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Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

Russell J Collighan , Martin J Woodward
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12645.x 207-213 First published online: 1 September 1997


The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (>75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

  • Salmonella enteritidis
  • Insertion element
  • Nucleotide sequence
  • Distribution

1 Introduction

Salmonella enteritidis has become the most common serotype associated with human food poisoning in the UK [1]. This is thought to be a direct result of an increase in contamination of poultry products with this serotype [2, 3]. As part of the effort to reduce the prevalence of salmonellas in poultry, research in this laboratory is ongoing into the role of fimbriae in the disease process, notably colonisation, invasion and persistence. Salmonellas express several classes of fimbriae including type 1 which mediate mannose-sensitive haemagglutination, type 2 which are morphologically similar to type 1 but do not agglutinate erythrocytes, and type 3 which mediate mannose-resistant haemagglutination [4]. Another class of fimbriae which has been shown to be highly immunogenic in the mouse model [5] is the SEF14 fimbriae. SEF14 fimbriae, first described in strains of S. enteritidis[6], comprise thin filamentous organelles composed of repeating 14.3 kDa protein subunits [7]. SEF14 is expressed solely by certain group D serotypes including all strains of S. enteritidis and some S. dublin strains [6, 8] although the gene encoding SEF14, designated sefA, is present in several other group D serotypes. Interestingly, the complete sef operon is present in S. gallinarum and S. pullorum but not expressed under the laboratory conditions tested [8]. With the exception of type 1 fimbriae, little is understood of the regulation of these fimbriae. Ogunniyi et al. [9] described a limited sequence upstream of the sefA gene of S. enteritidis which shared putative identity with IS3 and other instances of IS-like sequences adjacent to fimbrial operons have been described. For instance upstream of the CFA/I fimbrial operon of enterotoxigenic Escherichia coli is a region bearing homology to IS2 [10], whilst immediately downstream of the bfpTVW genes, responsible for regulating the expression of the bundle-forming pilus of enteropathogenic E. coli, is an IS-like sequence [11]. Interestingly, sequences with homology to IS3 have been located near invH of S. choleraesuis[12, 13]. In this paper we describe the determination of the nucleotide sequence upstream of the sefA gene as a first step to understanding the regulation of SEF14. We have identified an IS3-like element in close proximity to sefA.

2 Materials and methods

2.1 Bacterial strains, plasmids and media

Wild-type Salmonella isolates were obtained from the Salmonella reference laboratory, CVL, and were stored on Dorset egg slopes. Growth of salmonellae was either on nutrient agar or in nutrient broth. E. coli DH5α (MaxEfficiency competent cells, Life Technologies) was used as the recipient in all transformation experiments following the manufacturer's protocol. E. coli carrying recombinant plasmids were grown on Luria-Bertani medium supplemented with 100 μg ml−1 ampicillin (Penbritin, Beecham), 40 μg ml−1 X-gal (Sigma) and 100 μM IPTG (Sigma). The general cloning vector pUC18 was used in all cloning experiments and for nucleotide sequencing. Plasmid pSEFEX was a pUC18 clone containing a 5.5 kb insert encoding sefABC[8] and the complete IS1230 was subcloned from a sefA+ cosmid identified from a S. enteritidis cosmid library [14].

2.2 DNA sequencing and data manipulation

Nucleotide sequencing reactions were performed on 400 ng of purified plasmid template DNA using the Taq Terminator FS (Perkin-Elmer) fluorescent cycle sequencing kit according to the manufacturer's protocols. Reactions were analysed on an ABI model 373A automated DNA sequencer. Trace data were processed using DNASTAR software (DNASTAR Inc.) and assembled into a contiguous sequence.

2.3 Polymerase chain reaction (PCR)

Primers used to amplify the IS1230 sequence from plasmid pSEFEX for use as a probe were as follows: 5′-TGATCTTACCCAGCAAGAGTGG-3′ (ISF) binding at positions 1–22, and 5′-CTGGCGACTCCAATACTGTT-3′ (ISR) binding at positions 1364–1344 outside the IS1230 sequence. Primers used to assess the co-location of IS1230 and sefA were as follows: 5′-TTAGTTTTGATACTGCTGAAC GTA-3′ (SEFR) located towards the 3′ end of sefA, in combination with either ISR or 5′-TCCTCATCCCGTTCTGCCAGTT-3′ (ISR2) binding at positions 313–292 of IS1230. Taq polymerase, Thermo polymerase buffer and deoxynucleoside triphosphates were obtained from Promega. The conditions used for PCR were 95°C for 1.5 min, 60°C for 1.5 min, 72°C for 2 min for a total of 35 cycles using a thermal cycler (Biometra). PCR products were separated by agarose gel electrophoresis, the relevant DNA band was excised from the gel and the DNA purified using Wizard PCR Preps (Promega).

