REP code: defining bacterial identity in extragenic space

Through the analysis of 57 bacterial genomes we have detected repetitive extragenic palindromic DNA sequences (REPs) in 11 species. For a sequence to be considered as REP, the following criteria should be met: (i) It should be extragenic, (ii) palindromic, (iii) of a length between 21 and 65 bases and (iv) should constitute more than 0.5% of the total extragenic space. Species-specific REPs have been found in human pathogens such as Escherichia coli, Salmonella enterica, Neisseria meningitidis, Mycobacterium tuberculosis, Rickettsia conorii and Pseudomonas aeruginosa, the plant pathogen Agrobacterium tumefaciens and the soil bacteria Deinococcus radiodurans, Pseudomonas putida and Sinorhizobium meliloti.     

In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

Les BIMEs: un exemple de séquences répétées chez les entérobactéries

Bacterial interspersed mosaic elements (or BIMEs) are repeated sequences identified in several enterobacterial genomes. BIMEs are a mosaic combination of small sequence motifs. It has been estimated that 500 BIMEs are scattered over the bacterial genome. BIMEs have been identified in several enterobacteria: Escherichia coli, Salmonella typhimurium, Klebsiella sp. and relatives of these bacteria. BIME function is not known, but their interactions with specific proteins (DNA polymerase I, gyrase and integration host factor) suggest that they could be involved in functional organization of bacterial chromosomes. Four other families of interspersed repetitive sequences have been shown to exist in a variety of bacterial genomes. Like BIMEs, these sequences are rather small, contain a region of dyad symmetry, and are found in extragenic locations. Unlike BIMEs, IRU (or ERIC), box C sequences and RSA sequences occur in enterobacteria but also in other Gram-negative bacteria.     

Enterobacterial repetitive intergenic consensus (ERIC) sequences in Escherichia coli: Evolution and implications for ERIC-PCR

Enterobacterial repetitive intergenic consensus (ERIC) sequences are 127-bp imperfect palindromes that occur in multiple copies in the genomes of enteric bacteria and vibrios. Here we investigate the distribution of these elements in the complete genome sequences of nine Escherichia coli (including Shigella species) strains. There is a significant tendency for copies to be adjacent to more highly expressed genes. There is considerable variation among strains with respect to the presence of an element in any particular intergenic region, but some copies appear to have been conserved since before the divergence of E. coli and Salmonella enterica. In comparisons of orthologous copies between these species, ERIC sequences are surprisingly conserved, implying that they have acquired some function, perhaps related to mRNA stability. The relationships among copies within E. coli are consistent with a master copy mode of generation. Insertion of new copies seems to occur at, and involve duplication of, the dinucleotide TA. Two classes of inserts of about 70 bp each occur at different specific sites within ERIC sequences; these inserts evolve independently of the ERIC sequences. The small number of ERIC sequences in E. coli genomes indicates that a widely used bacterial fingerprinting method using primers based on ERIC sequences (ERIC-PCR) does not rely on the presence of ERIC sequences.     

Within-genome evolution of REPINs: a new family of miniature mobile DNA in bacteria

Repetitive sequences are a conserved feature of many bacterial genomes. While first reported almost thirty years ago, and frequently exploited for genotyping purposes, little is known about their origin, maintenance, or processes affecting the dynamics of within-genome evolution. Here, beginning with analysis of the diversity and abundance of short oligonucleotide sequences in the genome of Pseudomonas fluorescens SBW25, we show that over-represented short sequences define three distinct groups (GI, GII, and GIII) of repetitive extragenic palindromic (REP) sequences. Patterns of REP distribution suggest that closely linked REP sequences form a functional replicative unit: REP doublets are over-represented, randomly distributed in extragenic space, and more highly conserved than singlets. In addition, doublets are organized as inverted repeats, which together with intervening spacer sequences are predicted to form hairpin structures in ssDNA or mRNA. We refer to these newly defined entities as REPINs (REP doublets forming hairpins) and identify short reads from population sequencing that reveal putative transposition intermediates. The proximal relationship between GI, GII, and GIII REPINs and specific REP-associated tyrosine transposases (RAYTs), combined with features of the putative transposition intermediate, suggests a mechanism for within-genome dissemination. Analysis of the distribution of REPs in a range of RAYT-containing bacterial genomes, including Escherichia coli K-12 and Nostoc punctiforme, show that REPINs are a widely distributed, but hitherto unrecognized, family of miniature non-autonomous mobile DNA.     

Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.     

Palindromic units from E. coli as binding sites for a chromoid-associated protein

Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.     

Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene

Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.     

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.     

Cutting edge: natural DNA repetitive extragenic sequences from gram-negative pathogens strongly stimulate TLR9

Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-alpha production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9(tm1Aki) knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.     

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.     

Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria.

The distribution of dispersed repetitive DNA (repetitive extragenic palindromic [REP] and enterobacterial repetitive intergenic consensus [ERIC]) sequences in the genomes of a number of gram-negative soil bacteria was examined by using conserved primers corresponding to REP and ERIC sequences and the polymerase chain reaction (PCR). The patterns of the resulting PCR products were analyzed on agarose gels and found to be highly specific for each strain. The REP and ERIC PCR patterns of a series of Rhizobium meliloti isolates, previously ordered in a phylogenetic tree based on allelic variations at 14 enzyme loci (B. D. Eardly, L. A. Materon, N. H. Smith, D. A. Johnson, M. D. Rumbaugh, and R. K. Selander, Appl. Environ. Microbiol. 56:187-194), were determined. Isolates which had been postulated to be closely related by multilocus enzyme electrophoresis also revealed similar REP and ERIC PCR patterns, suggesting that the REP and ERIC PCR method is useful for the identification and classification of bacterial strains.     

Species-specific Typing of DNA Based on Palindrome Frequency Patterns

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Clusters of diverse genes existing as multiple, sequence-variable mosaics in a phytoplasma genome

Phytoplasmas are cell wall-less prokaryotes living as obligate parasites and pathogens of plants and insects, making them attractive subjects for studies to gain a greater understanding of transkingdom parasitism and pathogenicity. During a study of two phytoplasma genomes, we obtained evidence for previously unreported clustering of genes, pseudogenes, mobile genetic elements, intergenic repeat units, and repetitive extragenic palindromes that occur in multiple, homologous clusters in some phytoplasma genomes. The clusters represent previously unrecognized mosaics, possibly assembled through multiple events of targeted mobile element attack, duplication, recombination, and rearrangement. Multiple clusters could conceivably afford potential for genome reduction through homologous recombination. Differences in the sizes and multiplicity of such clusters possibly account for some of the previously reported but unexplained variations in genome size among closely related phytoplasma strains.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

Heterogeneity of<Emphasis Type="Italic">Escherichia coli</Emphasis> derived from artiodactyla animals analyzed with the use of rep-PCR fingerprinting

Genetic polymorphism of 83 isolates of E. coli, derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Swierkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers. The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates. The arithmetic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E. coli isolates into three similarity groups containing various numbers of isolates. The groups comprised isolates derived from two, three and four species of the source animals. The isolates derived from each source segregated in the dendrogram in a different way, both within the similarity groups and among them, indicating an individual repertoire of E. coli in the examined species of animals. The similarity relations among E. coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (< or = 20%), indicated an essential interstrain differentiation in terms of the distribution of REP sequences. Our results confirmed the hypothesis of the oligoclonal characters of populations obtained from particular sources. The rep-PCR fingerprinting method with REP primers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.     

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.     

Whole-genome sequence of the transformable Neisseria meningitidis serogroup A strain WUE2594

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.