The impact of insertion sequences on bacterial genome plasticity and adaptability

Abstract

Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.

Keywords:

• Insertion sequence
• transposition
• adaptation
• transposase

Introduction

Insertion sequences (IS) are among the simplest mobile genetic elements (MGEs) and widespread in all domains of life. They can occur in a wide range of copy numbers in a particular genome and can move within a genome (between different DNA molecules or within an individual DNA molecule) or horizontally between genomes as part of other MGE vectors such as phages and plasmids. More than 4500 IS belonging to 29 families have been identified to date (Siguier et al. 2006, 2014). These small and compact elements (typically less than 3 kb), capable of independent transposition, are generally flanked by short terminal inverted repeats (IR) and carry one or two open reading frames encoding gene products essential for their mobility (Mahillon & Chandler 1998; Siguier et al. 2014). DNA cleavage (at the transposon tips) and strand transfer (of the transposon DNA into the target) necessary for transposition are catalyzed by a transposon-specific enzyme, the transposase. The IR are necessary for transposase-binding, donor DNA cleavage and strand transfer. Often, short directly repeated sequences of the target DNA, from 2 to 14 bp long and whose length is specific for a given element, are generated at the point of insertion (for reviews see Mahillon & Chandler 1998; Siguier et al. 2014). IS vary in the degree of sequence specificity in choice of their DNA target-site. Depending on the element, this can vary from highly sequence-specific to near random insertion (Craig 1997). For some elements, the potential DNA structure of the target site is also important (Bender & Kleckner 1992; Hallet et al. 1994; Hu & Derbyshire 1998).

Although IS classification is based on a variety of characteristics, the amino acid sequence similarity of their transposases is the principal parameter used for classification (Mahillon & Chandler 1998; Siguier et al. 2006, 2014). Transposase types include DDE, DEDD, HUH (Y1), Tyrosine (Y) and Serine (S). These names reflect amino acids located at their catalytic sites and are related to their transposition chemistry. IS carrying the DDE transposases represent the majority of presently identified IS elements (Siguier et al. 2015).

IS elements have been reviewed thoroughly with a focus on their classification, transposition mechanism and structure (Mahillon & Chandler 1998; Chandler & Mahillon 2002; Curcio & Derbyshire 2003; Bennett 2004; Nagy & Chandler 2004; Siguier et al. 2006, 2014, 2015). In this review, we inventory the diverse impact of IS transposition on phenotypic traits in bacteria and the putative modulation of IS transposition by environmental signals and stressors.

IS-mediated gene inactivation affecting virulence, resistance and metabolism

Perhaps the most common effect of IS transposition is gene inactivation (Figure 1). Many cases have been described illustrating the modulation of resistance, virulence and metabolic activities by IS-mediated gene inactivation.

Figure 1. Possible effects of IS transposition in the function and expression of a target gene. The IS is shown as a blue rectangle delimited by short terminal inverted repeats (IR; blue triangles) and flanked by directed repeats (DR; yellow rectangles) and the target gene as a green rectangle. mRNA transcripts are represented by straight arrows, the target gene promoter sequence is represented by a curved arrow and detailed by −35 and −10 components, and transcriptional start site (+1). For IS elements carrying a complete outward-directed promoter (P_out), the target gene transcription start is altered and denoted as < +1>.

IS involvement in changing antimicrobial/xenobiotic resistance

A variety of cases where IS elements affect antibiotic/xenobiotic resistance by directly inactivating uptake determinants have been described. Examples, which are detailed in Table 1, include increased carbapenem resistance because of IS transposition into the porin-coding oprD gene of numerous Pseudomonas aeruginosa isolates (Wolter et al. 2004; Evans & Segal 2007; Ruiz-Martinez et al. 2011; Diene et al. 2013; Al Bayssari et al. 2014; Fowler & Hanson 2014; Rojo-Bezares et al. 2014; Al-Bayssari et al. 2015) and Pseudomonas putida (Kalantar-Neyestanaki et al. 2015), the porin-coding carO gene of Acinetobacter baumannii (Mussi et al. 2005); the porin-coding ompE36 gene of Enterobacter aerogenes (Chen et al. 2008b), and the porin-coding ompK36 gene of Klebsiella pneumoniae (Lee et al. 2007). Furthermore, IS-mediated inactivation of ompK36 also resulted in increased cefoxitin resistance (Hernandez-Alles et al. 1999). In contrast, inactivating efflux can result in hypersensitivity, e.g. to solvents due to insertional inactivation of acrB, part of the AcrAB-TolC efflux pump, by IS30 in Escherichia coli (Kobayashi et al. 2001).

Table 1. IS-mediated gene inactivation affecting virulence, resistance, and metabolism.

Phenotype affected	Gene	IS element	IS family	Organism	Reference
Antibiotic/xenobiotic resistance
β-lactam^R	ampD	IS1669	IS5	P. aeruginosa	Bagge et al. (2002)
	mexR	IS21	IS2	P. aeruginosa	Boutoille et al. (2004)
Carbapenem^R	carO	ISAba125	IS30	A. baumannii	Mussi et al. (2005)
		ISAba825	IS982	A. baumannii	Mussi et al. (2005)
	ompE36	IS903-like	IS903	E. aerogenes	Chen et al. (2008b)
	ompK36	IS5	IS5	K. pneumoniae	Lee et al. (2007)
	oprD	ISPa133	IS13	P. aeruginosa	Ruiz-Martinez et al. (2011)
		ISPa1328	IS256	P. aeruginosa	Al-Bayssari et al. (2015) and Wolter et al. (2004)
		ISPa46	IS256	P. aeruginosa	Diene et al. (2013)
		ISPpu22	IS3	P. putida	Kalantar-Neyestanaki et al. (2015)
		ISPa1635	IS4	P. aeruginosa	Wolter et al. (2004)
		ISPa45	IS4	P. aeruginosa	Rojo-Bezares et al. (2014)
		ISPa26	IS5	P. aeruginosa	Evans and Segal (2007)
		ISPa8	IS5	P. aeruginosa	Fowler and Hanson (2014)
		ISPre2-like	IS5	P. aeruginosa	Al Bayssari et al. (2014)
Cefoxitin^R	ompK36	IS26	IS6	K. pneumoniae	Hernandez-Alles et al. (1999)
		IS903	IS5	K. pneumoniae	Hernandez-Alles et al. (1999)
		IS1	IS1	K. pneumoniae	Hernandez-Alles et al. (1999)
		IS5	IS5	K. pneumoniae	Hernandez-Alles et al. (1999)
Colistin^R	lpxA/lpxC	ISAba11	IS701	A. baumannii	Moffatt et al. (2011)
	mgrB	IS1F-like	IS1	K. pneumoniae	Cannatelli et al. (2014)
		ISKpn14	IS1	K. pneumoniae	Cannatelli et al. (2014), Poirel et al. (2015)
		IS5-like	IS5	K. pneumoniae	Cannatelli et al. (2014) and Poirel et al. (2015)
		ISKpn13	IS5	K. pneumoniae	Poirel et al. (2015)
		ISKpn26-like	IS5	K. oxytoca	Jayol et al. (2015)
Methicillin^R	lytH	IS1182	IS1182	S. aureus	Fujimura and Murakami (2008)
Silver^R	ompR	IS3	IS3	E. coli	Graves et al. (2015)
Solvents^S	acrB	IS30	IS30	E. coli	Kobayashi et al. (2001)
Streptomycin^R	rsmG	ISTth7	IS5	T. thermophilus	Gregory and Dahlberg (2008)
Trimethoprim^R	thyA	ISDra2	IS200/IS605	D. radiodurans	Mennecier et al. (2006)
Toluene^R	srpS	ISS12	IS21	P. putida	Sun and Dennis (2009) and Wery et al. (2001)
Zinc^R	cnrYX	IS1086	IS30	C. metallidurans	Vandecraen et al. (2016)
		IS1087B	IS3	C. metallidurans	Collard et al. (1993), Tibazarwa et al. (2000), Vandecraen et al. (2016)
		IS1088	IS30	C. metallidurans	Vandecraen et al. (2016)
		IS1090	IS256	C. metallidurans	Vandecraen et al. (2016)
		ISRme3	IS3	C. metallidurans	Vandecraen et al. (2016)
		ISRme5	IS481	C. metallidurans	Vandecraen et al. (2016)
		ISRme15	IS3	C. metallidurans	Vandecraen et al. (2016)
Virulence
Biofilm formation	icaC	IS256	IS256	S. aureus	Kiem et al. (2004)
	icaC	IS256	IS256	S. epidermidis	Ziebuhr et al. (1999)
	rsbU	IS256	IS256	S. epidermidis	Conlon et al. (2004)
	sarA	IS256	IS256	S. epidermidis	Conlon et al. (2004)
Capsule biosynthesis	siaA	IS1301	IS5	N. meningitidis	Hammerschmidt et al. (1996)
Capsule O-acetylation	oatWY	IS1301	IS5	N. meningitidis	Claus et al. (2003)
Cell adhesion	rickA	ISRpe1	IS481	R. peacockii	Simser et al. (2005)
Cytotoxin expression	rot	IS256	IS256	S. aureus	Benson et al. (2014)
Eps biosynthesis	gumM	ISXo2	IS1595	X. oryzae	Rajeshwari and Sonti (2000)
		ISXo1	IS5	X. oryzae	Rajeshwari and Sonti (2000)
Gelatinase (biofilm)	fsrC	IS256	IS256	E. faecalis	Perez et al. (2015)
Pneumolysin	ply	IS1515	IS5	S. pneumoniae	Garnier et al. (2007)
Porin (antigen)	porA	IS1301	IS5	N. meningitidis	Newcombe et al. (1998)
Protease	prtP	IS195	IS982	P. gingivalis	Lewis and Macrina (1998)
Metabolism
Cellulose production	cel1	IS1031	IS5	A. xylinum	Coucheron (1991)
Fe^2+ oxidation	resB	ISTfe1	ISL3	T. ferrooxidans	Cabrejos et al. (1999)
Glucose utilization	rpoS	IS1	IS1	E. coli	Gaffe et al. (2011)
Indole production	tna operon	IS1	IS1	Shigella	Rezwan et al. (2004)
Lactocin S production	las operon	IS1163	IS3	L. sakei	Skaugen and Nes (1994)
		IS1520	IS3	L. sakei	Skaugen and Nes (2000)
Lactose utilization	lac operon	ISL4	IS110	L. delbrueckii	Lapierre et al. (2002)
Lipid A biosynthesis	lpxA/lpxC	ISAba11	IS701	A. baumannii	Moffatt et al. (2011)
Naphthalene dissimilation	nahH	ISPst9	ISL3	P. stutzeri	Christie-Oleza et al. (2008)
Poly-γ-glutamate production	comP	IS4Bsu1	IS4	B. subtilis	Nagai et al. (2000)
Protocatechuate catabolism	pcaH pobR	IS1236	IS3	A. calcoaceticus	Gerischer et al. (1996)
o-, m-, p-xylene catabolism	xyl/tou	ISPst2	ISL3	P. stutzeri	Bolognese et al. (1999)

