Genomic island excisions in Bordetella petrii

Abstract

Background: Among the members of the genus Bordetella B. petrii is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing B. petrii from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs). These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds.

Results: During in vitro culture of Bordetella petrii colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of B. petrii revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6) are highly related to ICEclc of Pseudomonas knackmussii sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5) we could detect circular intermediates. For the clc-like elements GI1 to GI3 of B. petrii we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from the bacterial population within about 100 consecutive generations. Furthermore, we show that GI3 is self transmissible and by conjugation can be transferred to B. bronchiseptica thus proving it to be an active integrative and conjugative element

Conclusion: The results show that phenotypic variation of B. petrii is correlated with the presence of genomic islands. Tandem integration of related islands may contribute to island evolution by the acquisition of genes originally belonging to the bacterial core genome. In conclusion, B. petrii appears to be the first member of the genus in which horizontal gene transfer events have massively shaped its genome structure.

Figure 1

A schematic presentation of the genomic islands described for B. petrii by bioinformatic analysis is shown [14]. Direct repeats (DR) flanking the islands and their sequence position in the B. petrii genome are indicated. Direct repeats with identical or nearly identical DNA sequence are shown in the same colour (see also Figure 4). The approximate location of several characteristic genes such as the parA, ssb and topB genes found on all clc-like elements, integrases (int), or some relevant metabolic functions encoded by the islands are shown. In case tRNA genes are associated with the islands these are shown with an arrow indicating their transcriptional polarity. Finally, the approximate sizes of the predicted islands are indicated.

Figure 2

The left panel shows genomic rearrangements of three spontaneous colony variants of B. petrii. Genomic DNA of B. petrii wild type (1), variant f (2), variant g (3) and variant k (4) was cut with BcuI and separated by pulsed field electrophoresis. The red arrows indicate three bands which are missing in the three variants as compared to the wild type. The right panel shows a representative pulsed field gel of wild type B. bronchiseptica PS2 (lane 1), B. petrii (lane 2) and the two GI3::tet^R transconjugants of B. bronchiseptica (lanes 3,4) after digestion with BcuI. The red arrows indicate the additional bands present in the transconjugants as compared to B. bronchiseptica wild type.

Figure 3

Schematic presentation of the genomic region comprising the genomic islands GI1, GI2 and GI3. The GIs are shown as a red lines, their flanking direct repeat regions (DR) by red boxes (dark and light red for identical or nearly identical sequences, respectively) (see also Figure 4). The sequence position of the direct repeats and the approximate size of the islands are shown below the elements. The position of tRNA genes is indicated. Some relevant or characteristic genes encoded by the islands are shown above the elements. The bars below the elements show the expected dimensions of the element after excision from the genome. Stars indicate predicted elements which may use alternative direct repeat sequences for excision or elements composed of more than one island. Arrows above the bars indicate the approximate position of PCR primers and their names (in blue) designed for the amplification of the respective circular intermediates of these elements (Tab. 3).

Figure 4

The direct repeats generated by the integration of the clc-like elements in the B. petrii genome are shown. Identical sequences are indicated in red or blue letters, respectively. Sequence identities are indicated by vertical bars. The positions of the sequences on the genome sequence are shown on the left and the right of the sequences. The core region identical in all repeats flanking the clc-like elements is indicated by the green box. In case the repeats are part of a tRNA gene, the respective gene is mentioned on the right side of the respective sequences.

Figure 5

Stability of the genomic island GI3 in the genome of B. petrii during culture grown without selective pressure. On the x-axis the number of consecutive generations of the bacteria culture and on the y-axis the proportion of tetracycline resistant bacteria in the culture is shown.

Figure 6

Comparison of the integration sites of GI1–GI3 in B. petrii (on the top) and of GI3::tet ^R in B. bronchiseptica PS2 (below). Above the respective DNA sequences a schematic presentation of the integration regions is shown. In B. petrii GI1–GI3 is integrated in a tRNA^Gly gene (tRNA11) leading to an 18 bp duplication of the 3'-end of the tRNA (red boxes). On the left side of the integration side an inverted repeat (IR) is indicated. Upstream of the IR a gene encoding a tRNA^Cys is located. In B. bronchiseptica GI3::tet^R is once more integrated in a gene encoding a tRNA^Gly (tRNA45) leading to a 18 bp duplication of its 3'-end. Much alike in B. petrii the direct repeat sequence is followed by an inverted repeat (IR). Below the schematic presentations of the integration regions the respective DNA sequences of the integration sites are shown. The start points of the tRNA genes are indicated by horizontal arrows indicating transcriptional polarity of the genes followed by a bar marked with a star which indicates the end of the tRNA gene. Vertical arrows indicate the integration sites of the GIs in the tRNA genes. Related inverted repeat sequences (IR) present in both species are boxed. In the case of B. bronchiseptica the sequence position indicated is taken from the genome sequence of strain RB50 [13].