Molecular Infectious Diseases Group, Institute of Molecular Medicine, University of Oxford, Headington, Oxford OX3 9DS, UK 1
Oxford University Bioinformatics Centre, Sir William Dunn School of Pathology, South Parks, Oxford OX1 3RE, UK2
Author for correspondence: Nigel J. Saunders. Tel: +44 1865 222347. Fax: +44 1865 222626. e-mail: saunders{at}molbiol.ox.ac.uk
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ABSTRACT |
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Keywords: uptake sequence, Helicobacter pylori , transformation
Abbreviations: US, uptake sequence
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INTRODUCTION |
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Most strains of Hel. pylori are naturally transformable (Nedenskov-Sorensen et al., 1990 ; Wang et al. , 1993
; Tsuda et al., 1993
). Naturally transformable bacteria are able to take up DNA readily from their environment and have the capacity to use it as a means of horizontal exchange of genetic information. Other naturally transformable bacteria have developed strategies to increase the likelihood that the substrate for recombination is derived from related bacteria (Solomon & Grossman 1996
; Saunders et al. , 1999
). In Gram-positive bacteria this is typically achieved through signals of growth phase and cell density that foster transformation only when the majority of the potential donor bacteria are the same species. In Gram-negative bacteria, as exemplified by Haemophilus and Neisseria spp., it is achieved by using uptake sequences (USs) which are involved in enhancing the binding and uptake of homospecific DNA.
The US of Haemophilus spp. is a sequence containing a 9 bp core motif, AAGTGCGGT, of which there are 1465 copies in the Haemophilus influenzae strain Rd genome (Smith et al., 1995 ). This US influences the process of DNA uptake into the recipient cell (Sisco & Smith 1979
; Danner et al., 1982
). Similarly, Neisseria spp. possess a 10 bp US (GCCGTCTGAA) that performs a similar role (Goodman & Scocca, 1988
; Elkins et al., 1991
). In both cases, USs often occur in inverted pairs and are typically located at the 3' end of ORFs where they are believed to act as transcriptional terminators a function which may account for their frequency and distribution in the chromosome. By using USs, related species provide a pool of genes that are available to the other members of the species.
Searching the complete Hae. influenzae genome sequence for frequent words readily identifies the US (Karlin et al., 1996 ) and this approach also identifies the US in those Neisseria meningitidis and Neisseria gonorrhoeae genome sequences that are available (N. J. Saunders, unpublished). We have used the approach of frequent word searching to demonstrate the absence of an US in the Hel. pylori strain 26685 genome sequence.
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METHODS |
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Where investigated, the sequence context of frequently occurring words was examined using ACEDB, a graphical user interface, to a whole genome analysis system (Saunders et al., 1998 ).
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RESULTS AND DISCUSSION |
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The most frequent words in Hel. pylori are composed of poly(A) and poly(T) repeats. Many of these have the potential to form hairpin loops that could form a structure with similarity to that formed by inverted pairs of USs in Hae. influenzae and N. meningitidis. In order to determine the distribution of these sequences and whether they are distributed similarly to other USs, the context of each 12-mer word (TTTTTTAAAAAA) was determined. They occur both intra and inter-genically (15 intergenically, 24 within probable coding sequence within ORFs, and in one case the central TAA is the stop codon of an ORF) and are therefore not located in a way similar to that of known USs.
Finally, a measure of functional selection for word 12 on the basis of Markov chain predictions (Table 2 ) was made. The analysis revealed that this word is present less frequently than the occurrence of its components would predict and that the largest discrepancy in the occurrence of its components is at the transition between 3- and 4-mers. This is indicated by the abundance of the heptamer sequences over that predicted on the basis of the occurrence of di- and trinucleotides (as indicated by the Markov chain n-3 prediction in Table 2
). In contrast, predictions based on longer words are much closer to observed results. Looking at the table as a whole, there would appear to be a bias towards the presence of two adenines after the run of thymidines. It is therefore clear that the abundance of the longer words is a consequence of the abundance of its component tetramers, rather than selection of the larger words. The abundance of the similar words is a product of the same phenomenon.
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The function of the targeting systems such as USs in Gram-negative bacteria and indicators of cell density in Gram-positive bacteria is to enhance the probability that the transforming DNA in donor and recipient is similar. This relative exclusivity increases the likelihood that products of recombination are going to be functional. There are several possible explanations for why Hel. pylori lacks an US. As described previously, a typical US of Neisseria and Haemophilus is located 3' of expressed ORFs where it is thought to act as a transcriptional terminator. At some point in the evolution of these species this sequence has been co-opted to a second function in transformation. This second function may have affected the sequence, structure and location of the US but it is hard to see how it could have developed without the pre-existing wide distribution of the sequence from which it arose. One possibility is that Hel. pylori has never possessed a suitable transcriptional terminator that could evolve this second function.
Another interesting possibility is that Hel. pylori has never been under selective pressure to develop a targeting system akin to those present in other species. Other naturally transformable bacterial species live in niches colonized by a mixed bacterial flora and would be exposed to DNA derived from unrelated bacteria much of the time. Hel. pylori resides for a large proportion of its life cycle and generations in the gastric mucosa. Colonization by multiple strains of Hel. pylori does occur (Taylor et al., 1995 ; van der Ende et al., 1996
; Jorgensen et al., 1996
; Berg et al., 1997
), so opportunity for horizontal exchange between strains exists in nature. However, the stomach is not colonized by many other bacterial species. It may be that this ecological separation makes an additional mechanism unnecessary. The potential for such separation to contribute to the maintenance of distinct populations is seen in the pathogenic Neisseria spp., which share a common US and are able to exchange DNA, but exist as clearly separate species (Vazquez et al. , 1993
).
Another factor acting to maintain the integrity of the species is the availability of substrates for homologous recombination. However, there are examples of the acquisition of whole genes for which the presence of a similar sequence in the recipient cannot be a pre- requisite (Vazquez et al., 1995 ). In the absence of a targeting system, Hel. pylori might be expected to have an increased likelihood of incorporating DNA from unrelated species when it is encountered. In this context, it is interesting to note that the sigma-factor-encoding rpoD gene of Hel. pylori has strongest similarity to those from Gram-positive rather than Gram- negative bacteria (Solnick et al., 1997
). Further, the cytochrome-c biogenesis system in Hel. pylori is a Type II system, which is typically found in Gram-positive bacteria, cyanobacteria and chloroplasts rather than Gram-negative bacteria, which normally have a Type I system (Goldman & Kranz, 1998
). Finally, it is striking that a large number of genes share an unusually high amount of sequence similarity with genes from other taxonomic groups although it is necessary to be circumspect about this interpretation given the bias in current databases used for the comparisons.
Horizontal transfer is undoubtedly important in the population biology and evolution of Hel. pylori. Although additional factors may exist, including a deoxyribonuclease resistance mechanism (Kuipers et al., 1998 ), natural transformation is probably an important means of genetic exchange in this species. Natural transformation can be observed in vitro, the nature of the genetic diversity observed in Hel. pylori is similar to that present in other naturally transformable species and co- colonization by multiple strains has been demonstrated. The absence of an US in such an organism is novel and we suggest reflects the environment in which Hel. pylori resides. Furthermore, it suggests that this species may have a greater propensity to be the recipient for interspecific horizontal transfer of genes than is the case for other naturally transformable bacteria.
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ACKNOWLEDGEMENTS |
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Received 4 May 1999;
revised 17 August 1999;
accepted 10 September 1999.