Control of expression of the ICE R391 encoded UV-inducible cell-sensitising function
© Armshaw and Pembroke; licensee BioMed Central Ltd. 2013
Received: 8 April 2013
Accepted: 28 August 2013
Published: 29 August 2013
Many SXT/R391-like enterobacterial Integrative Conjugative Elements (ICEs) have been found to express an atypical, recA-dependent, UV-inducible, cell-sensitising phenotype observed as a reduction in post-irradiation cell survival rates in host cells. Characterisation of a complete deletion library of the prototype ICE R391 identified the involvement of three core ICE genes, orfs90/91 encoding a putative transcriptional enhancer complex, and orf43, encoding a putative type IV secretion system, outer membrane-associated, conjugative transfer protein.
In this study, expression analysis of orf43 indicated that it was up-regulated as a result of UV irradiation in an orfs90/91-dependent manner. Induced expression was found to be controlled from a site preceding the gene which required functional orfs90/91. Expression of orfs90/91 was in turn found to be regulated by orf96, a λ cI-like regulator. Targeted construction of ICE R391 deletions, RT-PCR and qRT-PCR analysis confirmed a regulatory link between orfs90/91 and orf43 while site-directed mutagenesis of orf43 suggested an association with the cell membrane was a prerequisite for the cytotoxic effect.
Because of the recA-dependence of the effect, we hypothesise that UV induction of RecA results in cleavage of the cI-like ICE-encoded repressor protein, the product of orf96. This in turn allows expression of the transcriptional enhancer complex encoded by orfs90/91, which we conclude stimulates transcription of orf43, whose product is directly responsible for the effect.
Integrative Conjugative Elements (ICEs) are a class of bacterial mobile genetic elements that encode features necessary for their site-specific integration and excision from host genomes, self-circularisation and transfer by conjugation [1, 2]. ICEs are divided into families based on similarity between core genes (specifically the integrase gene) and the site of integration they utilise within host chromosomes. The SXT/R391 family share a highly similar integrase gene and integrate into the prfC gene of enterobacterial hosts [1, 3]. In addition to encoding host beneficial traits such as antibiotic resistance determinants [3–5], many SXT/R391 family ICEs express an unusual cell-sensitising function [6–8]. Preliminary characterisation of the UV-inducible, cell-sensitising function of the prototype, ICE R391, determined the effect to be recA-dependent , while further analysis based on construction of a deletion library of ICE R391 found that three core ICE genes, namely orfs90/91 and orf43 were involved . Deletion analysis also revealed that orf96, which encodes a putative λ cI-like repressor protein , could only be deleted in strains where orfs90/91 had previously been removed suggesting that the repressor protein may prevent lethal expression of orfs90/91. Additionally, cloning and controlled expression of both orfs90/91 and orf43 revealed that expression of orf43 alone was cytotoxic to wild type E. coli while expression of orfs90/91 was only cytotoxic to wild type E. coli cells harbouring the ICE R391. This indicated that orf43 was responsible for the observed UV-inducible cytotoxicity .
Here, a model is proposed (Figure 1) for the control of this unusual ICE R391 UV-inducible sensitising effect based on expression data examining the key genes involved and supported by a number of directed ICE R391 deletions.
Results and discussion
orfs90/91 stimulate orf43transcription after exposure to UV irradiation
We previously demonstrated that over-expression of orf43 when cloned into the arabinose inducible pBAD33-orf43 construct was responsible for the UV-inducible sensitisation observed in ICE R391 and other ICEs of the SXT/R391 family . Mutagenesis data also suggested that the putative transcriptional controller encoded by orfs90/91 was also involved, although not directly. To investigate the relationship between orfs90/91 and orf43, we utilised both qualitative and quantitative RT-PCR targeting these genes in different mutant backgrounds and with and without UV irradiation.
Cytotoxic orf43 transcription is regulated through a region directly upstream of orf43
Genotype of bacterial strains, plasmids and ICE R391 mutants used
F-, thr-1, araC14, leuB6, ∆(gpt-proA)62, lacY1, tsx-33, qsr’-0, glnV44, galK2, λ-, Rac-0, hisG4, rfbC1, mgl-51, rpoS396, rpsL31 (StrR), kdgK51, xylA5, mtl-1, argE3, thi-1
E. coli genetic stock centre (CGSC), Yale University, New Haven, Connecticut, USA
F-, mcrA0, ∆(mrr-hsdRMS-mcrBC), φ80dlacZ58(M15), ∆lacX74, recA1, araD139, ∆(araA-leu)7697, galU-, galK0, rpsL - (StrR), endA1, nupG -
Bio-Sciences, Dun Laoghaire, Dublin, Ireland
S. Enteritidis PT4 wild type (NCTC 13349), NalR
National Collection of Type Cultures (NCTC), Salisbury, UK
CmR, p15A ori, PBAD L-arabinose inducible, orf43
Armshaw and Pembroke, 2013 
CmR, p15A ori, PBAD L-arabinose inducible, orf43 containing mutation converting two leucines to prolines at a.a. position 47 and 48.
