Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation.

BACKGROUND: Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. "bavariensis" (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. "finlandensis" (1). RESULTS: Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. CONCLUSIONS: Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.

Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii.

It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B. valaisiana VS116, B. spielmanii A14S, and B. bissettii DN127.

Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: in vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA.

The discovery of 5-hydroxymethyl-cytosine (5hmC) in mammalian cells prompted us to look for this base in the DNA of Arabidopsis thaliana (thale cress), and to ask how well the Arabidopsis Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes this novel base. We found that the DNA of Arabidopsis' leaves and flowers contain low levels of 5hmC. We also cloned and expressed in Escherichia coli full-length VIM1 protein, the archetypal member of the five Arabidopsis VIM gene family. Using in vitro binding assays, we observed that full-length VIM1 binds preferentially to hemi-methylated DNA with a single modified 5mCpG site; this result is consistent with its known role in preserving DNA methylation in vivo following DNA replication. However, when 5hmC replaces one or both cytosine residues at a palindromic CpG site, VIM1 binds with approximately ≥10-fold lower affinity. These results suggest that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication.

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG.

The Escherichia coli McrA protein, a putative C(5)-methylcytosine/C(5)-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA's binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S. In addition to binding HpaII-methylated double-stranded DNA, rMcrA-S binds DNA containing a single, hemimethylated HpaII site; however, it does not bind if A, C, T or U is placed across from the m5C residue, but does if I is opposite the m5C. These results provide the first systematic analysis of McrA's in vitro binding specificity.

Whole-genome sequences of thirteen isolates of Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate.

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates.

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.

A new approach to a Lyme disease vaccine.

A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borrelia afzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B. afzelii species was shown in a tick-challenge model using feral ticks. In both models, as little as .03 µg of antigen, when administered in a 2-dose immunization schedule with aluminum hydroxide as adjuvant, was sufficient to provide complete protection against the species targeted. This proof of principle study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines requiring fewer OspA antigens and suggests that this approach may facilitate the development of an OspA vaccine for global use.

Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE).

Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome. Platelets have a unique and reproducible transcript profile, which includes approximately 1,600-3,000 individual transcripts. In this chapter, we will focus on platelet purification and on transcript profiling using an Affymetrix microarray platform and serial analysis of gene expression (SAGE). Platelet purification is described in detail. Large-scale platelet purification schema is designed to purify platelets from apheresis platelet bags (approximately 3-5 x 10(11) platelets/bag). Modification of this schema --small-scale platelet purification--is designed to isolate platelets from 20 ml of peripheral blood. This chapter provides detailed protocols for microarray and SAGE transcript profiling. We also discuss peculiarities of platelet purification, RNA isolation, and transcript profiling.

A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions.

The repressor element 1 (RE1) silencing transcription factor (REST) helps preserve the identity of nervous tissue by silencing neuronal genes in non-neural tissues. Moreover, in an epithelial model of tumorigenesis, loss of REST function is associated with loss of adhesion, suggesting the aberrant expression of REST-controlled genes encoding this property. To date, no adhesion molecules under REST control have been identified. Here, we used serial analysis of chromatin occupancy to perform genome-wide identification of REST-occupied target sequences (RE1 sites) in a kidney cell line. We discovered novel REST-binding motifs and found that the number of RE1 sites far exceeded previous estimates. A large family of targets encoding adhesion proteins was identified, as were genes encoding signature proteins of neuroendocrine tumors. Unexpectedly, genes considered exclusively non-neuronal also contained an RE1 motif and were expressed in neurons. This supports the model that REST binding is a critical determinant of neuronal phenotype.

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase.

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease.

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues.

Paired-end genomic signature tags: a method for the functional analysis of genomes and epigenomes.

Because paired-end genomic signature tags are sequenced-based, they have the potential to become an alternate tool to tiled microarray hybridization as a method for genome-wide localization of transcription factors and other sequence-specific DNA binding proteins. As outlined here the method also can be used for global analysis of DNA methylation. One advantage of this approach is the ability to easily switch between different genome types without having to fabricate a new microarray for each and every DNA type. However, the method does have some disadvantages. Among the most rate-limiting steps of our PE-GST protocol are the need to concatemerize the diTAGs, size fractionate them and then clone them prior to sequencing. This is usually followed by additional steps to amplify and size select for long (> or = 500) concatemer inserts prior to sequencing. These time-consuming steps are important for standard DNA sequencing as they increase efficiency approximately 20-30-fold since each amplified concatemer can now provide information on multiple tags; the limitation on data acqui- sition is read length during sequencing. However, the development of new sequencing methods such as Life Sciences' 454 new nanotechnology-based sequencing instrument (41) could increase tag sequencing efficiency by several orders of magnitude (> or = 100,000 diTAG reads/run), which is sufficient to provide in-depth global analysis of all ChIP PE-GSTs in a single run. This is because the lengths of our paired-end diTAGs (approximately 60 bp) fall well within the region of high accuracy for read lengths on this instrument. In principle, sequence analysis of diTAGs could begin as soon as they are generated, thereby completely bypassing the need for the concatemerization, sizing, downstream cloning steps and sequencing template purification. In addition, our protocol places any one of several unique four-base long nucleotide sequences, such as GATC, between each and every diTAG pair, which could be used to help the instrument's software keep base register and also provide a well-located peak height indicator in the middle of every sequence run. This additional feature could permit multiplexing of the data by simultaneous sequencing of several pooled libraries if each used a different linker sequence during diTAG formation (Figure 4).

