems from their ability khổng lồ replicate & hence be inherited in a growing population of host cells, which often requires a cadre of ren systems dedicated to lớn their efficient vertical inheritance. Conjugation or mobilization functions may also be present, allowing plinfobandarpkr.comids khổng lồ spread horizontally. Together the genes encoding these functions form a “backbone” (139) that represents a core of plinfobandarpkr.comid housekeeping functions to lớn which can be added “accessory” niche-adaptive sầu activities that might benefit the host cell (& hence the plinfobandarpkr.comid itself) in a particular environment. In resistance plinfobandarpkr.comids, these accessory regions are typically made up of one or more resistance genes & associated sản phẩm điện thoại elements of the types described above sầu (IS, Tn, and/or In). Closely related backbones may have sầu different insertions and/or resistance regions, và conversely, different backbones may house the same resistance genes & associated Smartphone elements. In this section, we first provide a summary of the main functions encoded by plinfobandarpkr.comid backbones before going on lớn describe the basic characteristics of known plinfobandarpkr.comid groups that have played a major role in the spread of antibiotic resistance in the species that are the focus of this nhận xét.


Replication Initiation và Copy Number ControlPlinfobandarpkr.comid replication initiates at a defined region, the origin (ori), triggered either by an RNA transcript or, more commonly, by the binding of an initiation protein (Rep), encoded by a rep ren on the plinfobandarpkr.comid, to proximal iterated DNA repeat sequences termed iterons. The ori và the (typically colocated) initiator gen khung the basic component of all plinfobandarpkr.comids, the minimal replibé. Thus, plinfobandarpkr.comids encode their own replication initiation but usually exploit the host"s chromosomally encoded replication machinery (helicase, primase, polymerase, etc.) for DNA synthesis itself. Interactions with & dependence on host-encoded DNA replication proteins are among muốn the factors that limit the host range of plinfobandarpkr.comids. Some plinfobandarpkr.comids are efficiently maintained only in closely related bacterial taxa and are hence termed narrow-host-range plinfobandarpkr.comids, whereas others are referred to lớn as broad-host-range plinfobandarpkr.comids because they have been found or shown to lớn replicate in quite diverse genera. Factors other than replication, particularly whether it is transmissible by conjugation or mobilization (see below), can also influence a plinfobandarpkr.comid"s host range. Conjugation can be an extraordinarily promiscuous process, capable of even transkingdom genetic exchange (140). Transfer of resistance plinfobandarpkr.comids inkhổng lồ hosts in which they cannot replicate is therefore likely khổng lồ be commonplace, with other MGE (e.g., IS, Tn, và In) providing intracellular mobility mechanisms that give sầu resistance genes an opportunity khổng lồ “escape” to other functional replicons (the chromosome or other resident plinfobandarpkr.comids). Thus, even narrow-host-range plinfobandarpkr.comids can act as suicide vectors for the horizontal spread of resistance genes into divergent hosts.

Replication initiation proteins often possess one of several ancient conserved domains (141), which define the type of replication system. Three modes of plinfobandarpkr.comid replication have been described for circular plinfobandarpkr.comids (142). Rolling circle (RC) replication is commonly used by small plinfobandarpkr.comids in Gram-positive and, less commonly, Gram-negative bacteria (143). It relies on a Rep protein nicking one DNA strvà at the double-stranded origin (dso), which provides a free 3′-OH to lớn prime leading-strand DNA synthesis that displaces the remainder of the nicked strvà. The displaced str& is then asymmetrically replicated from a second, distinct, single-stranded origin (sso). This mode of replication effectively limits plinfobandarpkr.comid form size, so RC plinfobandarpkr.comids are usually cryptic or carry only a single resistance gen.

The other modes of plinfobandarpkr.comid replication rely on initiator-mediated localized melting of double-stranded DNA (dsDNA) at the origin lớn trigger replication based on RNA primers. Theta-mode replication resembles circular chromosome replication & is widely used by small lớn very large plinfobandarpkr.comids. DNA synthesis is continuous on the leading strand & discontinuous via Okazaki fragments on the lagging str& (144). IncQ plinfobandarpkr.comids utilize the third mode of replication, termed str& displacement, where both DNA strands are replicated continuously in opposite directions from the origin (144); these plinfobandarpkr.comids are also usually small. IncQ plinfobandarpkr.comids exhibit an extremely broad host range, as they encode their own helicase và primase proteins in addition khổng lồ an initiator.

In order lớn balance the competing demands of effective sầu plinfobandarpkr.comid inheritance & metabolic impost on the host, plinfobandarpkr.comids control their copy number. The details of plinfobandarpkr.comid copy number control systems vary greatly between plinfobandarpkr.comid types, but two basic strategies have sầu been discerned. The first uses an antisense (countertranscript) RNA, constitutively expressed & hence proportional to plinfobandarpkr.comid copy number, which binds khổng lồ the complementary rep mRNA lớn repress its transcription and/or translation; in plinfobandarpkr.comids that use an RNA initiator, such as ColE1, countertranscript binding inhibits maturation of the RNA primer (145, 146). In the second mechanism, the ori sites on two plinfobandarpkr.comid molecules are “handcuffed” together by interactions between Rep proteins bound to lớn their iterons. This modulates Rep activity in response khổng lồ the concentration of iterons within the cell, which is directly proportional khổng lồ the plinfobandarpkr.comid copy number (147, 148).

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Plinfobandarpkr.comids with multiple replication regions are quite comtháng in both Gram-negative sầu & Gram-positive bacteria, suggesting that fusions/cointegrations between plinfobandarpkr.comids occur frequently. It would be expected that the rep region with the highest intrinsic copy number would initiate replication of a multireplicon plinfobandarpkr.comid. Additional replicons may unduly increase the fitness cost of a cointegrate plinfobandarpkr.comid and can be eliminated by mutations or deletions, but they may also be advantageous, e.g., being able khổng lồ use different replicons that can function in different host species may increase the plinfobandarpkr.comid host range. The presence of multiple replicons might also allow those that are not driving replication khổng lồ diverge, potentially changing incompatibility (149) (see below).