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Title: The Transposon-Like Correia Elements Encode Numerous Strong Promoters and Provide a Potential New Mechanism for Phase Variation in the Meningococcus}
Article:
Abstract:
Neisseria meningitidis is the primary causative agent of bacterial meningitis. The genome is rich in repetitive DNA and almost 2% is occupied by a diminutive transposon called the Correia element. Here we report a bioinformatic analysis defining eight subtypes of the element with four distinct types of ends. Transcriptional analysis, using PCR and a lacZ reporter system, revealed that two ends in particular encode strong promoters. The activity of the strongest promoter is dictated by a recurrent polymorphism( Y128) at the right end of the element. We highlight examples of elements that appear to drive transcription of adjacent genes and others that may express small non-coding RNAs. Pair-wise comparisons between three meningococcal genomes revealed that no more than two-thirds of Correia elements maintain their subtype at any particular locus. This is due to recombinational class switching between elements in a single strain. Upon switching subtype, a new allele is available to spread through the population by natural transformation. This process may represent a hitherto unrecognized mechanism for phase variation in the meningococcus. We conclude that the strain-to-strain variability of the Correia elements, and the large number of strong promoters encoded by them, allows for potentially widespread effects within the population as a whole. By defining the strength of the promoters encoded by the eight subtypes of Correia ends, we provide a resource that allows the transcriptional effects of a particular subtype at a given locus to be predicted.
Introduction:
Neisseria meningitidis is an encapsulated Gram-negative diplococcus commensal of the human nasopharyngeal tract. Although carried asymptomatically by 10–15% of the population, it occasionally crosses the epithelial cell barrier, causing bacterial meningitis and septicemia. Vaccination against serogroup A and C strains limits the impact of the disease in developed countries. However, the disease remains a significant problem in the meningitis belt of sub-Saharan Africa, where epidemics begin at the start of the dry season and may affect up to 1% of the population[1],[2]. Even in the UK there are about 3000 cases each year. The disease has a rapid onset and is almost always fatal if untreated. The N. meningitidis genome contains a relatively large amount of repetitive DNA. The repeats range in size from single nucleotide homopolymeric tracts, which mediate antigenic phase variation, to larger repeats of unknown or uncertain function[3]–[6]. One of the most abundant repeats is a miniature inverted-repeat transposable-element( MITE) first identified by Correia and colleagues in 1986[7],[8]. We refer to the repeat as the Correia element( CE), but it has also been known as NEMIS( Neisseria miniature insertion sequence) and CREE( Correia repeat enclosed element). The archetypal genome sequences for the serogroup A, B and C strains of N. meningitidis( Z2491, MC58 and FAM18, respectively) each contain about 250 intact CEs[3],[5],[6]. Insertion of the element is accompanied by duplication of a TA dinucleotide at the target site[9]. This is the hallmark of the mariner transposons, represented in the eubacteria by the IS630 family[10]–[13]. Short dispersed repeats, such as the CE, are dispersed and amplified by transposition and DNA recombination. However, their persistence in large populations of free-living bacteria, where natural selection is strong, has prompted frequent speculation that they directly benefit their hosts eg.[14]. Early chemostat experiments, in which strong nutritional selection was applied, revealed that most successful mutations were not in structural genes, but in their regulatory regions[15]. Many of these changes were due to transposons. This phenomenon is not restricted to bacteria: for example, transposon insertions upstream of the Cyp6g1 gene in Drosophila melanogaster have spread to high frequency in response to the use of insecticides[16]. There are a number of ways in which transposons can change the pattern of gene expression and alter host cell physiology[15],[17]. In the simplest case, an insertion may inactivate the gene encoding a transcriptional inducer or repressor. Insertions may also increase the distance between regulatory elements, interfering with activation or relieving repression. Transposons also have more direct mechanisms to control transcription. Many have constitutive promoters that drive transcription outwards from one end of the element[18]–[21]. Indeed, it is transposon-encoded promoter activity that is responsible for the successful chemostat take-over events and the spread of Cyp6g1 alleles in Drosophila mentioned above. CEs appear to influence their hosts in multiple ways. At the DNA-level, CEs