Abundant Oligonucleotides Common to Most Bacteria

Background

Bacteria show a bias in their genomic oligonucleotide composition far beyond that dictated by G+C content. Patterns of over- and underrepresented oligonucleotides carry a phylogenetic signal and are thus diagnostic for individual species. Patterns of short oligomers have been investigated by multiple groups in large numbers of bacteria genomes. However, global distributions of the most highly overrepresented mid-sized oligomers have not been assessed across all prokaryotes to date. We surveyed overrepresented mid-length oligomers across all prokaryotes and normalised for base composition and embedded oligomers using zero and second order Markov models.

Principal Findings

Here we report a presumably ancient set of oligomers conserved and overrepresented in nearly all branches of prokaryotic life, including Archaea. These oligomers are either adenine rich homopurines with one to three guanine nucleosides, or homopyridimines with one to four cytosine nucleosides. They do not show a consistent preference for coding or non-coding regions or aggregate in any coding frame, implying a role in DNA structure and as polypeptide binding sites. Structural parameters indicate these oligonucleotides to be an extreme and rigid form of B-DNA prone to forming triple stranded helices under common physiological conditions. Moreover, the narrow minor grooves of these structures are recognised by DNA binding and nucleoid associated proteins such as HU.

Conclusion

Homopurine and homopyrimidine oligomers exhibit distinct and unusual structural features and are present at high copy number in nearly all prokaryotic lineages. This fact suggests a non-neutral role of these oligonucleotides for bacterial genome organization that has been maintained throughout evolution.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.     

Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization.

Oligonucleotide microchips are manufactured by immobilizing presynthesized oligonucleotides within 0.1 x 0.1 x 0.02 mm or 1 x 1 x 0.02 mm polyacrylamide gel pads arranged on the surface of a microscope slide. The gel pads are separated from each other by hydrophobic glass spacers and serve as a kind of 'microtest tube' of 200 pl or 20 nl volume, respectively. Fractionation of single-stranded DNAs is carried out by their hybridization with chip pads containing immobilized 10mers. DNA extracted separately from each pad is transferred onto a sequencing chip and analyzed thereon. The chip, containing a set of 10mers, was enzymatically phosphorylated, then hybridized with DNA and ligated in a site-directed manner with a contiguously stacked 5mer. Several cycles of successive hybridization-ligation of the chip-bound 10mers with different contiguously stacked 5mers and hybridized with DNA were carried out to sequence DNA containing tetranucleotide repeats. Combined use of these techniques show significant promise for sequence comparison of homologous regions in different genomes and for sequence analysis of comparatively long DNA fragments or DNA containing internal repeats.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Genome-wide selection of unique and valid oligonucleotides

Functional genomics methods are used to investigate the huge amount of information contained in genomes. Numerous experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization forms the underlying principle for these methods. For all these techniques, the probes should be unique for analyzed genes. In addition to being unique for the studied genes, the probes should fulfill a large number of criteria to be usable and valid. The criteria include for example, avoidance of self-annealing, suitable melting temperature and nucleotide composition. We developed a method for searching unique and valid oligonucleotides or probes for genes so that there is not even a similar (approximate) occurrence in any other location of the whole genome. By using probe size 25, we analyzed 17 complete genomes representing a wide range of both prokaryotic and eukaryotic organisms. More than 92% of all the genes in the investigated genomes contained valid oligonucleotides. Extensive statistical tests were performed to characterize the properties of unique and valid oligonucleotides. Unique and valid oligonucleotides were relatively evenly distributed in genes except for the beginning and end, which were somewhat overrepresented. The flanking regions in eukaryotes were clearly underrepresented among suitable oligonucleotides. In addition to distributions within genes, the effects on codon and amino acid usage were also studied.     

Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems

In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20–22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2–5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius . The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius , as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.     

Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.     