2.4 Hybridisation

The lysis of bacterial colonies for colony hybridisation and the capillary transfer of DNA from agarose gels onto nylon membrane (Hybond N, Amersham) were performed according to methods described by Maniatis et al. [15]. The IS1230 probe was radiolabelled by the incorporation of [α-32P]dCTP using a random-primed labelling kit (Ready Reaction dCTP labelling mix, Pharmacia). Hybridisations and post-hybridisation washes were carried out using the RapidHyb (Amersham) system according to the manufacturer's protocols. Low and high stringency hybridisations were performed by washing at 65°C and 75°C respectively.

3 Results and discussion

3.1 IS1230 is a member of the IS3 family of insertion sequences

In order to investigate the regulation of expression of the sef operon, sequence analysis of the region of DNA upstream of the sefA promoter revealed the presence of an insertion sequence sharing identity with the IS3 family of insertion elements. A homology search of the GenBank and EMBL nucleotide sequence databases revealed that a 1230 bp portion of the sequence designated IS1230 shared sequence identity with database entries for Serratia odorifera IS3 (81%), E. coli IS3 (79%), Edwardsiella fergusonii IS3 (77%) and Shigella dysenteriae IS3 (75%). The nucleotide sequence of the S. enteritidis IS1230 (Fig. 1) showed several characteristic features common to members of the IS3 family of insertion elements. IS1230 possessed imperfect terminal inverted repeats of approximately 40 bp which matched those of E. coli IS3 (90% identity). However, unlike other IS3 elements which possess two open reading frames the second of which is shifted in frame by −1 with respect to the first to encode the transposase after ribosomal translational frameshifting [16], IS1230 appears to lack these open reading frames. A region of 27 nucleotides corresponding to positions 458–484 of the E. coli IS3 sequence was absent from IS1230, although this did not result in a frameshift. However, an insertion of T at position 332 of the IS1230 sequence and single base pair deletions after positions 147, 677 and 895, not present in other IS3 sequences studied, resulted in frameshifts which interrupted the putative open reading frames. This apparent lack of ability to produce an intact transposase may indicate that IS1230 is an inactive insertion element in the S. enteritidis genome or that the expression of the IS1230 transposase is more complicated than a single ribosomal frameshift to couple the translation of two open reading frames. The probable lack of production of an intact transposase raises the possibility that IS1230 is no longer an active insertion element, but does not detract from the possibility that IS1230 has some influence in the regulation of sefA expression. The effect of IS1230 on the expression of sefA will be the subject of further investigation. Of the known sequence of the sef operon and adjacent regions of approximately 5.5 kb including IS1230 [8, 17], the GC content averages 33%. For the known sequence 3′ to IS1230 (this work, data not shown), the GC content averages 45% over 1.6 kb. Regions of DNA with a reduced GC content have been linked to the presence of pathogenicity islands which are large stretches of DNA associated with virulence that are thought to originate from another species [18]. The possibility that the sef operon lies at one end of a pathogenicity island suggests that SEF14 fimbriae may be a virulence determinant although as yet there is little evidence for these fimbriae having a role in the pathogenesis of S. enteritidis[19].


Nucleotide sequence and amino acid translation of IS1230. The amino acid sequence shown is the one which corresponds to the putative transposase of E. coli IS3. Frameshifts with respect to orfA and orfB of IS3 are denoted by a change of line. The putative ribosome binding site at positions 55–58 is underlined. Stop codons are represented by asterisks, inverted repeats are denoted by arrows above the sequence. The complete IS1230 nucleotide sequence has been submitted to the GenBank database and has been assigned the accession number U91789.