In addition to IS influence on transport, antibiotic/xenobotic processing or target sites can also be affected by IS transposition (Table 1). For instance, IS-mediated inactivation (ISAba11, IS701 family) of lipid A biosynthesis genes in Acinetobacter baumannii results in high-level colistin resistance because of complete loss of lipopolysaccharide production, the initial binding target of colistin (Moffatt et al. 2011). Insertional inactivation of the thymidylate synthase-encoding thyA gene in Deinococcus radiodurans, the 16S rRNA methyltransferase-encoding rsmG gene in Thermus thermophilus HB8 and the lytH gene involved in the biogenesis of the bacterial cell and the membrane protein-coding tcaA gene in Staphylococcus aureus results in increased trimethoprim (Mennecier et al. 2006), streptomycin (Gregory & Dahlberg 2008), methicillin (Fujimura & Murakami 2008) and glycopeptide resistance (Maki et al. 2004), respectively.

Next to a direct effect, IS elements can also affect regulatory pathways, thereby modulating expression of determinants leading to resistance (Table 1). IS-mediated derepression of efflux pumps has been observed in P. aeruginosa, by IS21 (IS21 family) insertion into the mexR repressor resulting in increased transcription of the mexAB-oprM efflux pump and increased β-lactam resistance (Boutoille et al. 2004); in P. putida, by ISS12 (IS21 family) insertion into the srpS repressor resulting in increased expression of the srpAC efflux pump and constitutive tolerance to toxic organic solvents (Wery et al. 2001; Sun & Dennis 2009); and in Cupriavidus metallidurans AE126, by inactivation of cnrY or cnrX, coding, respectively, for a membrane-bound anti-sigma factor (CnrY) and a sensor protein (CnrX), resulting in release of the extracytoplasmic function family (ECF) sigma factor CnrH that directs transcription of the cnrCBAT efflux pump and increased (non-specific) Zn²⁺ efflux (Collard et al. 1993; Grass et al. 2000; Tibazarwa et al. 2000; Vandecraen et al. 2016). Other examples are the insertional inactivation of the regulatory mgrB gene in K. pneumoniae and K. oxytoca resulting in colistin resistance (Cannatelli et al. 2014; Jayol et al. 2015; Poirel et al. 2015), of the ompR regulator in E. coli resulting in decreased porin expression and increased silver resistance (Graves et al. 2015; Randall et al. 2015) and of the amidase-coding ampD gene resulting in high-level β-lactam resistance (Bagge et al. 2002).

IS involvement in modulating virulence

The impact of IS transposition on virulence via modulation of biofilm formation and the production of extracellular polymeric substances has been described for different bacteria (Table 1). In Staphylococcus epidermidis and S. aureus, reversible IS256 transposition into the icaADBC locus, encoding the polysaccharide intercellular adhesin (a poly-β(1–6)-N-acetylglucosamine), results in phenotypic biofilm variation (Ziebuhr et al. 1999; Arciola et al. 2004; Kiem et al. 2004). Importantly, in S. epidermidis, the presence of the ica locus together with phenotypic evidence of biofilm production is used as a virulence marker (Arciola et al. 2015). IS256 has also been found to indirectly affect ica expression by inactivating rsbU (a positive regulator of the global stress response sigma factor σ^B) or sarA (encoding the staphylococcal accessory regulator) (Conlon et al. 2004). In Enterococcus faecalis, IS256 transposition mediated inactivation of gelatinase activity, which is essential in the initial steps of biofilm formation (Perez et al. 2015). Loss of extracellular polysaccharide production was also observed in Xanthomonas oryzaei via IS-mediated inactivation of the gumM gene, which is part of the gum cluster involved in the biosynthesis of extracellular polysaccharides (Rajeshwari & Sonti 2000). In Neisseria meningitidis, capsule expression and endogenous lipooligosaccharide sialylation, as well as an important antigen are affected by IS1301 (IS481 family) inactivation of essential biosynthesis genes such as siaA (sialic acid biosynthesis) and oatWY (O-acetyltransferase), and porA (porin), respectively (Hammerschmidt et al. 1996; Newcombe et al. 1998; Claus et al. 2003). In Rickettsia peacockii, ISRpe1 (IS481 family) transposition mediated the disruption of two genes involved in actin-tail polymerization and cell adhesion (Simser et al. 2005). In Ralstonia solanacearum, ISRso4 (IS5 family) transposition mediated the inactivation of the global regulatory gene phcA, which in turn modulated expression of extracellular polysaccharides (Jeong & Timmis 2000).

Next to biofilm formation and the production of extracellular polymeric substances, IS transposition also affected other virulence factors, including the above described phcA inactivation as the global regulator PhcA also modulates other virulence factors such as β-1,4-endoglucanase, pectin methylesterase, endopolygalacturonase A and motility (Clough et al. 1997; Jeong & Timmis 2000). Other examples include IS195 (IS982 family)-mediated inactivation of the protease-coding prtP gene in Porphyromonas gingivalis (Lewis & Macrina 1998), the IS1515 (IS5 family)-mediated inactivation of the ply gene coding for the virulence factor pneumolysin in a clinical isolate of Streptococcus pneumoniae (Garnier et al. 2007) and IS256-mediated modulation of S. aureus virulence by insertion into the rot (repressor of toxins) promoter, which drives expression of Rot, resulting in the derepression of cytotoxin expression and increased virulence (Benson et al. 2014).

