Biomedical subjects

Neil G Stoker

Publications and source records attributed to Neil G Stoker.

10 recordsLinked to original sources

PubMed2003

Analysis of whole-genome microarray replicates using mixed models.

MOTIVATION: Microarray experiments are inherently noisy. Replication is the key to estimating realistic fold-changes despite such noise. In the analysis of the various sources of noise the dependency structure of the replication needs to be taken into account. RESULTS: We analyzed replicate data sets from a Mycobacterium tuberculosis trcS mutant in order to identify differentially expressed genes and suggest new methods for filtering and normalizing raw array data and for imputing missing values. Mixed ANOVA models are applied to quantify the various sources of error. Such analysis also allows us to determine the optimal number of samples and arrays. Significance values for differential expression are obtained by a hierarchical bootstrapping scheme on scaled residuals. Four highly upregulated genes, including bfrB, were analyzed further. We observed an artefact, where transcriptional readthrough from these genes led to apparent upregulation of adjacent genes. AVAILABILITY: All methods and data discussed are available in the package YASMAhttp://www.cryst.bbk.ac.uk/wernisch/yasma.html for the statistical data analysis system R (http://www.R-project.org).

Algorithms↗

PubMed2003

The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence.

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

Animals↗

PubMed2003

Gene replacement in mycobacteria by using incompatible plasmids.

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.

Bacterial Proteins↗

PubMed2003

Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis.

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devRdelta) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.

Animals↗

PubMed2002

Crystal structure of inositol 1-phosphate synthase from Mycobacterium tuberculosis, a key enzyme in phosphatidylinositol synthesis.

Phosphatidylinositol (PI) is essential for Mycobacterium tuberculosis viability and the enzymes involved in the PI biosynthetic pathway are potential antimycobacterial agents for which little structural information is available. The rate-limiting step in the pathway is the production of (L)-myo-inositol 1-phosphate from (D)-glucose 6-phosphate, a complex reaction catalyzed by the enzyme inositol 1-phosphate synthase. We have determined the crystal structure of this enzyme from Mycobacterium tuberculosis (tbINO) at 1.95 A resolution, bound to the cofactor NAD+. The active site is located within a deep cleft at the junction between two domains. The unexpected presence of a zinc ion here suggests a mechanistic difference from the eukaryotic inositol synthases, which are stimulated by monovalent cations, that may be exploitable in developing selective inhibitors of tbINO.

Amino Acid Sequence↗

PubMed2002

The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis.

Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroKDelta::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.

Amino Acids, Aromatic↗

PubMed2000

Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement.

Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyADelta plcABCDelta. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PAC:I restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PAC:I site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

Bacterial Proteins↗

PubMed1999

Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination.

The ability to generate mutants of Mycobacterium tuberculosis will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of M. tuberculosis using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into M. tuberculosis, and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the metB gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were obtained for argF and glnE. Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.

Amino Acids↗

PubMed1999

Enhanced gene replacement in mycobacteria.

Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.

Alcohol Oxidoreductases↗

PubMed1998

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

Amino Acid Sequence↗