Robert A. Cox

Physiology of Mycobacteria

Mycobacterium tuberculosis is a prototrophic, metabolically flexible bacterium that has achieved a spread in the human population that is unmatched by any other bacterial pathogen. The success of M. tuberculosis as a pathogen can be attributed to its extraordinary stealth and capacity to adapt to environmental changes throughout the course of infection. These changes include: nutrient deprivation, hypoxia, various exogenous stress conditions and, in the case of the pathogenic species, the intraphagosomal environment. Knowledge of the physiology of M. tuberculosis during this process has been limited by the slow growth of the bacterium in the laboratory and other technical problems such as cell aggregation. Advances in genomics and molecular methods to analyze the M. tuberculosis genome have revealed that adaptive changes are mediated by complex regulatory networks and signals, resulting in temporal gene expression coupled to metabolic and energetic changes. An important goal for bacterial physiologists will be to elucidate the physiology of M. tuberculosis during the transition between the diverse conditions encountered by M. tuberculosis. This review covers the growth of the mycobacterial cell and how environmental stimuli are sensed by this bacterium. Adaptation to different environments is described from the viewpoint of nutrient acquisition, energy generation, and regulation. To gain quantitative understanding of mycobacterial physiology will require a systems biology approach and recent efforts in this area are discussed.

The dissociation of reticulocyte polysomes into subunits and the location of messenger RNA

Reticulocyte polysomes were dissociated with EDTA into 20 s and 40 s subunits, and also by brief exposure to dilute alkali into 30 s and 50 s subunits. Fractions were assayed for the capacity to stimulate the incorporation of amino acids into protein in a reticulocyte cell-free system. Neither the unfractionated subunits, nor any of the fractions were active. RNA was then isolated from pooled fractions and assayed in the cell-free system. The full messenger activity of the original polysomes was recovered in the RNA from the subunits. Three-quarters of the total messenger activity remains associated with the larger subunit, and one-quarter with the smaller subunit. These results show that messenger RNA of about 8 s, the estimated value based on the triplet code hypothesis, for a single chain of haemoglobin, is not released from the subunits by EDTA or dilute alkali. Analysis of the results reveals that messenger RNA is bound to ribosomal RNA. Experiments are presented which show that the linkage probably does not involve either magnesium bridges or base pairing alone. It is therefore suggested that the messenger RNA is covalently linked to ribosomal RNA.

Primary structure of an α-tubulin gene of Physarum polycephalum

Abstract An α-tubulin gene of Physarum was isolated as a phage-λ NM1149 recombinant (designated phage-λ NαTu). Phage-λ NαTu contained a 4700 base-pair Hind III nuclear DNA fragment of an allele of the alt B locus of Physarum (one of four unlinked α-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-λ NαTu were cloned into phage M13 and the nucleotide sequence was determined by the dideoxy chain termination method. The start point of transcription was identified by primer extension and a putative polyadenylation site was located by S 1 nuclease analysis. The 4650 base-pair Hind III insert into phage-λ NαTu spans the complete gene; sequences upstream from the 5′ end contain the RNA transcription promoter elements (the TATA and CCAAT boxes). The nucleotide sequence encoding α-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum α-tubulin cDNA clone, except for three base changes, one leading to a Val codon in place of a Met codon, another leading to a Glu codon in place of an Asp codon, and the third change is silent. The genomic clone provides the nucleotide sequence coding for the last 26 amino acid residues missing from the cDNA clone. The new sequence data indicate that the α-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all α-tubulin genes sequenced to date.

Quantification of global transcription patterns in prokaryotes using spotted microarrays

We describe an analysis, applicable to any spotted microarray dataset produced using genomic DNA as a reference, that quantifies prokaryotic levels of mRNA on a genome-wide scale. Applying this to Mycobacterium tuberculosis, we validate the technique, show a correlation between level of expression and biological importance, define the complement of invariant genes and analyze absolute levels of expression by functional class to develop ways of understanding an organisms biology without comparison to another growth condition.

Growth Regulation in the Mycobacterial Cell

A framework was developed to provide an integrated view of mycobacterial growth and its regulation. The topics reviewed include the properties of cell cultures and their relation to properties of individual cells, cell sizes and macromolecular compositions, uptake of nutrients through the cell envelope, protein biosynthesis, core metabolic pathways, generation of an electrochemical gradient of protons, ATP synthesis and the control of energy generation.