IS involvement in modulating metabolic activities

Other cases were described that illustrate the modulation of metabolic activities such as carbon source utilization, electron donor usage, and production of primary and secondary metabolites by IS-mediated gene inactivation (Table 1). For instance, the ability to grow on different aromatic compounds can be modulated by insertion and precise excision of ISL3-family ISs in the o-, m- and p-xylene, and naphthalene catabolic pathway of Pseudomonas stutzeri OX1 (ISPst2) (Bolognese et al. 1999) and AN10 (ISPst9) (Christie-Oleza et al. 2008), respectively. Other examples are inactivation of protocatechuate catabolism by IS1236 (IS3 family) in Acinetobacter calcoaceticus (Gerischer et al. 1996) and constitutive expression of the lac genes in Lactococcus delbrueckii (Lapierre et al. 2002). In E. coli, IS1 insertion within rpoS, the master regulator of gene expression under stress conditions, confers higher fitness in glucose- and phosphate-limited chemostats thanks to loss of RpoS function mutation alleviating competition with other sigma factors, such as RpoD, presumably improving nutrient scavenging by increasing RpoD-dependent gene expression (Ferenci 2003; Gaffe et al. 2011). The ability of Thiobacillus ferrooxidans to oxidize Fe²⁺ (electron donor) can be affected by ISTfe1 (ISL3 family) insertion into the resB gene (Cabrejos et al. 1999).

IS-mediated inactivation of primary and secondary metabolite production has been observed in Acinetobacter baumannii for lipid A biosynthesis (Moffatt et al. 2011) and in Lactobacillus sakei for lactocin S production (Skaugen & Nes 1994, 2000), and in Bacillus subtilis for poly-γ-glutamate production (Nagai et al. 2000) and in A. xylinum for cellulose production (Coucheron 1991), respectively. Noteworthy, the production of indole, often used as a biochemical characteristic for species differentiation, is inactivated in many Shigella strains via IS transposition into the tna operon (Rezwan et al. 2004).

IS-mediated modulation of expression of neighbouring genes

IS transposition into non-coding regions can lead to altered expression of adjacent genes (Figure 1). IS elements can provide promoter sequences, which are either entirely included within the IS (Table 2) or created by the correct placement of an outward-directed -35 promoter component (Table 3), often present within the IS ends, relative to a suitable -10 box in the flanking genomic region to form a hybrid promoter. These IS-provided promoter sequences can be oriented towards the right or the left IR (defined relative to the transcriptional orientation of the transposase gene).

Table 2. Modulation of expression of neighbouring genes by IS elements carrying a complete outward-directed promoter.

IS family	IS element	IRx^a	Phenotype affected	Gene	Organism	Reference
IS1380	ISEcp1	R	β-Lactam^R	blaCTX-M-15	Enterobacteriaceae	Karim et al. (2001)
				blaCTX-M-17	K. pneumoniae	Cao et al. (2002)
				blaCTX-M-19	K. pneumoniae	Poirel et al. (2003)
				blaCTX-M-2	K. ascorbata	Lartigue et al. (2006)
			Aminoglycoside^R	rmtC	E. coli	Wachino et al. (2006)
	IS1170	L	5-Nitroimidazole^R	nimC	Bacteroides	Soki et al. (2006) and Trinh et al. (1995)
	IS1188	L	Carbapenem^R	cfiA	B. fragilis	Podglajen et al. (2001)
	IS612	L		cfiA	B. fragilis	Kato et al. (2003)
	IS613	L		cfiA	B. fragilis	Kato et al. (2003)
	IS614	L		cfiA	B. fragilis	Kato et al. (2003)
	IS942	L		cfiA	B. fragilis	Podglajen et al. (2001)
IS3	ISPst4	R	Effective host range expression	oriV	P. stutzeri	Coleman et al. (2014)
	IS3	R	Arginine biosynthesis	argE	E. coli	Charlier et al. (1982)
		R	Citrate utilization	citT		Blount et al. (2012)
		R	Acetate utilization	acs		Treves et al. (1998)
	IS407	R	Lactose utilization	lac	B. cepacia	Wood et al. (1991)
	IS6110	R	Genes activated during infection	Rv2280/Rv1468c	M. tuberculosis	Safi et al. (2004)
IS4	ISAba1	L	Ceftazidime^R	blaampC	A. baumannii	Corvec et al. (2003), Heritier et al. (2006) and Segal et al. (2004)
			Carbapenem^R	blaOXA-51-like	A. baumannii	Turton et al. (2006)
				blaOXA-23	A. baumannii	Turton et al. (2006)
	ISPa12	L	Ceftazidime^R	blaPER-1	A. baumannii	Poirel et al. (2005a)
					P. aeruginosa	Poirel et al. (2005a)
					S. enterica	Poirel et al. (2005a)
	IS10	L	Fluoroquinolone^R	acrEF pump	S. enterica	Olliver et al. (2005)
			Histidine biosynthesis	hisD	S. enterica	Wang and Roth (1988)
	IS1999	L	Ceftazidime^R	blaVEB-1	P. aeruginosa	Aubert et al. (2003, 2006)
				blaOXA-48	K. pneumoniae	Aubert et al. (2003, 2006)
	IS942	L	β -lactam^R	ccrA	B. fragilis	Rasmussen and Kovacs (1991)
IS481	ISRme5	R	Zinc^R	cnrCBAT	C. metallidurans	Vandecraen et al. (2016)
	IS481a	L	Catalase activity	katA	B. pertussis	DeShazer et al. (1994)
			Type III cytotoxic effector protein	bteA	B. pertussis	Han et al. (2011)
IS5	ISBf6	L	5-nitroimidazole^R	nimE	Bacteroides	Soki et al. (2006)
	IS1168	R		nimA nimB	Bacteroides	Haggoud et al. (1994), Soki et al. (2006)
	IS1169	R		nimD	B. fragilis	Trinh et al. (1995)
	IS1186	R	Carbapenem^R	cfiA	B. fragilis	Podglajen et al. (1994, 1995)
IS6	ISS1	L	Restriction-Modification system	abi-416	Lactobacillus lactis	Cluzel et al. (1991)
	IS257	L	Tetracycline^R	tetA(K)	S. aureus	Simpson et al. (2000)
IS982	IS1187	L	Carbapenem^R	cfiA	B. fragilis	Podglajen et al. (2001)
ISL3	IS1411	L	Phenol degradation	pheBA	P. putida	Kallastu et al. (1998)

^a Orientation of the outward-direct promoter oriented, with R (IRR) right inverted repeat and L (IRL) left inverted repeat.

Phenotype affected, R: resistance.

Table 3. Modulation of expression of neighbouring genes by IS elements carrying an outward-directed -35 promoter component.