Investigations of oligonucleotide usage variance within and between prokaryotes

Oligonucleotide usage in archaeal and bacterial genomes can be linked to a number of properties, including codon usage (trinucleotides), DNA base-stacking energy (dinucleotides), and DNA structural conformation (di- to tetranucleotides). We wanted to assess the statistical information potential of different DNA ‘word-sizes’ and explore how oligonucleotide frequencies differ in coding and non-coding regions. In addition, we used oligonucleotide frequencies to investigate DNA composition and how DNA sequence patterns change within and between prokaryotic organisms. Among the results found was that prokaryotic chromosomes can be described by hexanucleotide frequencies, suggesting that prokaryotic DNA is predominantly short range correlated, i.e., information in prokaryotic genomes is encoded in short oligonucleotides. Oligonucleotide usage varied more within AT-rich and host-associated genomes than in GC-rich and free-living genomes, and this variation was mainly located in non-coding regions. Bias (selectional pressure) in tetranucleotide usage correlated with GC content, and coding regions were more biased than non-coding regions. Non-coding regions were also found to be approximately 5.5% more AT-rich than coding regions, on average, in the 402 chromosomes examined. Pronounced DNA compositional differences were found both within and between AT-rich and GC-rich genomes. GC-rich genomes were more similar and biased in terms of tetranucleotide usage in non-coding regions than AT-rich genomes. The differences found between AT-rich and GC-rich genomes may possibly be attributed to lifestyle, since tetranucleotide usage within host-associated bacteria was, on average, more dissimilar and less biased than free-living archaea and bacteria.     

Sequences downstream of the start codon and their relations to G + C content and optimal growth temperature in prokaryotic genomes

The mechanism of translation initiation is responsible for shaping the mRNA sequences downstream of the start codon. However, this region has not been systematically analyzed in prokaryotes. We used sequence logos and statistic methods to analyze the patterns of overrepresented sequences in this region for 125 species of bacteria and 23 species of archaea. The specific positions are compared to the first 33 amino acids in the proteins. At the 2nd amino acid position, Lys, Ser or Thr is highly overrepresented for 68% to 84% of the genomes examined and Ala is highly overrepresented for 57% of the genomes. Overrepresentation of Lys2 is negatively correlated with the G + C content and overrepresentation of Ser2 or Thr2 is positively correlated with the G + C content of genomes. Ile at the 4th to the 8th positions were found to be overrepresented for 91% of the genomes analyzed and this seemed to be conserved for both bacteria and archaea. Organisms growing at high temperatures have relatively low extent of nucleotides bias at 5′ termini of open reading frames (ORFs). The extent of overrepresenting A and underrepresenting G at ORF 5′ termini is reduced in thermophiles and hyperthermophiles for both archaea and bacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection and Identification of Brenneria nigrifluens, the Causal Agent of the Shallow Bark Canker of Walnut by, PCR Amplification

A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1–C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens . Several strains of B. nigrifluens were assessed with F1–C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies.     

Think big--giant genes in bacteria

Long genes should be rare in archaea and eubacteria because of the demanding costs of time and resources for protein production. The search in 580 sequenced prokaryotic genomes, however, revealed 0.2% of all genes to be longer than 5 kb (absolute number: 3732 genes). Eighty giant bacterial genes of more than 20 kb in length were identified in 47 taxa that belong to the phyla Thermotogae (1), Chlorobi (3), Planctomycetes (1), Cyanobacteria (2), Firmicutes (7), Actinobacteria (9), Proteobacteria (23) or Euryarchaeota (1) (number of taxa in brackets). Giant genes are strain-specific, differ in their tetranucleotide usage from the bulk genome and occur preferentially in non-pathogenic environmental bacteria. The two longest bacterial genes known to date were detected in the green sulfur bacterium Chlorobium chlorochromatii CaD3 encoding proteins of 36 806 and 20 647 amino acids, being surpassed in length only by the human titin coding sequence. More than 90% of bacterial giant genes either encode a surface protein or a polyketide/non-ribosomal peptide synthetase. Most surface proteins are acidic, threonine-rich, lack cystein and harbour multiple amino acid repeats. Giant proteins increase bacterial fitness by the production of either weapons towards or shields against animate competitors or hostile environments.     

Identification of novel intergenic repetitive units in a mycobacterial two-component system operon

Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two-component system, named senX3-regX3 . Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae . These could be grouped into three major families, containing elements of 77–101 bp, 46–53 bp and 58–101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40–50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two-component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.     