CmR, p15A ori, PBAD L-arabinose inducible, orf43 containing mutation converting glutamine at position 115 to asparagine.
Ts, PBAD-gam-bet-exo cat (CmR)
Dr. P. Latour-Lambert, Institut Pasteur, 25 rue du Dr Roux, Paris, France
AmR template for deletion mutant construction
Sigma-Aldrich, Arklow, Wicklow, Ireland
ZeR template for deletion mutant construction
Invitrogen, Bio-Sciences, Dun Laoghaire, Dublin, Ireland
Dr R.W. Hedges, Royal Postgraduate Medical School, London, UK
AB1157 R391 ∆14 (∆orf43)
ICE R391 orf43 deletion strain, AmR, UV-, tra-
Armshaw and Pembroke, 2013 
AB1157 R391 ∆26 (∆orfs90/91)
ICE R391 orfs90/91 deletion strain, AmR, UV-, tra-
Armshaw and Pembroke, 2013 
AB1157 R391 ∆11 (∆orfs40/41)
ICE R391 orfs40/41 deletion strain, AmR, tra-
Armshaw and Pembroke, 2013 
AB1157 R391 ∆25AmR∆14ZeR
ICE R391 orf90 – orf94 and orf43 deletion strain, AmR, ZeR, UV-, tra-
AB1157 R391 KOA
ICE R391 orf32 - orf42 (29575 bp – 41491 bp) deletion strain, AmR, tra-
AB1157 R391 KOB
ICE R391 orf32 - orf42 (29575 bp – 41527 bp) deletion strain, AmR, UV-, tra-
AB1157 R391 KOC
ICE R391 orf32 - orf42 (29575 bp – 41491 bp) and orfs90/91 deletion strain, AmR, ZeR, UV-, tra-
Site-directed mutagenesis of Orf43
Hierarchical control of the ICE R391 UV-inducible sensitising effect
Many SXT/R391-like ICEs reduce post UV survival rates of E. coli host cells through the action of a recA-dependent process [6, 20]. Mutational analysis of the ICE R391 determined that the core genes orfs90/91 and orf43 were required for expression of the cell-sensitising function  while bioinformatic analysis indicated that orf96 likely encodes a λ cI-like repressor similar to RecA substrates in other phage systems that are cleaved following SOS induction . Initial attempts to delete orf96 proved fruitless and no deletion could be isolated. However a Δorf96 (Δ28) deletion  could be isolated in an ∆orfs90/91 mutant background suggesting that orf96 may control expression of orfs90/91 which we have shown here directly control expression of orf43, the ultimate instigator of the cytotoxicity associated with ICE R391. The data presented here and in Armshaw and Pembroke (2013)  have led to the development of a model to explain the control of UV-inducible sensitisation (Figure 1). We hypothesise that UV irradiation of E. coli induces the host RecA protein which results in cleavage of the ICE R391 encoded product of orf96, the phage λ434 cI-like ICE repressor. We propose that cleavage of Orf96 in turn leads to expression of orfs90/91 which in turn leads to up-regulation of orf43 and other ICE R391 genes such as orf4 (jef) . We have previously demonstrated that up-regulation of orf4 (jef) leads to increased ICE R391 transfer . In the related ICE SXT, Beaber et al., (2004)  demonstrated that SetR, the SXT homolog of Orf96, acted as a repressor of ICE SXT transfer and that it is bound to ICE operators that controlled setC/D, SXT homologs of orfs90/91, in a similar way to our proposal for ICE R391. They also proposed that repression was lifted by induced RecA protein cleaving the SetR repressor in a similar manner to our proposal for orfs90/91. The recA dependence for the ICE R391 UV-sensitising effect , the similarity to the SXT system , the deletion data and qRT-PCR data presented here support the model presented.
It would thus appear that UV irradiation is the instigator of the control loop leading to over expression of orf43 which leads to cytotoxicity. Since normal levels of Orf43 are required for ICE transfer and play a key part in formation of the conjugative transfer system of ICE R391, it appears that the associated cytotoxicity is related to the induced overexpression. Evidence in support of this comes from data showing that overexpression of orf43 from the arabinose inducible clone pBAD33-orf43 leads directly to cytotoxicity . The UV-inducible sensitising effect is conserved amongst many SXT/R391 ICE family members [6, 20]. A sophisticated control system is in place to control this effect yet the exact nature and reason for conservation of such an unusual apparently ‘evolutionary negative’ effect remains to be elucidated. We are currently examining the nature of the cytotoxicity and developing theories for its function and retention.