IS family	IS element	Phenotype affected	Gene	Organism	Reference
IS1	IS1	Erythromycin^R		E. coli	Barany et al. (1982)
		Extended-spectrum β-lactamase	blaTEM-1		Prentki et al. (1986)
	IS1	Fluoroquinolone^R	acrEF pump	S. enterica	Olliver et al. (2005)
	IS1-like	Extended-spectrum β-lactamase	blaT-6	E. coli	Goussard et al. (1991)
IS21	ISBf1	High-level β-lactam ^R	cepA	B. fragilis	Rogers et al. (1994)
	ISKpn7	Extended-spectrum β-lactamase	blaKPC	K. pneumoniae	Naas et al. (2012)
IS256	IS1490	2,4,5-trichlorophenoxyacetic acid catabolism	tftA	B. cepacia	Hubner and Hendrickson (1997)
	IS256	Methicillin^R	llm	S. aureus	Maki and Murakami (1997)
			mecA	S. sciuri	Couto et al. (2003)
	IS406	Lactose utilization	lac	B. cepacia	Wood et al. (1991)
IS3	IS2	Ampicillin^R	ampC	E. coli	Jaurin and Normark (1983)
		Fluoroquinolone^R	acrEF pump	E. coli	Jellen-Ritter and Kern (2001)
		Erythromycin^R			Barany et al. (1982)
		Galactose utilization	gal	E. coli	Saedler et al. (1974)
		Arginine biosynthesis	argE		Nevers and Saedler (1977) Glansdorff et al. (1981)
			xis/int	Bacteriophage λ	Mosharrafa et al. (1976), Pilacinski et al. (1977)
	IS981	Lactate production	ldhB	L. lactis	Bongers et al. (2003)
IS30	IS18	Aminoglycoside^R	aac(6’)-lj	Acinetobacter	Rudant et al. (1998)
	IS30	Spectinomycin^S	cmr/mdfA	E. coli	Bohn and Bouloc (1998)
		Galactose utilization	galK	E. coli	Dalrymple (1987), Dalrymple and Arber (1985)
IS6	IS1008	Carbapenem^R	blaOXA-58	A. baumannii	Chen et al. (2008a)
	IS140	Gentamycin^R	aac(3)-III and IV		Brau et al. (1984b)
	IS257	Tetracycline^R	tetA(K)	S. aureus	Simpson et al. (2000)
		Trimethoprim^R	thyE-dfrA-orf140	S. aureus	Leelaporn et al. (1994)
	IS26	Aminoglycoside^R	aphA7, blaSHV-2a	K. pneumoniae	Jones et al. (2005) and Lee et al. (1990)
		SHV-type extended-spectrum β-lactamase	blaSHV-2a	P. aeruginosa	Naas et al. (1999)
IS5	ISVa1	Ferric anguibactin transport and biosynthesis	fatDCBAangRT	V. anguilarum	Di Lorenzo et al. (2003), Tolmasky and Crosa (1995)
IS982	IS982	Citrate transport	citQRP	L. lactis	Lopez de Felipe et al. (1996)

R : resistance;

S : sensitivity.

IS elements carrying a complete outward-directed promoter

IS1380 family

ISEcp1-like elements are one of the most important genetic elements associated with increased expression of bla_CTX-M genes that code for expanded-spectrum β-lactamases of the CTX-M type (Lartigue et al. 2004). They are often located at the 5′ ends of β-lactamase genes and may provide both −35 and −10 promoter sequences, located within the IS proximal to its right IR (IRR), for expression. Examples of genes which are activated in this way are: bla_CTX-M-2, blaCTX_-M-15, bla_CTX-M-17 and bla_CTX-M-19 carried by plasmids from Kluyvera ascorbata (Lartigue et al. 2006), enterobacterial isolates (Karim et al. 2001), K. pneumoniae BM4493 (Cao et al. 2002) and a clinical K. pneumoniae isolate (Poirel et al. 2003), respectively. In addition to driving β-lactamase gene expression, an ISEcp1-like element was also found to drive expression of rmtC, a 16S rRNA methyltransferase gene located on plasmid pARS68 from the aminoglycoside-resistant clinical Proteus mirabilis strain ARS68 (Wachino et al. 2006). RmtC methylates 16S rRNA, which decreases the affinity of aminoglycosides for their target, and thus prevent the antibiotic interfering with mRNA decoding. IS1380-family members in Bacteroides include multiple IS elements with putative internal promoter sequences, which are, however, oriented towards their IRL. These are presumed to promote expression of β-lactam and 5-nitroimidazole resistance by providing a promoter sequence for the silent carbapenemase gene cfiA (Podglajen et al. 2001; Kato et al. 2003; Soki et al. 2006) and nimC gene (Haggoud et al. 1994; Soki et al. 2006), respectively. Molecular data (e.g. transcription start mapping) evidencing a mobile promoter has been provided for ISEcp1, IS1170, IS1188 and IS942 (Trinh et al. 1995; Podglajen et al. 2001; Poirel et al. 2003; Kamruzzaman et al. 2015).

IS3 family

In E. coli, arginine biosynthesis and utilization of citrate and acetate were activated by an outward-directed promoter located on IS3 (Charlier et al. 1982; Treves et al. 1998; Blount et al. 2012). In Burkholderia cepacia isolates, lactose could be utilized thanks to IS407-promoted expression of the otherwise silent lac operon (Wood et al. 1991). In Mycobacterium tuberculosis, some evidence has been presented that IS6110 directed the expression of multiple genes (Safi et al. 2004). In ampicillin-resistant transformants of P. stutzeri, ISPst4 inserted in pUS23 and presumably promoted transcription of the ori region resulting in robust replication in P. stutzeri (Coleman et al. 2014). All outward-directed promoters from IS3-family members are oriented towards their IRR and molecular data (e.g. transcription start mapping, deletion and reporter strategies) evidencing a mobile promoter has been provided for IS3, IS6110 and ISPst4 (Charlier et al. 1982; Safi et al. 2004; Coleman et al. 2014).

IS4 family

Control of antibiotic resistance has been shown for a number of IS4-family members. In A. baumannii isolates, insertion of ISAba1 (bla_ampC and bla_OXA-51-like genes) resulted in ceftazidime- and carbapenem-resistant isolates, respectively (Corvec et al. 2003; Segal et al. 2004; Poirel et al. 2005c; Heritier et al. 2006; Turton et al. 2006). ISPa12 induced expression of extended-spectrum β-lactamase (ESBL) PER-1 in a series of Gram-negative isolates, including Salmonella enterica, P. aeruginosa, Providencia stuartii and A. baumannii, resulting in resistance to penicillins, cefotaxime, ceftibuten, ceftazidime and aztreonam (Poirel et al. 2005a). IS1999-mediated constitutive expression of alternative ESBL VEB-1 and OXA-48 genes resulted in antibiotic-resistant clinical isolates of P. aeruginosa and K. pneumoniae, respectively (Aubert et al. 2003, 2006). IS4-family members in Bacteroides spp. such as B. fragilis promoted expression of β-lactam and 5-nitroimidazole resistance by providing a promoter sequence for the metallo-β-lactamase gene ccrA (Rasmussen & Kovacs 1991) and nimC genes (Trinh et al. 1995), respectively. In acrB-inactivated S. enterica serovar Typhimurium DT204 mutants, missing the functional AcrAB RND efflux pump, high-level resistance to fluoroquinolone can be restored by IS10-mediated increased expression (via pOUT) of the alternative multidrug efflux AcrEF transporter (Olliver et al. 2005). In addition to resistance, IS10 transposition has also been found to activate histidine biosynthesis (Wang & Roth 1988). All outward-directed promoters from IS4-family members are oriented towards their IRL and molecular data (e.g. transcription start mapping) evidencing a mobile promoter has been provided for ISAba1, ISPa12, IS1999, IS10 and IS942 (Simons et al. 1983; Podglajen et al. 2001; Aubert et al. 2003; Segal et al. 2004; ; Poirel et al. 2005a Kamruzzaman et al. 2015).

IS5 family

In Bacteroides spp., expression of carbapenem and 5-nitroimidazole resistance determinants (Haggoud et al. 1994; Podglajen et al. 1994, 1995) can be promoted via an outward-directed promoter oriented by IS1186 (Podglajen et al. 1994, 1995) and IS1168 (Haggoud et al. 1994; Soki et al. 2006), and IS1169 (Trinh et al. 1995) and ISBf6 (Soki et al. 2006), respectively. All outward-directed promoters from IS5-family members, except ISBf6, are oriented towards their IRR and transcription start mapping evidencing a mobile promoter has been provided for IS1168 and IS1186 (Podglajen et al. 1994, 2001).

Other families

IS elements from other families have also been found to carry complete outward-directed promoters. IS481 family members include IS481a in Bordetella pertussis, driving the expression of katA and bteA coding, respectively, for a catalase and a type III secretion system effector (IS481 family) (DeShazer et al. 1994; Han et al. 2011), and ISRme5 in C. metallidurans, driving the expression of the cnrCBAT efflux pump (Vandecraen et al. 2016). IS6 family members include Iso-ISS1 in L. lactis supsp. lactis, driving expression of a restriction modification system responsible for phage resistance (Cluzel et al. 1991), and IS257 in S. aureus, resulting in tetracycline resistance (Simpson et al. 2000). Singular examples of other IS families include IS1187 (IS982 family) in B. fragilis, driving the expression of carbapenem resistance, and IS1411 (ISL3 family) in P. putida, directing the expression of the phenol degradation genes pheBA (Kallastu et al. 1998).