Mapping markers linked to porcine salmonellosis susceptibility

The goal of this study was to identify pig chromosomal regions associated with susceptibility to salmonellosis. Genomic DNA from pig reference populations with differences in susceptibility to Salmonella enterica serovar Choleraesuis as quantified by spleen and liver bacterial colonization at day 7 post-infection (dpi; Van Diemen et al. 2002 ) was used. These samples belonged to the offspring of a sire thought to be heterozygous for genes involved in susceptibility to salmonellosis. Amplified fragment length polymorphism (AFLP) markers were created and used to determine associations with spleen or bacterial counts at 7 dpi. To position linked markers, two mapping populations, the Roslin and Uppsala PiGMaP pedigrees were used to create an integrated map which included the AFLP markers associated with salmonellosis. Twenty-six AFLP markers located in 14 different chromosomal regions in the porcine genome were found to be significantly associated with susceptibility (Chi-square P  < 0.05). More than one linked marker was found on chromosomes 1, 7, 13, 14 and 18. It is likely that these regions contain genes involved in Salmonella susceptibility. Regions on chromosomes 1, 7 and 14 were significantly associated with Salmonella counts in the liver and regions on chromosomes 11, 13 and 18 with counts in spleen. The identification of these chromosomal regions highlights specific areas to search for candidate genes that may be involved in innate or adaptive immunity. Further investigation into these chromosomal regions would be useful to improve our understanding of host responses to infection with this widespread pathogen.     

Demonstration of a word design strategy for DNA computing on surfaces.

A strategy for DNA computing on surfaces using linked sets of 'DNA words' that are short oligonucleotides (16mers) is proposed. The 16mer words have the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in 8 variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. Using a template and map strategy, a set of 108 8mers each of which possesses at least a 4 base mismatch with the complements to all the other members of the set (4bm complements) are identified for use as a variable base sequence set. In addition, sets of 4 and 12 word labels of the form ABCD....DCBA that are respectively 8bm and 6bm complements with each other are identified. The 16mers are chosen to have a G/C content of 50% in order to make the thermodynamic stability of the perfectly matched hybridized DNA duplexes similar; a simple pairwise additive method is used to estimate the perfect match and mismatch hybridization thermodynamics. A series of preliminary experiments are presented that use small arrays of 16mers attached to chemically modified gold surfaces and fluorescently labeled complements to study the hybridization adsorption and enzymatic manipulation of the oligonucleotides.     

Interrupted coding sequences in <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>: authentic mutations or sequencing errors?

Background  

In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with undetected frameshifts and in-frame stop codons. These interrupted coding sequences (ICDSs) may really be present in the organism or may result from misannotation based on sequencing errors. The reality or otherwise of these sequences has major implications for all subsequent functional characterization steps, including module prediction, comparative genomics and high-throughput proteomic projects.     

A new species of Astragalus L. (Leguminosae) from India based on morphological and molecular markers

A new species, Astragalus himachalensis , is described and illustrated from the Himalaya in India. The species grows occasionally in different parts of the cold desert of Lahul-Spiti (Himachal Pradesh). It resembles A. himalayanus , but differs chiefly in the small size of the plants (length, 5–15 cm), diadelphous stamens, and small pods (length, 5–6 mm) with the stipe length (2–3 mm) more or less equal to the calyx length. The similarities and significant differences between A. himachalensis and A. himalayanus were confirmed by analyses of random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite DNA (DAMD) markers. RAPD and DAMD methods clearly distinguished between the two species and were in congruence with morphological markers. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 27–34.     

Parallel multiplex thermodynamic analysis of coaxial base stacking in DNA duplexes by oligodeoxyribonucleotide microchips

Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotides were immobilized in an array of three-dimensional polyacrylamide gel pads of microchips (MAGIChips‘). The stacking effect was studied for all combinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to microchip-immobilized 10mers. For any given interface, the effect was studied for perfectly paired bases, as well as terminal mismatches, single base overlaps, single and double gaps, and modified terminal bases. Thermodynamic parameters of contiguous stacking determined by using microchips closely correlated with data obtained in solution. The extension of immobilized oligonucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, or by phosphate, decreased the stacking effect moderately, while extension with FITC or Texas Red virtually eliminated stacking. The extension of the immobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measurements of stacking parameters were performed in different melting buffers. Although melting temperatures of AT- and GC-rich oligonucleotides in 5 M tetramethylammonium chloride were equalized, the energy of stacking interaction was significantly diminished.