Alex C. Jeffries

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.

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.

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 β‐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 N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation.

The RASSF (Ras-association domain family) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. The present review will summarize what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins with the classical RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members or imposters in the RASSF family.

Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum

Those aerobic archaea whose genomes have been sequenced possess a single 4‐gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2‐oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero‐tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched‐chain 2‐oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.

The growing utility of microbial genome sequences

A report from the Genome Biology session of the 4th annual conference on microbial genomes, Virginia, February 12-15, 2000.

Multienzyme Complexes in the Archaea: Predictions from Genome Sequences

Aerobic Bacteria and Eukarya possess a family of 2-oxoacid dehydrogenase multienzyme complexes that catalyse the general reaction:

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).