All these outward-directed promoters are oriented towards their IRL, except ISRme5, and molecular data (e.g. transcription start mapping) evidencing a mobile promoter has been provided for all (Cluzel et al. 1991; DeShazer et al. 1994; Kallastu et al. 1998; Simpson et al. 2000; Podglajen et al. 2001; Han et al. 2011; Vandecraen et al. 2016).

Hybrid promoters

In addition to IS elements that provide entire promoter sequences, IS elements can also activate downstream genes via the formation of a hybrid promoter in which the -35 promoter sequence at the element's border is properly positioned close to the -10 promoter sequence of the flanking gene. It is noteworthy that some IS elements possess, in addition to a -35 hexamer at the right end, an inward directed -10 hexamer in their IRL. These elements all appear to transpose through a circular dsDNA intermediate and it has been demonstrated that they generate relatively strong promoters if two ends of such an element are juxtaposed, by formation of head-to-tail dimers or by circularization. This arrangement can lead to high transposase expression and consequent increased transposition activity (Mahillon & Chandler 1998; Prudhomme et al. 2002; Szeverenyi et al. 2003).

IS1 family

Insertion of IS1 into the blaT-1 promoter of plasmid pBR322 resulted in an active, although less efficient, hybrid promoter (Prentki et al. 1986), transposition in front of the emr gene resulted in enhanced erythromycin resistance (Barany et al. 1982) and fluoroquinolone resistance was acquired via activation of the acrEF efflux pump in S. typhimurium (Olliver et al. 2005). Similarly, in E. coli strain HB251, an IS1-like element upstream of blaT-6, coding for ESBL TEM-6, resulted in the creation of a hybrid promoter in which the -10 region was that of the TEM-6 promoter whereas the -35 canonical sequence TTGACA was provided by the right end of the IS1-like element (Goussard et al. 1991). In this case, the newly constructed promoter was 10 times more active and conferred higher levels of antibiotic resistance (Goussard et al. 1991).

IS256 family

In S. aureus and S. sciuri, IS256 transposition into an upstream region of, respectively, the llm and mecA gene resulted in a hybrid promoter that enhanced their transcription and elevated methicillin resistance (Maki & Murakami 1997; Couto et al. 2003). In Burkholderia cepacia isolates, the ability to utilize lactose and the herbicide 2,4,5-trichlorophenoxyacetic acid as a sole carbon source was promoted by IS406 and IS1490, respectively. IS406 promoted expression of the otherwise silent lac operon by formation of a hybrid promoter comprising a -35 promoter box at the right IR of IS406 and a duplicated -10 box from the native lac promoter (Wood et al. 1991). Whereas insertion of IS1490 110 bp upstream of tftA, coding for 2,4,5-trichlorophenoxyacetic acid oxygenase, created a fusion promoter responsible for high-level constitutive transcription (Hubner & Hendrickson 1997).

IS3 family

Insertion of IS2 in front of the emr gene resulted in enhanced erythromycin resistance (Barany et al. 1982) and fluoroquinolone resistance was acquired via activation of the acrEF efflux pump in E. coli. Likewise, insertion of IS2 into the E. coli ampC promoter resulted in a hybrid promoter with a 20-fold increased strength compared with the original ampC promoter (Jaurin & Normark 1983). Besides the formation of hybrid promoters involved in antibiotic resistance, IS2 was also found to modulate expression of the gal and arg operons (Saedler et al. 1974; Nevers & Saedler 1977; Glansdorff et al. 1981), and the xis and int genes of bacteriophage lambda (Mosharrafa et al. 1976; Pilacinski et al. 1977).

Another IS3-family member was described in L. lactis NZ9010, which displays a retarded growth rate under anaerobic conditions due to replacement of the las operon-encoded ldh genes with an erythromycin resistance gene cassette. Normal growth rate and the ability to produce lactate could be recovered by “unsilencing” ldhB with site-specific and directional IS981 insertions. Characterization of the upstream region of the ldhB gene in ten independently isolated lactate-producing derivatives of L. lactis NZ9010 indicated that IS981-mediated lactate production represents the predominant mechanism of the observed recovery to produce lactate (Bongers et al. 2003).

IS30 family

In E. coli, IS30 transposition upstream of cmr/mdfA in the cmlA1 strain creates a hybrid promoter region increasing Cmr/MdfA efflux pump expression leading to exclusion of the synthethic, non-metabolizable β-galactoside isopropyl-beta-d-thiogalactopyranoside (IPTG) and protects the cell against effects of plasmid-encoded IPTG-induced lethal genes.

Although overexpression of the efflux pump caused spectinomycin hypersensitivity, little is known concerning spectinomycin uptake. Possibly, CmlA1 overproduction may contribute to the increase of the intracellular concentration of spectinomycin (Bohn & Bouloc 1998). The latter exemplifies that, instead of inducing antibiotic resistance, IS-mediated expression of multidrug efflux pumps can also promote hypersensitivity to other antibiotics through an increased uptake.

The potential -35 transcriptional promoter at the right end of IS30, which after transposition may create a putative σ⁷⁰ promoter, was already detected upstream of galK (Dalrymple & Arber 1985; Dalrymple 1987). In Acinetobacter, aminoglycoside resistance could be promoted by IS18-mediated expression of the otherwise silent aac(6’)-lj gene (Rudant et al. 1998).

IS6 family

IS140 mediated the expression of gentamycin resistance in R-plasmids encoding genes for aminoglycoside acetyltransferase enzymes (Brau et al. 1984a). The latter is another example of the involvement of IS elements in plasmids conferring antibiotic resistance to their host. Expression of ESBL SHV-2a in P. aeruginosa and of both aphA7, mediating resistance to aminoglycosides, and ESBL SHV-2a in K. pneumoniae clinical isolates was directed by a hybrid promoter made from the IRL of IS26 and the -10 region from the native promoter (Lee et al. 1990; Naas et al. 1999; Jones et al. 2005). In S. aureus, IS257 upstream the thyE-dfrA genes encoded on plasmids pSK1 and pSK818, and the tetA(K) gene encoded by a cointegrated copy of a pT181-like plasmid resulted in a hybrid promoter that induced high-level trimethoprim and tetracycline resistance, respectively (Leelaporn et al. 1994; Simpson et al. 2000). Interestingly, trimethoprim resistance could be abolished due to deletions just next to the IS257 copy as seen in other plasmids that induced only a low-level trimethoprim resistance, indicating that an optimal spacing between the outward-directed -35 promoter hexamer and the resident -10 hexamer is necessary for high-level resistance to trimethoprim (Leelaporn et al. 1994). Increased carbapenem resistance was described in A. baumannii via the formation of a hybrid promoter upstream the bla_OXA-58 gene. Similarly, the -35 sequence was provided by the IRR of IS1008. However, the -10 sequence was not provided by the promoter of the bla_OXA-58 gene but by an upstream ISAba3-like element in which IS1008 was transposed (Chen et al. 2008a).

Other families

IS elements from other families have also been found to form hybrid promoters. IS21 family members include ISBf1 in Bacteriodes fragilis, promoting high-level β-lactam resistance (Rogers et al. 1994), and ISKpn7 in K. pneumoniae, contributing to extended-spectrum β-lactamase expression (Naas et al. 2012). Singular examples of other IS families include IS982 (IS982 family) on the lactococcal plasmid pCIT264 from L. lactis biovar diacetylactis, increasing the expression of the citrate utilization citQRP cluster (Lopez de Felipe et al. 1996), and ISVa1 (IS5 family) on the virulence plasmid pJM1from the fish pathogen Vibrio anguilarum, increasing the expression of the fatDCBAangRT operon, thereby causing septicemic disease in salmonids and other fishes (Tolmasky & Crosa 1995; Di Lorenzo et al. 2003).