are hotspots for DNA recombination and rearrangement[9],[22]. At the RNA-level, CEs that are co-transcribed with adjacent genes are often targets for cleavage by RNase III[23]–[25]. Such processing may either stabilize or destabilize transcripts, potentially altering gene expression levels. CEs have also been proposed to act as transcriptional terminators, a consequence of their stem-loop structures and frequent presence near the 3′ end of genes[26]. The Correia terminal inverted repeat( TIR) also contains a sequence resembling a −35 box for the σ70 class of promoters located 17 nucleotides upstream of a TATA sequence that forms at the end of the element as a result of insertion into a target site( Figure 1)[9],[27]. Consequently, CEs have the potential to form outward-facing promoters at their insertion sites. In fact, CEs have been shown to contribute to the transcription of the meningococcal lst and hemO genes and the gonococcal uvrB gene[27]–[29]. Although such studies have identified transcripts emanating from individual CEs, a detailed examination of CE transcription, taking into account the variation that exists amongst copies, has not hitherto been performed. The large number of CEs in the genome, their potential to influence gene expression patterns, and the variation in their complement between different strains, raises the question as to whether they are significant determinants of meningococcal physiology. This is important because most cases of meningococcal disease are caused by a few persistent hyper-invasive lineages and the physiological differences between these strains remain unclear. Bioinformatic analysis alone is not sufficient to settle such questions. We have therefore taken an experimental approach by measuring the strength of the promoters encoded by the CEs. We identify eight different subtypes, some of which have much higher promoter activity than others. The activity of the strongest promoter is dictated by a recurrent single polymorphism in the −35 box of the TIR. We present a genome-wide analysis of the elements with the strongest activity, focusing on their flanking sequences and distribution in the population.
Discussion:
The promoter activity of the α-rightY128T repeat is 10 to 20-fold higher than that of α-rightY128C( Table 1 and Table 2). The thymidine responsible for this dramatic difference is also present within the α-left end and it seems likely to contribute to the strong transcription from this end as well. In previous studies, the putative −35 box was positioned one nucleotide closer to the end of the CE[9],[28]. However, the large effect of the Y128 polymorphism on transcription argues in favor of the new position illustrated in Figure 1. Interestingly, the Y128T mutation in the β TIR does not raise promoter activity as much as it does at the α TIR. This difference is probably due to the greater spacing between the −10 and −35 boxes of the β end relative to the α end( 18 versus 17 nucleotides). Black and colleagues predicted a stationary-phase, σS( rpoS)-dependent “gearbox” promoter in the prime version of the CE, but not in the full-length version, where a 50 bp insertion separates the −10 and −35 boxes[27]. Subsequent genome sequencing revealed that rpoS is absent in the meningococcus and the gonococcus[6],[42]. However, a gearbox promoter could have affected our transcriptional analysis in E. coli, which does encode rpoS. This does not appear to be the case because a comparison of transcription from the right end of the full-length and prime elements shows no discernable effect of the putative gearbox promoter on the activity of any of the reporter constructs( Table 2). An intriguing aspect of the structure of the CE is the presence of an IHF binding site in the full-length element. IHF is a histone-like protein which bends DNA by 180° upon binding[43]. In E. coli, it has a role as an accessory protein in a variety of cellular processes including replication, recombination and transcription[44]. The Correia IHF-binding site has been shown to bind IHF protein from E. coli and N. gonorrhoeae in gel shift mobility assays[9],[45]. Consequently, we hypothesized that IHF might modulate the activity of the Correia promoter. However, β-galactosidase assays performed with CE-lacZ reporter constructs indicated that IHF has no significant effect on CE transcription( Table 2 and Table 3). We therefore wonder whether the primary effect of IHF may be on genomic architecture and compaction of the nucleoid. In this capacity, it may alter the expression of genes at a distance by bringing distil regulatory elements together. A comparison of CEs from Z2491, MC58 and FAM18 reveals several conversion events in which one class of Correia repeat at a given locus is replaced by another. For example, the αL-αRY128T element in Z2491 on line 3 of Table 4 has been converted to αL-αRY128C in MC58. This should have the effect of reducing CE-driven transcription of the adjacent threonine tRNA