Identification of new IS711 insertion sites in Brucella abortus field isolates
© Mancilla et al; licensee BioMed Central Ltd. 2011
Received: 29 April 2011
Accepted: 3 August 2011
Published: 3 August 2011
Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated). Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains.
We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far.
Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.
Brucella is a genus of bacteria causing brucellosis, a zoonosis that affects a large variety of mammals and that is readily transmitted to humans. The genus includes several classical species that can be distinguished by their preferential host range, surface structure, biochemical and physiological features, and genetic markers. This classification is reflected in some degree of genetic polymorphism, one of the main sources of which is the copy number and distribution of IS711 (IS6501) [1, 2]. B. melitensis and B. suis contain seven complete IS711 copies . B. abortus carries six complete and one truncated IS711 copies , B. ceti and B. pinnipedialis more than 20 copies [5, 6] and B. ovis 38 copies . IS711 is very stable: its mobility has been demonstrated only by using a "transposon trap" in vitro in B. ovis and B. pinnipedialis, but not in B. melitensis and B. abortus . Based on this stability, polymorphism at the alkB locus  is used to differentiate B. abortus from B. melitensis, B. ovis and B. suis in the AMOS multiplex PCR assay .
IS711 stability is not only relevant for Brucella typification: its mobility is implicated in the generation of genetic diversity and speciation, as shown by the distribution of IS711 among the extant Brucella species. Here we report that IS711 transposition and the generation of the associated polymorphism takes place in B. abortus under natural conditions, when genetic drift should be limited by the selective pressure imposed by the host.
Results and discussion
Brucella strains used
Genetic profile by:
RFLP IS711 AvaI-ClaI a
AMOS enhanced PCR b
B. abortus 544
Reference strain of biovar 1
B. abortus 2308
USDA challenge strain; biovar 1
B. abortus RB51
Vaccine rough derivative from 2308
B. abortus B51 c
Biovar 1; milk isolate (Río Bueno, Chile; 2004)
B. abortus B12 c
Biovar 1; milk isolate (Río Bueno, Chile; 2004)
B. abortus B16 d
Biovar 1; aborted fetus isolate (Osorno, Chile; 2002)
B. abortus B49 d
Biovar 1; aborted fetus isolate (Osorno, Chile; 2000)
B. abortus B50 d
Biovar 1; aborted fetus isolate (Osorno, Chile; 2004)
B. ovis 23/290
B. ovis reference strain
B. ceti NCTC 12891T
B. ceti type strain
B. pinnipedialis NCTC 12890T
B. pinnipedialis type strain
B. abortus 2308 NalR
Nalidixic acid resistant derivative of 2308 strain
Primers used in this work
IS transposition can disrupt genes and produce negative polar effects, but also cause beneficial changes by remodeling genomes through long range recombination . In the case of strain B12, it is uncertain whether the intergenic position of IS711 disturbs the expression of nearby genes. Most IS711 studied in detail (1a, 2a, 3a, 5a, 6a, xa and x-08) are also located within intergenic regions showing that transposition is mostly viable when occurring into neutral sites. However, the extra IS711 copy in B16, B49 and B50 interrupts a putative transcriptional regulator that is expressed during the late-logarithmic phase of growth in B. melitensis (BMEII0520)  and, interestingly, these strains did not show urease activity, a factor that has been proposed to favor Brucella gastrointestinal infections in mice . We investigated whether the marR mutation was involved in the urease-negative phenotype by constructing a B. abortus 2308 ΔmarR mutant. This mutant displayed urease activity (not shown), suggesting that the absence of urease in B16, B49 and B50 is probably caused by mutation(s) in ure genes . The fact that these urease negative marR mutant strains were repeatedly isolated from aborted fetuses for at least four years questions the relevance of this factor in placental colonization and abortion induction. Research is in progress to characterize the genetic background of this urease negative phenotype.
In this report, we have provided evidence that IS711 polymorphism occurs in B. abortus field strains. The fact that such polymorphism can take place in sites shared with related species points out the relevance of a multiple-marker approach in molecular typing of Brucella species. In addition, our results suggest that the extra IS copies might originate from what seems to be the most active IS711 copy. Although the environmental signals involved in the activation of the transposase remain unknown, host-pathogen interactions may play a role. Further work is needed to elucidate if changes promoted by IS transposition are associated with virulence fluctuations in this pathogen.