Co-transcription

Modulation of neighbouring genes is also possible via read-through transcription from the transposase promoter (Figure 1). For instance, in the cyanobacterium Tolypothrix sp. PCC 7601, the gltX gene, coding for a glutamyl-tRNA synthetase, is located downstream of and co-transcribed with ISTosp1 (IS200/IS605 family) (Luque et al. 2006). For Francisella tularensis, the causative agent of tularemia, spermine-induced expression of the ISFtu1 (IS630 family) and ISFtu2 (IS5 family) transposase genes also resulted in increased expression of their adjacent genes in broth culture and in macrophages (Carlson et al. 2009). In the plasmid-encoded LlaKR21 restriction-modification (R-M) system of L. lactis supsp. lactis biovar diacetylactis KR2, an IS982 element (IS982 family) inserted in the intergenic region between the endonuclease llaKR2IR gene and the methyltransferase llaKR2IM gene. The latter was co-directionally transcribed with the IS982 transposase gene. The R-M system may originally have been more efficient at restricting phage proliferation when the IS element was absent. However, it is credible that if methylation activity was weakened in the presence of an active endonuclease also autodigestion of the hosts genomic DNA will occur. This stress imposed on the cell by the aberrant restriction-modification activity may have provoked the insertion of IS982, permitting enhancement of modification activity (Twomey et al. 1998).

Other cases

Topology

Insertion of a specific IS element can result in topological changes in DNA and transcriptional activation of adjacent genes. In E. coli, the normally cryptic, silent β-glucoside (bgl) catabolic operon is activated by the insertion of either IS5 or IS1 upstream or downstream of the promoter, probably disrupting the flanking silencer element sequences (Reynolds et al. 1981; Schnetz & Rak 1992). Moreover, it was shown for IS5 that activation was abolished by internal deletions but again restored by providing an IS5-encoded gene product in trans, necessary for transposition, indicating that interaction of IS5 with its ends in some way changes the topology of the bgl promoter region (Schnetz & Rak 1992). A second example is the enhanced motility of poorly motile E. coli after extended incubation in motility agar via IS1 or IS5 insertion at various locations upstream of the flhDC promoter, without altering the transcriptional start site. It was postulated that IS element insertion might activate transcription of the flhDC operon by reducing transcriptional repression (Barker et al. 2004). The role of regional topology in the regulation of flhDC expression was further confirmed by the fact that insertion of IS elements or a kanamycin gene far upstream the promoter or repressor binding sites also stimulated motility (Fahrner & Berg 2015). Another example is the mutation of E. coli crp (cAMP receptor protein) deletion strains, which carry a silent glpFK operon, to utilize glycerol. Interestingly, all mutants contained an IS5 element upstream of the glpFK promoter, always in the same position and orientation (Zhang & Saier 2009a). Insertion of IS5 activated the otherwise cryptic expression of the glpFK operon. The activation was caused by a short region at the 3′ end of IS5 that harbours a permanent bend and an overlapping nucleoid protein binding site for the integration host factor (IHF), both of which are required for maximal gene expression. Expression was still activated when a 10 bp fragment was inserted between IS5 and the glpFK promoter, but was completely abolished by insertion of a 5 bp fragment. Thus, positioning of the upstream IS5 at the correct phase angle relative to the promoter sequence of glpFK is essential for full promoter activity, emphasizing the important role of DNA topology (Zhang & Saier 2009b).

Furthermore, the lacZYA operon was also subject to IS5-mediated activation in the absence of CRP (Zhang & Saier 2009b). Finally, IS5 has also been shown to activate the cryptic ade gene encoding an adenine deaminase catalyzing deamination of adenine to hypoxanthine (Petersen et al. 2002). The activation was proposed to be caused by a relief of HNS-mediated normal repression by IS5 insertion (Petersen et al. 2002; Barker et al. 2004).

Attenuation

Several IS elements carry mobile attenuators at their 3′ end providing transcription terminators that may be adapted as 5′ regulators leading to repression of downstream genes (Naville & Gautheret 2010). Previously, in E. coli, rho-dependent termination sites were detected in IS1 and IS2, respectively, which block expression of downstream genes (Hinton & Musso 1983; Hubner et al. 1987).

Not determined

Several other cases of IS-mediated modulation of expression of neighbouring genes have been described for which the actual mechanism has not been determined (Supplementary Table 1). For instance, insertion of ISAac2 upstream of the orfA-ltxCABD operon increased its transcription and concomitant leukotoxin expression in Aggregatibacter actinomycetemcomitans (He et al. 1999). Although ISAac2 carries an outwardly directed -35 sequence at its IRR, which is positioned upstream from a potential -10 sequence, this putative promoter does not influence transcription of the ltx genes (Mitchell et al. 2003). ISAac2 carries in addition a -10-like element at its IRL that is apparently required for increased transcription of the ltx promoter (Schaeffer et al. 2008). However, no transcript spanning the ISAac2 transposase and orfA was detected (Mitchell et al. 2003; Schaeffer et al. 2008). Schaeffer et al. (2008) concluded that either this -10 sequence has an indirect effect on the expression of orfA, which codes for an unknown protein that is required for the increased ltxCABD expression and interacts with the ltx promoter, or the transcript undergoes rapid post-transcriptional processing.

IS-mediated mobilization of neighbouring genes

It has been known for many years that IS can, under certain circumstances, mobilize neighbouring genes. In early studies, so-called compound (or composite) transposons were identified in which two flanking IS in inverted or directly repeated orientations could together mobilize one or more interstitial genes. Initial examples were limited to antibiotic resistance genes since this type of phenotypic trait was relatively easy to identify. Initial examples included the classical models Tn5 (flanking IS50), Tn10 (flanking IS10) as well as Tn9 (flanking IS1) (Alton & Vapnek 1979; Bennett 2008; Haniford & Ellis 2015). However, many other examples are now known with interstitial genes other than those coding for antibacterial traits (Siguier et al. 2014). In some instances, mutations have arisen in one or both flanking IS, which render them less autonomous and facilitate their recruitment in the transposon.

A number of TE, which are very closely related to known ISs, carry passenger genes involved in a variety of processes not linked to transposition, e.g. transcriptional regulation, methylation and antibiotic resistance (Siguier et al. 2014). Siguier et al. (2009) coined the term transporter IS (tIS), since these elements are simpler than known transposons. Although the mechanisms involved in the acquisition of passenger genes are unclear, tISs do not appear to carry programmed recombination systems (as for instance some members of the Tn3 family seem to do). One route for the formation of tIS is from compound transposons in which one of the IS has undergone deletion. Several structures have been identified which could represent intermediates. These include an entire IS copy together with a surrogate IR located at some distance. Examples are IS elements such as IS911 (IS3 family) (Prere et al. 1990; Polard et al. 1994), IS102 (IS5 family) (Machida et al. 1982) and Ecp1-like elements (Poirel et al. 2003, 2005b). The latter (also referred to above) are often located upstream of several β-lactamase genes and their promiscuous transposases can recognize a variety of related DNA sequences as a functional IR. This enables the transposition and mobilization of the adjacent bla_CTX-M genes (Poirel et al. 2005b). Other potential tISs of this type, containing a single IS and a short flanking sequence resembling an additional IR, harbour passenger genes involved in the resistance to bromoacetate (IS1380-family member IS1247 in Xanthobacter autotrophicus; van der Ploeg et al. 1995), triclosan (IS1182-family member IS1272 in Staphylococcus haemolyticus; Furi et al. 2016) and possibly colistin (IS30-family member ISApl1; Snesrud et al. 2016). tIS could be derived from such structures by mutation (deletion) of the internal end of the complete IS rendering the entire structure transposable. tIS might then undergo further deletion to generate minimal insertion cassettes (MICs) in which the IS transposase is removed leaving the transposable gene flanked by correctly oriented IR copies. At present, little is known concerning the evolutionary relationship between these structures and, indeed, between these structures and miniature inverted repeat transposable elements (MITES) in which two IR flank a short section of non-coding DNA (Siguier et al. 2014).