Bacterial strains, elements and media
The bacterial strains, plasmids and ICE R391 deletion mutants utilised as part of this study are listed in Table 1. Strains were stored at −80°C in either Luria-Bertani (LB) broth or M9 minimal media containing 50% (v/v) glycerol. Media was supplemented with appropriate antimicrobial agents: nalidixic acid, 30 μg ml-1; ampicillin, 100 μg ml-1; chloramphenicol, 25 μg ml-1, kanamycin, 30 μg ml-1, streptomycin, 100 μg ml-1; mercuric chloride, 20 μg ml-1; zeocin, 25 μg ml-1 as required. For growth and analysis of strains containing pBAD33-orf43, M9 minimal media containing 0.4% (v/v) glycerol was used with either 0.4% (w/v) glucose or 0.02%-0.2% (w/v) L-arabinose to repress or induce gene expression respectively as previously described .
Directed deletions of ICE R391 and subsequent deletion mutant screening
ICE R391 specific deletions were generated as previously described . Screening of resulting ICE R391 deletion mutants for loss of cell-sensitising function by qualitative and quantitative UV survival assays were carried out as described . Screening of ICE R391 deletion mutants’ conjugative transfer ability to recipient Salmonella enterica serotype Enteritidis strain P125109 was performed as described .
Qualitative reverse transcriptase PCR
Cells were collected by centrifugation, washed twice with diethyl pyrocarbonate-treated distilled water and resuspended in 10 mM Tris, [pH8.0]. Total RNA was isolated using the Absolutely RNA Miniprep kit (Agilent Technologies) according to the manufacturer’s protocol. Absence of contaminating DNA was verified by PCR. Qualitative reverse transcriptase PCR was performed using the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Agilent Technologies) according to the manufacturer’s protocol. Resulting cDNA was analysed immediately by PCR using gene-specific primers or stored at −20°C.
Quantitative reverse transcriptase PCR (qRT-PCR)
Quantitative UV assays were carried out as described . Unirradiated and irradiated cells were collected by centrifugation and total RNA isolated as described. Absence of contaminating DNA was verified by PCR. qRT-PCR was performed using the Brilliant III Ultra-Fast SYBR Green qRT-PCR Master Mix (Agilent Technologies) according to the manufacturer’s protocol using the Stratagene Mx3000P Real Time PCR System and appropriate gene-specific PCR primers with the following temperature profile: 1 cycle at 42°C for 30 minutes to convert RNA to cDNA, 1 denaturation cycle at 95°C for 3 minutes followed by 40 cycles at 95°C for 30 seconds, 54°C for 60 seconds, 72°C for 30 seconds followed by melting curve analysis from 65°C to 95°C to determine specificity of the PCR reaction. Specificity of the PCR reaction was verified by SYBR safe staining on a 2% (w/v) agarose gel. The internal standard curve using the unirradiated RNA sample to estimate the change in target RNA quantity consisted of: undiluted RNA, a 1 in 2 dilution, a 1 in 4 dilution and a 1 in 10 dilution of unirradiated RNA. A no template negative control was also included. In addition, qRT-PCR was also carried out on the known endogenous housekeeping gene proC as an internal control to quantify the relative change in transcription of the gene of interest .
Site-directed mutagenesis of pBAD33-orf43
Site-directed mutagenesis of pBAD33-orf43 was performed using specifically designed complementary mutagenic primers to linearly amplify pBAD33-orf43 to generate a mutated nicked DNA product. Non-mutated methylated template DNA was eliminated by incubation with the DpnI restriction enzyme. Mutated DNA products were then transformed into TOP10 and plated on appropriate media containing chloramphenicol, 25 μg ml-1. Resulting TOP10 colonies were cultured, had plasmid content extracted using the QIAprep Spin Miniprep Plasmid extraction kit from QIAGEN (West Sussex, RH10, 9NQ, UK) according to the manufacturer’s protocol and screened for the presence of pBAD33-orf43 by restriction enzyme digestion. Mutated pBAD33-orf43 was verified by DNA sequencing to contain the desired mutation without additional mutations. Mutated pBAD33-orf43 was confirmed to still transcribe orf43 specific mRNA by RT-PCR as described. Determination of the effect of induction of mutated pBAD33-orf43 on host cell growth rate was carried out as described .
This work was funded by the Irish Research Council for Science, Engineering and Technology (IRSCET) to PA. The authors would like to thank Dr. P. Latour-Lambert for providing the pKOBEG plasmids and Drs. John O’Halloran and Michael P. Ryan for helpful discussion.
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