gene in MC58. Clearly, Correia end subtype switching has the potential to act as a mechanism for phase variation, in which the transcription of genes under the influence of a CE is modulated by the recombination-mediated switching of Correia promoters. Indeed, we have surveyed CE class switching in the meningococcal reference collection and find that the differences are highly correlated with the various clonal complexes( to be presented elsewhere). Class switching also has the potential to affect gene expression by altering the sensitivity of CE-containing transcripts to cleavage by RNase III. RNase III targets CE-derived stem-loop structures in transcripts, and is sensitive to point mutations that enhance or diminish the stem-loop[24]. In our analysis of the Z2491 genome we focused on the α-rightY128T repeat, which provides the strongest promoter activity in our assay( Table 2). However, one should note that other classes of element, particularly the α-left repeat, also provide significant promoter activity and may be linked to important functions. In each of the three strains studied there are over 100 α-rightY128T and α-left promoters. Could they substantially impact gene expression in the organism? We provide evidence for the transcription of gidA, a tRNA modification gene, from a nearby α-rightY128T promoter( Figure 3). gidA mutants have pleiotropic effects in bacteria and include virulence defects in Streptococcus pyogenes and Aeromonas hydrophila.[46],[47]. In this example, the α-rightY128T end is retained in all three meningococcal strains( Table 4, line 2; Table S3, line 1; Table S4, line 2). However, it will undoubtedly be of much interest to consider whether natural variation in the distribution of CEs contributes to the development or persistence of hypervirulent lineages that are the source of most global meningococcal disease. During our analysis of Correia α-rightY128T ends in Z2491, we observed that several Correia promoters oriented towards the 3′-end of adjacent ORFs were located within short distance of a downstream transcriptional terminator. RT-PCR analysis detected RNA transcripts from two of the promoters. N. meningitidis does not have an extensive protein-based regulatory network for transcription and small non-coding RNAs might play a role in helping to bolster or expand this relatively skeletal network. Certain CEs might also produce transcripts that read into the 3′-end of genes at some loci. These “antisense” transcripts could act in cis to modulate expression of the adjacent gene( s). Although cis-acting regulatory RNAs in bacteria are typically associated with extra-chromosomal and mobile elements[48], the plasticity of the meningococcal genome may favor this type of regulation. CEs are not simply the degenerate remnants of transposition events that have accumulated over long periods of time. The homogeneity of CE sequences suggests that they were created relatively recently in a burst of transposition. It is not possible at present to say whether the transposition events took place in a single lineage, and were spread subsequently by genetic exchange, or whether they are the result of separate amplification events in multiple lineages. The picture is further complicated by the evidence for gene conversion between elements, as exemplified by the inter-conversion of different subtypes of CEs. These issues make it difficult to know whether CEs are under selection or evolving neutrally. However, under any model, functional elements may arise occasionally by chance. In identifying the strongest CE promoters the present work provides a way to assess the potential importance of specific CEs at loci of interest.
Transposons are mobile DNA elements that can jump from one location in the genome to another . They have had a profound influence on the evolutionary history of most , if not all , organisms by rearranging the order of genes and changing their expression patterns . The mariner family of transposons is probably the most successful group if judged by the breadth and depth of its phylogenetic distribution . One example is the Correia element , which has been amplified to a few hundred copies in Neisseria meningitidis . Transposons often encode promoters that drive the expression of adjacent genes . This raises the question of whether the large numbers of Correia elements in N . meningitidis have a significant genome-wide role in the control of gene expression . This is an interesting issue because N . meningitidis has evolved recently , having been first recognized in the 19th century , and is probably undergoing a period of rapid adaptation . Here we present a systematic analysis that defines eight sub-classes of Correia elements . We show that two subtypes encode strong promoters . The differential distribution of the strongest Correia promoter in the three strains provides a snapshot of evolution in action and sheds new light on the role of dispersed repeats in bacterial genomes .