Bacterial strains, growth conditions, plasmids and DNA manipulation
The Brucella strains studied are listed in Table 1 and the E. coli strains and plasmids used are in the Additional file 2. Bacteria were stored in tryptic soy broth (Becton Dickinson, Sparks, Md) with 20% glycerol at -70°C and, for routine use, grown on tryptic soy agar (when necessary under a 5% CO2 atmosphere) for 24-48 h at 37°C. Plasmids were obtained with Qiaprep (Qiagen, Hilden, Germany). PCR products and genomic DNA were purified with a QiaexII kit (Qiagen) or by standard protocols .
Molecular typing techniques
AMOS PCR was carried out as described before . For IS711 Southern blots, genomic DNA (1-2 μg) was digested with AvaI and ClaI (Fermentas Inc, Burlington, Canada) at 37°C overnight, the fragments resolved in 1.0% agarose at 15 mA for 10 h, blotted on nylon, fixed at 80°C for 30 min and probed with a biotin-labelled IS711 fragment obtained by PCR with primers 711u and 711d (Table 2). Hybridization was performed at 42°C for 2 h, and detected by chemiluminescence (KPL, Gaithersburg, MD) .
Genome mapping of new IS711insertion sites
For IS-anchored PCR, we adapted a protocol previously described . IS711-bound primers RB51 and IS711out in combination with an arbitrary primer P5 (Table 2) were used to generate a pattern of PCR products specific for diverse IS positions. The reaction mixture contained 0.2 μM of RB51 or IS711out primers and P5 decamer, 5.0 μl of 10X enzyme buffer, 2 mM of MgCl2, 0.4 mM of dNTP, 1 U of Taq polymerase (Invitrogen) and 10 ng of genomic DNA. The amplification conditions were: 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec; annealing at 55°C for 30 sec; extension at 72°C for 2 min; final extension at 72°C for 5 min. Amplicons were electrophoresed in 1.5% agarose in 20 mM Tris, 20 mM acetic acid, 1 mM EDTA, and detected with ethidium bromide.
Cloning and sequence analysis
Specific IS-anchored and flanking PCR products purified from gels were cloned into the pCR2.1 vector (Invitrogen) and sequenced by fluorescence-labeled dideoxynucleotide technology (Macrogen Inc, Seoul, South Korea). Sequences were analyzed by BLASTN (http://www.ncbi.nlm.nih.gov/). Comparison of the IS711 sequences in the B. abortus 9-941 genome (accession numbers AE017223 and AE017224)  and the new IS711 was performed with ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2). Sequences of new IS711 were deposited under GenBank accession numbers: JF345125 and JF345126.
Construction of B. abortus 2308 ΔmarRmutant
A B. abortus 2308 NalR ΔmarR non polar mutant was constructed by allelic exchange  with primers designed on the sequence of marR (BAB2_0468, the marR homologous). Briefly, two fragments generated with primer pairs marR-F1, R2 and marR-F3, R4 (Table 2) were ligated by overlapping PCR and the resulting fragment (containing a ΔmarR lacking the nucleotides corresponding to amino acids 13-120) was cloned into pCR2.1 to produce plasmid pMM19 (Additional file 2). The BamHI-NotI fragment of pMM19 was subcloned into plasmid pJQK  to generate the pMM21 suicide vector (Additional file 2), which was transferred to B. abortus 2308 NalR by conjugation with a suitable E. coli strain . Nalidixic acid and sucrose resistant clones were screened by PCR, and tested for urease .
Acknowledgements and funding
We thank Servicio Agrícola y Ganadero de Chile (SAG) for providing Brucella strains.This work was funded by FONDEF D02I 1111, CONICYT-FIC-R-EQU18, the Department of Research and Development at Universidad Austral de Chile, project S-2009-33 and Ministerio de Ciencia y Tecnología of Spain (AGL2008-04514). MM was supported by CONICYT-Ph.D. fellowship (Chile) and PIUNA grant (Universidad de Navarra).
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