IS elements recognized by the cell's recombination machinery

As mentioned above, transposition of IS elements can locally destroy or alter gene function or expression. However, by amplification of their copy number within a genome, IS elements can also cause more extensive/general loss of genetic information by recombination events between identical individual copies (Cole et al. 2001; Preston et al. 2004). In an evolution experiment involving 12 populations of E. coli B, which have been propagated for more than 20,000 generations by daily serial transfers in minimal medium supplemented with glucose, multiple insertion, deletion and inversion events were detected (Papadopoulos et al. 1999; Schneider et al. 2000; Cooper et al. 2001). One deletion event was found in all 12 independent lines providing a fitness advantage in glucose medium and an example of parallel phenotypic and genomic evolution. This deletion was generated by a first transposition of IS150 (IS3 family) close to the rbs operon, encoding genes required for ribose utilization, followed by a recombination event with a second IS150 element, leading to deletion of (part of) the rbs operon (Cooper et al. 2001).

In another evolution experiment, where E. coli was adapted to extended starvation in liquid medium (Zambrano et al. 1993), mutants with increased fitness during stationary phase were isolated and an IS5-mediated gene loss involving the exchange of regulatory sequences was detected. First, transposition of an IS5 element occurred between the CRP-binding site and promoter of cstA, encoding for an oligonucleotide permease. Second, an inversion event occurred between the transposed IS5 copy and an IS5 copy located at 60 kb distance and upstream of the ybeJ-gltJKL-ybeK operon, involved in aspartate-glutamate transport. This inversion led to the inactivation of the cstA gene and CRP-dependent induction of the latter operon, conferring a new ability to grow on aspartate as a sole carbon source and grow faster with glutamate, asparagine and proline as carbon sources (Zinser et al. 2003). IS5-mediated deletion was also detected in E. coli populations evolved in glucose- and phosphate-limited chemostats (Gaffe et al. 2011). Here, a chromosomal deletion including mutY, involved in DNA repair, providing a mutator phenotype was identified in independent chemostats. As in the rbs deletion, a first transposition event was necessary before the subsequent recombination and deletion.

IS can also be involved in plasmid rearrangements. For instance, the large pAsa5 plasmid from the fish pathogen Aeromonas salmonicida encodes a type three secretion system, which is an important factor for virulence, but also harbours 11 IS elements, including three copies of ISAs11 (IS256 family). Stressful conditions such as growth at 25 °C caused ISAs11-mediated recombination events that resulted in pAsa5 rearrangements and loss of the type three secretion system and virulence (Tanaka et al. 2012).

As described above, IS elements can play an important role in the (in)activation of genes through transposition and recombination events. Furthermore, IS elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements (detailed in Siguier et al. 2014). This is especially the case for pathogens which are dependent on their host for survival, since the host provides nutrition and protection. For example, comparative genetic analysis of Yersinia pestis, the causative agent of plague, has revealed that this organism has lost approximately 13% of its genome through first successive horizontal gene transfer of virulence factors and IS enrichment followed by IS-mediated massive genome decay (Chain et al. 2004). Yersinia pestis is not a unique example. Although this phenomenon would be detrimental in the free environment, it might be required for further niche adaptation among bacteria living in nutritionally protected environments such as S. enterica serovar Typhi (Parkhill et al. 2003), Mycobacterium leprae (Cole et al. 2001) and Mycobacterium ulcerans (Stinear et al. 2007).

IS transposition affected by host factors

Transposition rates per generation differ from element to element and host to host, depend on the type of donor molecule, and vary widely from 10⁻³ to 10⁻⁷ (Shen et al. 1987; Craig et al. 2002). Recently, Sousa et al. (2013), using mutation accumulation and the vectorette PCR method, showed that the spontaneous transposition rate of multiple IS elements from one locus to another locus in the chromosome of E. coli K12 MG1655 srl::Tn10 mutS, without selective pressure during the analysis, ranged from 10⁻⁵ to 10⁻⁶. Transpositional activity of IS elements is strongly controlled, presumably to limit their detrimental effects to the host cell. IS transposition is regulated by the element itself, including transcriptional repressors (Zerbib et al. 1990; Hu et al. 1994), translational inhibitors (Kleckner et al. 1996), ribosome frame shifting (Escoubas et al. 1991), impinging of transcription by secondary mRNA structures (Beuzon et al. 1999), methylation sites (Roberts et al. 1985), transposase instability (Derbyshire et al. 1990) and target site preference (Olasz et al. 1998; Kiss et al. 2007). Furthermore, IS transposition can also be modulated by host factors, including, but not limited to, the DNA chaperones IHF, HU, HNS and FIS, the replication initiator DnaA, the protein chaperone/protease ClpX/P/A, the SOS control protein LexA, the DNA methylase Dam and GTP levels (Chandler & Mahillon 2002; Nagy & Chandler 2004; Coros et al. 2005; Twiss et al. 2005). In general, these effects are specific for each element (Mahillon & Chandler 1998). Recently, bacterial transposition reactions have also been shown to be regulated by Hfq, a well-studied RNA chaperone that binds small regulatory RNA (sRNA) and plays a central role in complex post-transcriptional regulatory networks in many bacteria (Vogel & Luisi 2011; Sobrero & Valverde 2012). For instance, IS10/Tn10 transposase translation is repressed by Hfq. When a single IS10 copy is present, Hfq directly binds to the translation initiation region of the transposase mRNA (RNA-IN) and blocks ribosome binding, when more IS10 copies are present, translation of RNA-IN is more efficiently inhibited by antisense pairing between RNA-IN and the cis-encoded sRNA, RNA-OUT (Ross et al. 2010; Ellis et al. 2015b). In addition, increased expression of the ChiX sRNA, for instance in stationary phase (Vogel et al. 2003), titrated Hfq away from RNA-IN, thereby derepressing RNA-IN translation (Ellis et al. 2015b). Another example is the post-transcriptional regulation of the expression of the IS200 transposase, an IS ubiquitous in Enterobacteriaceae (Beuzon et al. 2004). All three independent mechanisms, which include the tnpA mRNA secondary structure, Hfq binding to the tnpA mRNA and base-pairing of the cis-encoded sRNA, art200, with the tnpA mRNA, prevent ribosome binding and repress translation (Ellis et al. 2015a).

IS transposition affected by environmental stimuli

IS elements can be induced by environmental signals resulting in the creation of new genetic variability that can be useful to withstand stressful conditions. Multiple examples are described below; however, it is important to note that increased transposase transcription does not necessarily result in an increase in transposase.

Host environment

In F. tularensis, a highly virulent bacterial pathogen, environmental signals such as the presence of host cell-produced polyamines, including spermine and spermidine, dramatically alter cytokine induction in their host cells establishing a successful infection. Carlson et al. (2009) showed that two classes of Francisella IS elements, ISFtu1 and ISFtu2, which are the most frequent in overall occurrence of six defined classes in F. tularensis (Rohmer et al. 2007), harboured spermine-responsive promoters leading to increased expression of their transposase genes and of adjacent genes via co-transcription. This increased expression was both observed in macrophages and in broth culture, where some of the cues encountering a mammalian host could be mimicked, including the presence of polyamines (Carlson et al. 2009).

A more general approach in M. tuberculosis indicated that IS6110 directed the expression of multiple genes via an outward-directed promoter in a 110 bp fragment, located near its IRR (Safi et al. 2004). Interestingly, this promoter is responsive to certain stress conditions, including the macrophage environment and the late phases of growth in broth, and thus has the ability to activate potential virulence genes during infection (Safi et al. 2004). In addition, IS6110 expression is also increased under microaerobic conditions (Ghanekar et al. 1999). Survival and growth in human monocytes and macrophages are major factors in the success of M. tuberculosis as a pathogen. Therefore, a mobile promoter, responsive to the macrophage environment, provides a mechanism for generating variants with potentially advantageous phenotypes.