3.2 Distribution of IS1230 amongst salmonellae

The distribution of IS1230 amongst salmonellae was tested by colony hybridisation with a DNA probe prepared by PCR amplification of IS1230 from pSEFEX plasmid DNA using primers ISF and ISR. The probe consisted of the entire IS1230 sequence with an extra 32 bases of DNA at the 5′ end. This extra DNA was considered to provide an insignificant contribution to the IS1230 hybridisation signal. The specificity of the probe was tested by Southern hybridisation with E. coli K12 genomic DNA digests where, even at low stringency, the probe failed to hybridise with any of the five E. coli IS3 copies (data not shown). Since E. coli IS3 and S. enteritidis IS1230 share 79% nucleotide sequence identity, any hybridisation with the IS1230 probe was attributable to the presence of IS1230 homologous sequences.

At high stringency all S. dublin (100), S. enteritidis (150), S. berta (20), S. blegdam (20), S. gallinarum (20), S. moscow (20), S. pullorum (20), S. rostock (20) and S. serambam (20) isolates hybridised strongly with the IS1230 probe and weak hybridisation was observed with most other serotypes. However, at low stringency, strong hybridisation was observed as for high stringency but in addition, strong hybridisation signals were observed in other serotypes tested, including S. manhattan, S. virginia, S. wangata, and S. waral, but the distribution did not appear to correlate with group classification. Notably, all strains of S. typhimurium hybridised strongly with the IS1230 probe at high stringency, although none hybridised with sefA[8].

The distribution of IS1230 amongst different Salmonella serotypes as observed by colony hybridisation at high stringency correlates well with the known distribution but not expression of the sefA gene. Those group D1 members which failed to hybridise sefA (S. canastel, S. dar es salaam, S. durban, S. eastbourne, S. kapemba, S. miami, S. napoli) also failed to hybridise IS1230 at high stringency.

The co-location of IS1230 and sefA in different strains of S. enteritidis was shown by PCR using IS1230 and sefA specific primers (Fig. 2). All strains produced amplification products of the expected size and the negative controls (S. durban and S. typhimurium) failed to generate products.


PCR products generated from salmonellae using primer combinations SEFR and ISR (A), SEFR and ISR2 (B). Lane 1: S. durban (IS1230sefA); lanes 2–8: S. enteritidis (IS1230+sefA+); lane 7: S. typhimurium DT4 (IS1230+sefA). M indicates DNA size markers (1 kb ladder).

3.3 Copy number of IS1230 in the genome

The relative strength of the hybridisation signal with IS1230 at different post-hybridisation wash stringencies indicated the presence of IS1230-like sequences with reduced homology, located elsewhere in the genome. Southern hybridisation with IS1230 of total genomic DNA digested to completion with HindIII was used to investigate this possibility. HindIII was chosen for the digests to allow differentiation of IS1230 from other hybridisation signals because of the characteristic three-banded pattern produced with this enzyme (Fig. 3). All serotypes tested possessed at least one fragment which hybridised whilst some had multiple fragments which hybridised. S. enteritidis gave the expected pattern deduced from restriction mapping of a sefA+ cosmid clone, but possessed at least one more weaker fragment which hybridised. This showed that IS1230 was present as a single copy in the genome of S. enteritidis and gave evidence for the presence of another sequence which shared considerable identity located elsewhere in the genome in S. enteritidis and other serotypes. The variable patterns of IS1230-hybridising fragments observed with different Salmonella serotypes has potential as a tool to aid the classification of these organisms and will be investigated further.


Southern analysis of restriction endonuclease digests of Salmonella genomic DNA probed with an IS1230 probe at low stringency. Lanes 1–36 contain HindIII digests of DNA extracted from the following salmonellae respectively: S. essen, S. heidelberg, S. wien, S. typhimurium DT4, S. typhimurium DT8, S. typhimurium DT12, S. infantis, S. choleraesuis, S. nigeria, S. oslo, S. blockley, S. dusseldorf, S. hadar, S. berta, S. blegdam, S. gallinarum, S. miami, S. moscow, S. pullorum, S. wangata, S. dublin 3443, S. dublin 3468, S. dublin 3517, S. enteritidis pt1, S. enteritidis pt4, S. enteritidis pt9a, S. fresno, S. london, S. drypool, S. thomasville, S. aberdeen, S worthington, S. cerro, S. urbana, S. adelaide, S. johannesburg. IS1230 produces three HindIII fragments based on the restriction map (arrowed). Extra bands are attributed to homologous sequences located elsewhere in the genome.


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