Enzymatic activity of the SARS coronavirus main proteinase dimer.

The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents.

Structure-based design of a second-generation Lyme disease vaccine based on a C-terminal fragment of Borrelia burgdorferi OspA.

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi elicits protective immunity that blocks transmission of Borrelia from the tick vector to the vaccinated animal, and thus has been a focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer beta-sheet. All anti-OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of using the C-terminal domain alone as a recombinant protein-based vaccine. The removal of ineffective parts from the OspA antigen may reduce side effects and lead to a safer vaccine. We prepared a C-terminal fragment of OspA by removing approximately 45% of residues from the N terminus. Although the fragment retained the native conformation and affinity to a protective antibody, its vaccine efficacy and conformational stability were significantly reduced with respect to full-length OspA. We successfully stabilized the fragment by replacing amino acid residues involved in buried salt-bridges with residues promoting hydrophobic interactions. The mutations promoted the vaccine efficacy of the redesigned fragment to a level comparable to that of the full-length protein, demonstrating the importance of the antigen stability for OspA's vaccine efficacy. Our strategy should be useful for further refining OspA-based vaccines and developing recombinant vaccines for other diseases.

Characterization of a unique borreliacidal epitope on the outer surface protein C of Borrelia burgdorferi.

The outer surface protein C (OspC) of the Lyme disease agent, Borrelia burgdorferi, is an immunoprotective antigen in laboratory models of infection. However, to understand its protective effects, it is important to identify the key epitopes of this protein. We produced a borreliacidal anti-OspC monoclonal antibody specific to the B31 strain and identified its binding site. The specificity of MAb 16.22 was determined by Western blot reactivity using OspC derived from different Borrelia isolates which had varying amino acid sequences. Comparison of the OspC sequences and binding data suggested that MAb 16.22 binds to amino acids 133-147 of the OspC protein. To test this hypothesis, we synthesized a 15-amino acid peptide containing the target sequence and, using competition enzyme-linked immunosorbent assay (ELISA), we found that this peptide included the epitope of MAb 16.22. In addition, we determined that MAb 16.22 is able to kill of B. burgdorferi in a complement-independent fashion.

Evidence for two commonly deleted regions on mouse chromosome 2 in gamma ray-induced acute myeloid leukemic cells.

OBJECTIVE: The objective of this study was to delineate a precise molecular map of the commonly deleted region (CDR) on mouse chr2 in radiation-induced mouse acute myeloid leukemic (AML) cells. MATERIALS AND METHODS: We used a PCR-based loss of heterozygosity (LOH) assay to map the chr2-CDR in AML cells isolated from F1 hybrid mice (BALB/cJ x CBA/CaJ) which developed AML following exposure to a single dose of 3 Gy of 137Cs gamma rays. A total of 30 polymorphic microsatellite markers, mapping within or close to chr2(D-E), were used under optimized PCR conditions that generate a single major band for each marker on a nondenaturing polyacrylamide gel. RESULTS: Detailed LOH mapping identified two distinct AML-CDRs: one localized to a 4.6 centiMorgan (cM) interval between markers D2Mit272 and D2Mit394; the other mapped to a 0.8 cM interval between markers D2Mit276 and D2Mit444. Both CDRs span the mouse chr2E region. CONCLUSION: The data present, for the first time, evidence for two distinctly noncontiguous CDRs on mouse chr2 harboring gene(s) involved in AML development. These CDRs are orthologous to human chromosomes 11p11-13 and 15q11-15 that have been implicated in subsets of AML. This finding indicates the region of mouse chr2 that must be searched for candidate genes involved in radiation-induced AML.

A simple modification to the luminometric methylation assay to control for the effects of DNA fragmentation.

Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently described methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequencing technology for quantification. Here we provide evidence suggesting that the global methylation level reported by LUMA is affected by the integrity of the DNA being analyzed. The less intact the DNA, the lower the global methylation levels reported by LUMA. In order to overcome this problem, we propose the use of undigested DNA alongside digested samples. Finally, we demonstrate that this results in a more accurate assessment of global DNA methylation levels.

Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids.

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

Enzyme activity in dialkyl phosphate ionic liquids.

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.