Repetitive DNA Sequence Elements Govern Neisserial Biology

One of the most striking and unique characteristics of the annotated neisserial genomes [12, 13] is the abundance and diversity of repetitive DNA. The genomes are replete with an unprecedented number of genetic elements that contribute to both genome fluidity and physical variability, attesting to the adaptability of the Neisseriae and their potential to evade the human immune system. DNA Uptake Sequences, Horizontal Gene Transfer, and Antigenic Diversity

An example of the abundance of repetitive DNA sequence elements is the neisserial DNA uptake sequence which is involved in the recognition and uptake of DNA from the environment [33]. There are nearly 2000 copies of the 10-bp uptake sequence in each genome, which either occurs alone or in inverted repeats as part of a transcriptional terminator [34]. During colonization of the nasopharynx the bacteria are exposed to DNA bearing the appropriate uptake sequence from lysed strains of the same [35, 36] or related species [37], and natural competence of the Neisseriae facilitates uptake of this cognate DNA. Thus, the high competence for natural transformation of Neisseria species together with DNA recombination results in mosaicism of many chromosomal genes [38, 39] as well as in an epidemic population structure [40]. In fact, genetic changes in N. meningitidis occur even more frequently through DNA recombination than by mutation [41]. Recombination is also an important genetic mechanism in the generation of new meningococcal clones and alleles [42] and particularly affects several bacterial structures involved in virulence and/or transmission. For example, it has been established that capsule switching from a serogroup B to a serogroup C capsule phenotype was the result of the substitution of the serogroup B polysialyltransferase with the serogroup C polysialyltransferase after transformation and horizontal transfer of a serogroup C capsule biosynthetic operon [43, 44]. However, the natural frequency of this process is still a matter of debate.

Of note, there was a significantly higher density of DNA uptake sequences within genes involved in DNA repair, recombination, restriction modification, and replication than in any other annotated gene group in these organisms [45]. This finding might reflect facilitated recovery from DNA damage caused by an adverse environment such as oxygen or modification of the pH as occurs inside phago-cytic vacuoles. Additionally, it was shown that induction of a gene implicated in DNA repair (xseB) during the initial step of adhesion increased the ability of the bacteria to repair their chromosome, suggesting that some of the DNA repair mechanisms could be controlled by the interaction of meningococci with host cells [46].

H.2.2.2 Simple Sequence Repeats and Phase Variation

The sequenced genomes of the pathogenic Neisseriae are also littered with a vast number of polymorphic simple sequence repeats, ranging from homopolymeric tracts to pentanucleotide repeats [47]. The length of the homopolymeric or hetero-polymeric tracts, i.e., the number of tandemly repeated motifs, can be modified during replication due to slipped-strand mispairing, and can consequently influence translation or transcription [48-50]. In Neisseria species, phase variation is consistently associated with reversible changes within such simple DNA repeats.

The repertoire of putative phase variable genes was originally determined by the identification of simple DNA repeats in the MC58 genome sequence using criteria based upon the length of the repeat, its position within the gene, and knowledge of tracts in established phase-variable genes in Neisseria. This analysis identified 65 potentially phase-variable genes [51]. Currently, based on the MC58 genome sequence there is experimental evidence of phase variation for 14 genes encoding opacity proteins [52], lipopolysaccharide biosynthesis proteins [53], proteins involved in the biosynthesis and modification of pili [54, 55], hemoglobin receptors [56, 57], PorA outer membrane protein [58], Opc outer membrane protein [48], capsular polysaccharide biosynthesis proteins [49], and a putative adhesin (nadA) [59]. Further computational analysis of the remaining 51 putative phasevariable genes identified in the MC58 genome sequence predicted that 33 genes could be considered phase-variable and 18 nonvariable [59].

In a complementary approach, the results of independent analyses of the complete genome sequences of N. meningitidis strain Z2491 and N. gonorrhoeae strain FA1090 were combined with the previous analysis of N. meningitidis strain MC58 [51] and a comprehensive genomic comparison was carried out to determine the repertoires of potentially phase-variable genes [47]. This comparative whole-genome approach identified 68 phase-variable gene candidates in N. meningitidis strain Z2491, 83 candidates in N. gonorrhoeae strain FA1090, and 82 candidates in N. meningitidis strain MC58 [47, 51]. The number of potentially phase-variable genes in N. meningitidis is substantially greater than that for any other species studied to date [51]. In addition to the sheer number of genes identified, another noteworthy feature is the diversity of functions observed in these genes. While a significant number of genes (31 of 61 with known functions or homologies) fall within the functional categories traditionally associated with phase variation and virulence - e.g., surface proteins, lipopolysaccharide and sugar metabolism genes, toxins and restriction modification genes - a large number of the remainder appear to have other, as yet unknown functions.