Radiation and oxidative stress

Using a mutagenic assay to detect any ThyA inactivation events in D. radiodurans, five IS elements (ISDra2, ISDra3, ISDra4, ISDra5 and IS2621), belonging to different families, were found to be transpositionally active. In the presence of an active mismatch repair system, 75% of the mutations to trimethoprim resistance (Tmp^R) resulted from an IS insertion into the thyA coding region. ISDra2, which was rarely found among the spontaneous Tmp^R mutants, became the predominant IS among damage-induced mutants. Transposition of ISDra2 was induced by γ-irradiation (approximately 100-fold) and by UV-irradiation (approximately 50-fold) (Mennecier et al. 2006). It was later postulated that transposition of at least ISDra2 required single-strand DNA substrates and that its transposition thus could be triggered by the increase in its single-strand DNA substrate during irradiation (Pasternak et al. 2010). Induced transposition of ISDra2 by DNA damage is consistent with previous research where expression of the ISDra2 transposase was induced following a heat shock or treatment with H₂O₂ (Lipton et al. 2002; Schmid et al. 2005).

In E. coli, a mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid seemed to have identified intermolecular IS10 transposition from the chromosome to the target plasmid inactivating cI. Moreover, this transposition appeared to be induced by UV-light in a SOS-induced manner. However, most of the UV-induced transposition of IS10 was accompanied by the integration of the target plasmid into the chromosome and by the production of extra IS10 copies (Eichenbaum & Livneh 1998).

Fingerprinting profiles of clinical isolates of B. cenocepacia showed substantial band variability that resulted from genomic rearrangements mediated by insertion sequences. Furthermore, transposition activity of an ISBcen20 homolog appeared to be induced when the ST-32 isolate CZ1238 was exposed to oxidative stress (Drevinek et al. 2010).

Metals and antibiotics

Subinhibitory zinc and cadmium concentrations induced the promoter activity of ISRme5 (IS481 family), IS1087B (IS3 family) and IS1088 (IS30 family) in C. metallidurans AE126 (Vandecraen et al. 2016). All three were involved in gene inactivation resulting in increased zinc resistance (see above and Vandecraen et al. 2016).

In addition, subinhibitory concentrations of antibiotics can also promote transposition of IS elements. ISEcp1-like elements are one of the most important genetic elements associated with bla_CTX-M genes, which code for expanded-spectrum β-lactamases of the CTX-M type (Lartigue et al. 2004). Subinhibitory concentrations of several β-lactams, including cefotaxime, ceftazidime and piperacillin induced ISEcp1B-mediated transposition (Lartigue et al. 2006). However, amoxicillin/clavulanic acid, cefquinome and cefuroxime did not enhance ISEcp1B-mediated transposition (Nordmann et al. 2008). Transposition of ISEcp1B was also not promoted by nalidixic acid, a quinolone antibiotic, agreeing with previous work of Cattoir et al. (2008) who found that ISEcp1-like elements mediated mobilization but not expression of qnrB genes coding for quinolone resistance. In addition, Lartigue et al. (2006) showed that transposition might be temperature dependent as an enhanced transposition was detected at 40 °C compared with 37 °C.

Temperature

In B. subtilis, using an intermolecular transposition assay it seemed that the transposition frequency of IS4Bsu1 was increased under high temperature (4.4-fold, 49 °C compared with 37 °C), as well as under competence-inducing conditions (15.7-fold) (Takahashi et al. 2007).

Increased transposition of IS elements at high temperature (42 °C) was also observed in B. multivorans ATCC 17616 by using an IS trap with a positive sacB-based selection system (Ohtsubo et al. 2005). From the 13 active IS elements in B. multivorans ATCC 17616 (IS401 to IS408, IS411, IS415, ISBmu1, ISBmu2 and ISBmu3) (Lessie et al. 1990; Ohtsubo et al. 2005), eight were detected with this particular IS trap and transposition of IS401, IS402, IS406 and ISBmu3 was only detected under a high-temperature condition (Ohtsubo et al. 2005). In addition to high temperature, oxidative stress and starvation conditions were also evaluated but did not affect transposition activity in B. multivorans ATCC 17616 (Ohtsubo et al. 2005). Since, multiple IS elements (belonging to four different IS families) increased their transposition at high temperature, Ohtsubo et al. (2005) suggested that this induction was not elicited by intrinsic transposase properties but by common host factor(s). In contrast, in E. coli, the transposition activities of several mobile elements (e.g. Tn3, IS1, IS30 and IS911) exhibit decreased transposition at 42 °C (Nagy & Chandler 2004) and the sensitivity of transposition to temperature has been considered to be an intrinsic feature of each transposase (Haren et al. 1997). For IS911, this decreased transposition resulted from an increased production of truncated transposase, devoid of the catalytic domain (Gueguen et al. 2006). Thus, environmental signals can also further repress the expression of transposases. For instance, in the cyanobacterium Tolypothrix sp. PCC 7601, expression of the ISTosp1 transposase is subjected to metabolic repression by ammonium ions via the presence of binding sites for the global regulator NtcA in the tnpA promoter (Luque et al. 2006).

Conjugation

Finally, conjugative interaction, possibly even without transfer, can also induce transposition of IS elements as this was demonstrated for the transposition of ISPst9. The genomic dose and/or distribution of ISPst9 copies was changed when P. stutzeri AN10 cells were put in contact with a (plasmidless) conjugative strain compared with a non-conjugative E. coli strain (Christie-Oleza et al. 2009). The role of conjugational transfer in adaptive mutability was first shown by the recombination-mediated high frequency of complete Tn10 loss among Lac⁻ and Lac⁺ bacteria that have experienced conjugational transfer of the F’128 episome harbouring a Tn10 linked to the mutated lacI33 allele (Godoy & Fox 2000).

Evolutionary dynamics of ISs

The many cases detailed above illustrate the impact of ISs on the bacterial genome. Depending on the target gene, IS movement can have positive, neutral or negative effects on cell fitness. Under selective conditions, e.g. antibiotic pressure, IS-mediated beneficial mutations are favored and fixation of the IS at that particular target site depends on its exerted effect (e.g. gene inactivation or modulation of expression).

On an evolutionary timescale, an interesting question is how ISs are maintained in a genome. This issue can be approached from different angles. One approach focuses more on the parasitic nature of these elements and their ability to self-replicate (Doolittle & Sapienza 1980), a second takes into account the fact that ISs can also provide selective benefits to the host, for example, by generating occasional beneficial mutations in promoting genetic variation (Blot 1994; Schneider & Lenski 2004; De Palmenaer et al. 2008; Bichsel et al. 2013). A third hypothesis was postulated by Iranzo et al. (2014), who concluded via a large-scale genomic analysis that IS abundance and distribution are selectively neutral. However, these hypotheses are not necessarily mutually exclusive. Recently, Wu et al. (2015) showed that transposition bursts accelerate the extinction of IS elements and that this process could be decelerated by mechanisms suppressing transposition activity, which ISs as well as their hosts have developed (see above). The latter corresponds with the findings of Iranzo et al. (2014) that ISs and their hosts tend to coexist in a dynamic equilibrium. In addition, this still allows generating some genetic variability and occasional IS-induced mutations are beneficial to overcome environmental challenges or to adapt to new environmental niches.

Conclusions

The examples described here, highlighting the effect and regulation of IS transposition in bacteria, clearly show that IS elements can have an important impact on the evolution of their hosts. IS transposition can have different outcomes, from simple gene inactivation to constitutive expression or repression of adjacently located genes by delivering IS-specified (partial) promoter or terminator sequences, respectively. Furthermore, multiple copies of an identical IS element dispersed over a genome promote various genomic rearrangements such as inversion, deletion, duplication and fusion of two replicons. Additionally, sequences flanked by two identical IS elements, properly orientated, can become mobilized and dispersed across different strains, even to unrelated bacteria. For example, the dissemination of antibiotic resistance genes has become a major health problem. Moreover, the activity of, at least some, IS elements can be modulated by environmental signals to promote genome plasticity and genetic variability offering occasional beneficial mutations to overcome environmental challenges or to adapt to new environmental niches. Thus, whether or not IS elements are selfish and parasitic elements, their impact on the architecture of microbial genomes is undeniable.

Disclosure statement

The authors report no declarations of interest.