Experimentally revised repertoire of putative contingency loci in Neisseria meningitidis strain MC58: evidence for a novel mechanism of phase variation.

Analysis of the genome sequence of Neisseria meningitidis strain MC58 revealed 65 genes associated with simple sequence repeats. Experimental evidence of phase variation exists for only 14 of these 65 putatively phase variable genes. We investigated the phase variable potential of the remaining 51 genes. The repeat tract associated with 20 of these 51 genes was sequenced in 26 genetically distinct strains. This analysis provided circumstantial evidence for or against the phase variability of the candidate genes, based on the sequence and the length of the repeated motif. These predictions of phase variability were substantiated for three of these candidate genes using colony immunoblotting or beta-galactosidase as a reporter. This investigation identified a novel phase variable gene (NMB1994 or nadA) associated with a repeat tract (TAAA) not previously reported to be associated with phase variable genes in N. meningitidis. Analysis of the nadA transcript revealed that the repeat tract was located upstream of the putative -35 element of the nadA promoter. Semiquantitative RT-PCR showed that variation in the number of repeats was associated with changes in the level of expression of nadA, findings consistent with a model whereby the variable number of (TAAA) repeats modulates the promoter strength.

The complete genome of the crenarchaeon Sulfolobus solfataricus P2.

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Genome sequencing and annotation.

The availability of complete microbial genome sequences enormously facilitates experimental molecular investigations of the respective organisms by providing complete lists of genes, their genetic contexts, and their predicted functions. This can be used in a number of ways to focus studies on bacterial pathogenesis and also vaccine development (1,2). The complete genome sequences from two unrelated strains of Neisseria meningitidis, a derivative of isolate MC58 which originally expressed serogroup B capsule and strain Z2491, which is serogroup A, are now available (3,4). The genome sequences of both these strains were determined using the whole genome shotgun approach (5). In this approach, randomly sheared chromosomal DNA is cloned to make a small insert library (1.5-2.0 kb for MC58, 0.5-0.8 kb and 1.0-1.5 kb for Z2491), then each insert is sequenced from both ends using plasmidspecific primers. For the MC58 genome sequence, a large insert lambda library (8-24 kb) was also used. In the initial sequencing phase, 6-8 times coverage of the estimated size of the genome is generally achieved. The DNA sequences are linked together (assembled) into large contigs (a derivative of the word contiguous). Polymerase chain reaction (PCR) and sequencing of large insert libraries are then used to join the contigs, close gaps, and resolve ambiguities (see ref. 6 for a review).

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58.

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes.

Completing the sequence of the Sulfolobus solfataricus P2 genome.

The Sulfolobus solfataricus P2 genome collaborators are poised to sequence the entire 3-Mbp genome of this crenarchaeote archaeon. About 80% of the genome has been sequenced to date, with the rest of the sequence being assembled fast. In this publication we introduce the genomic sequencing and automated analysis strategy and present intial data derived from the sequence analysis. After an overview of the general sequence features, metabolic pathway studies are explained, using sugar metabolism as an example. The paper closes with an overview of repetitive elements in S. solfataricus.

Phylogenetic analysis of coccidia based on 18S rDNA sequence comparison indicates that Isospora is most closely related to Toxoplasma and Neospora.

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa, continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis, and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.

Identification of synapomorphic characters in the genus Sarcocystis based on 18S rDNA sequence comparison.

In order to further investigate synapomorphic characters in the genus Sarcocystis, the small subunit ribosomal RNA gene sequences of Sarcocystis capracanis and Sarcocystis moulei were determined and used to infer the phylogenetic position of these two organisms within the cyst-forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated that S. capracanis groups with Sarcocystis tenella and Sarcocystis arieticanis as a clade that shares the characteristic of using canids as their definitive host. S. moulei was shown to group with Sarcocystis gigantea and Sarcocystis fusiformis as a clade that shares the characteristic of using fields as their definitive host.

Genetic diversity in Sarcocystis gigantea assessed by RFLP analysis of the ITS1 region.