An additional mechanism by which Neisseria species adapt to their niche is the variation in the number of the coding tandem repeats within protein coding genes [60]. Coding tandem repeats are those which do not alter the reading frame with copy number, and the changes in copy number of these repeats may then potentially alter the function or antigenicity of the protein encoded. A total of 28 genes were identified in the three complete neisserial genomes containing coding tandem repeats that had structural consequences for the encoded protein [60]. Fourteen genes encode predicted and known surface proteins with coding repeat copy number variation that might result in antigenic variation, including pilQ several putative lipoproteins, the Lip/H.8 antigen, AniA, and a putative adhesin (NMB0586), amongst others. Remarkably, some of the genes identified encode proteins with cytoplasmic functions, including sugar metabolism, DNA repair, and protein production, in which repeat length variation may have other functions.

This large repertoire of genes potentially subject to phase variation results in a stochastically dynamic population of bacteria that maximizes the likelihood of successful establishment in new hosts. Individual neisserial colonies are therefore genetically and phenotypically heterogeneous as a result of the presence of these phase-variable genes. Insertion Sequences and the Regulation of Gene Expression

The genomes are also peppered by several insertion sequences (IS). Most of the insertion sequences vary in size from 0.5 kbp to 1.3 kbp and are members of at least four specific DNA families: IS1016, IS1106, IS1655, and IS4351 [12, 13]. For example, the genome of strain MC58 contains 22 intact and 29 remnant IS [13]. Other repetitive sequence elements in the N. meningitidis genome are concentrated within intergenic repeat arrays of 0.2-2.7kbp. These repeat arrays are composed of several different repeat types including larger units such as so-called "Correia elements" (CEs). They have been described in both N. meningitidis and N. gonorrhoeae [61] and represent about 2% of the N. meningitidis genomes [62]. While the number of CEs is comparable in the N. meningitidis genomes, it is considerably reduced in the N. lactamica and N. gonorrhoeae genomes [63, 64], although estimates of the absolute numbers of CEs vary depending on the methodologies applied [12, 13, 63, 64]. However, the abundance of CEs and the percentage of nucleotides contained in these repetitive elements in the neisserial genomes are higher than those described for comparable intergenic repeats of other prokaryotes [63]. CEs are sequence indels comparable to small insertion sequences 100-155 bp in length, but in contrast to conventional IS elements do not encode a transposase [63-65]. They carry transcription initiation signals [66, 67] as well as functional integration host factor (IHF) binding sites [65, 68], and hence may play a role in modulating gene expression. For example, in gonococci IHF has been shown to bind proximal of the three pilE promoters and function as a transcriptional coactivator [69], whereas it had a negative impact on mtrC transcription in meningococci [68]. In addition, there is growing evidence that CEs may influence gene expression at the posttranscriptional level [64, 70]. The abundance of CEs in the different Neisseria genomes suggests that they may have played a major role in genome organization, function, and evolution. Their differential distribution in different Neisseria strains may also contribute to the distinct behaviors of each Neisseria species.

Another class of repetitive extragenic palindromic (REP) sequence called REP2 was present in 26 copies in the Z2491 genome [12]. Like CE, it was found to contain promoter as well as ribosome binding sites and to influence the expression of a set of genes such as pilCl and crgA which are necessary for the efficient interaction of N. meningitidis with host cells [71].

Taken together, three mechanisms of repeat-mediated antigenic variation operate within the N. meningitidis genome: (1) on/off switching and transcriptional modulation of gene expression by slipped-strand mispairing of short tandem repeats or by reversible insertion of IS elements; (2) intragenomic recombination of localized repeats leading to the use of different carboxy termini for surface-exposed proteins; and (3) intergenomic gene conversion of specific surface-associated genes associated with large arrays of global repeats, mediated by the inter-nalization of related DNA through the highly repetitive DNA uptake sequence. Phase and antigenic variation not only facilitate microbial evasion of immune responses, but the resulting polymorphisms influence all aspects of gonococcal and meningococcal biology.

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