Dissociation properties of ribonucleic acid. I. Titration of rat-liver RNA and model polynucleotides.

Abstract High-molecular-weight RNA from rat liver was titrated with acid and alkali at 0.4°. The dissociation curve (I) obtained on titration from pH 7 to pH 3 was different from the curve (II) obtained on titration from pH 3–7 although Curves I and II were accurately repeated on subsequent cycles. About 50% of the titratable groups dissociated over the range pH 7–11.6. DNA from calf thymus was titrated at 25°. Few, if any, of the titratable groups were ionized at pH 11.4 but ionization was complete at pH 11.6 (Curve I). A broader dissociation curve (II) which was reversible over the range pH 3–11.6 was obtained on titration with acid from pH 11.6. A partly denatured sample was found to have a dissociation curve characteristic of both Curves I and II; about 50% of the groups dissociated over the range pH 8–11.4 and the remainder dissociated over the range pH 11.4–11.6. The complex poly-(A+U) formed between polyadenylic acid and polyuridylic acid was found to be stable over the range pH 4–10.7 at 20°. At more extreme pH values dissociation into component polynucleotides took place and the conformation of polyadenylic acid was determined by pH and temperature. It was found that whereas the alkaline form of polyadenylic acid interacted with polyuridylic acid reversibly, hysteresis was found in acidic solution. It was noticed that both the acid form of polyadenylic acid and poly-(A+U) were stable over the range pH 4.2–5.5 at 22° but that thermodynamic equilibrium was not attained. The results, which may be summarized in a phase diagram, suggest an explanation for the hysteresis found on titration of RNA in acidic solutions.

Protein Synthesis in the Plastid of Plasmodium falciparum

The plastid organelle of malarial and other apicomplexan parasites contains ribosome-like particles as well as a genome dedicated largely to specifying components of a protein expression system. We have identified plastid ribosomes using hybridization studies and show that in erythrocytic stages of Plasmodium falciparum a subset of polysomes carries plastid-specified rRNAs and mRNA, supporting the idea that protein synthesis is active in the plastid.

STRUCTURE AND FUNCTION OF PROKARYOTIC AND EUKARYOTIC RIBOSOMES

Publisher Summary The integration of components needed for protein biosynthesis with other metabolic events is illustrated by the versatility of tRNA. This molecular species has not only a pivotal role in the synthesis of proteins but is also implicated in the regulation of the operon for enzymes associated with the biosynthesis of its own particular and specific amino acid. tRNA functions as a primer for reverse transcriptase and is involved in the synthesis of the bacterial cell wall through special tRNA species as well as in the translation of mRNA. The recognition of the sophisticated interplay between elements of protein synthesizing and other systems was made possible by increasingly detailed studies of the structural components themselves, based on the mastery of the skills of nucleotide sequencing and of protein chemistry. This chapter discusses aspects of recent progress in delineating the fundamentals of ribosome structure and function—bacterial ribosomes and eukaryotic ribosomes. The development of the tRNA field reveals the steps that are needed for the confor-mation of an RNA species to be established. These include (1) the determination of nucleotide sequence; (2) the elucidation of secondary structure (the clover leaf model in the case of tRNA) mainly through the accumulation of sequences of different species; and (3) the production of single crystals suitable for crystallographic studies that allow the three-dimensional structure to be established at high resolution.

A single‐step procedure for the isolation of individual mRNA species from crude lysates of Physarum polycephalum

An individual mRNA species was isolated directly from lysates of Physarum polycephalum by hybridization to immobilised complementary DNA. The solvent for lysis was a 6 M‐guanidinium salt solution, and the hybridization reaction was carried out at 42°C in 4 M guanidinium salt/33% (v/v) formamide buffer. The mRNA species isolated by this procedure was active in cell‐free protein synthesis and directed the synthesis of a polypeptide of M r 25 500 ± 750.

Determination of pyridoxal phosphate levels in the brains of audiogenic and normal mice.

The concentrations of pyridoxal-5′-phosphate in the brains of audiogenic and normal mice were measured fluorimetrically. The brain of the audiogenic mouse (DBA/2J) contains 25% more pyridoxal-5′-phosphate than the brain of a control mouse. Intraperitoneal injection of this substance causes a transient increase of its concentration in the brain, lasting a few hours. The substance thereafter is degraded to pyridoxal and pyridoxic acid.