The genetic diversity among Sarcocystis gigantea isolates derived from individual cysts within a given infected animal at two abattoirs in Australia and one abattoir in Germany was studied using Restriction Fragment Length Polymorphism (RFLP) analysis of the intergenic transcribed spacer region 1 (ITS 1) of the ribosomal RNA gene operon. S. gigantea isolates were obtained from infected sheep from Blayney (New South Wales), Katanning (Western Australia), and Detmold (North-Rhine Westfalia, Germany) in order to assess the level of diversity among isolates from different geographic locations. Polymerase chain reaction amplification and RFLP analysis of the ITSI region with the restriction enzymes HaeIII, NlaIII, and Sau3AI found no genetic variation among the isolates within one animal or among animals in the same or different locations. To our knowledge, such a field study has not yet been performed on a species in the genus Sarcocystis.

The growing importance of the plastid-like DNAs of the Apicomplexa.

Organisms in the phylum Apicomplexa possess, in addition to their mitochondrial genome, an extrachromosomal DNA that possesses significant similarities with the extrachromosomal genomes of plastids. To date, the majority of data on these plastid-like DNAs have been obtained from the human malarial organism, Plasmodium falciparum. In common with plastid DNAs, the plastid-like DNA of P. falciparum possesses genes for DNA-dependent RNA polymerase subunits beta and beta 1 and for organellar-like large- and small-subunits ribosomal RNAs. Both the polymerase subunit and ribosomal RNA gene sequences share a number of features with those from plastid DNAs. In addition, the ribosomal RNA genes are organised in an inverted repeat arrangement, reminiscent of plastid DNAs. Additional molecular features shared between the 2 genomes are discussed. Plastid-like DNAs have also been identified in other Plasmodium species as well as Toxoplasma gondii, Eimeria tenella, Babesia bovis and a number of Sarcocystis species. A cryptic organelle often observed in apicomplexans has been proposed as the organelle that harbours the plastid-like DNAs, but conclusive evidence for this has not yet been obtained. Although approximately 1/2 of the plastid-like DNA of P. falciparum has been sequenced to date, no function has yet been ascribed to this DNA or its putative organelle. Phylogenetic inferences based on sequence data from this DNA have indicated an evolutionary origin from photosynthetic organisms, but the true provenance of the plastid-like DNAs remains to be determined. Because of the specific nature of the plastid-like DNAs, they may prove useful as effective targets for chemotherapeutics.

A RAPD-PCR derived marker can differentiate between pathogenic and non-pathogenic Sarcocystis species of sheep.

Random amplified polymorphic DNA (RAPD)-PCR was used to differentiate among four cyst-forming coccidia of sheep, Sarcocystis tenella, Sarcocystis gigantea, Sarcocystis arieticanis, and Toxoplasma gondii. Genomic DNA of the four parasite species was amplified using RAPD-PCR and the DNA fragments were separated on agarose gels. A RAPD-PCR band derived from S. tenella was isolated from the gel and subcloned into pUC18. The insert was sequenced and found to be 1278 nucleotides long. This sequence appeared to be cryptic in nature as it showed no significant sequence peculiarities or similarity with any other known sequences either at the nucleotide or derived amino acid levels. The recombinant plasmid was radiolabelled and used as a probe in Southern hybridization. This probe, termed pSTF10, hybridised to Mbo I restricted genomic DNA of S. tenella and S. arieticanis, but not to DNA of S. gigantea, T. gondii, mouse, or sheep. It is likely that STF10 will become a valuable diagnostic tool for Sarcocystis infections in sheep to differentiate between pathogenic species of this genus and S. gigantea or T. gondii.

A catalytic 13-mer ribozyme.

A 13-mer oligoribonucleotide can act as a ribozyme for the specific self-cleavage of a 41-mer oligoribonucleotide substrate in the presence of Mg2+. The two sequences involved correspond to the self-cleavage hammerhead structure of the virusoid of lucerne transient streak virus. The Michaelis-menten kinetic parameters for the reaction were; Km 1.3 microM, Vmax 0.012 microM min-1, kcat 0.5 min-1. The 13-mer RNA is the smallest ribozyme so far reported. A DNA analogue of the 13-mer can not substitute for the RNA in the reaction.

Self-cleaving viroid and newt RNAs may only be active as dimers.

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.