Robert E. Glass

Escherichia coli rpoA mutation which impairs transcription of positively regulated systems

The rpoA341 (phs) mutation of Escherichia coli results in decreased expression of several positively regulated operons and has been mapped to within or very near the rpoA gene encoding the α subunit of RNA polymerase. We have shown that plasmid‐directed synthesis of the wild‐type α subunit can complement the defective phenotypes associated with this mutation consistent with its proposed location within rpoA. This mutation was mapped by marker rescue to within a 182bp region near the 3’end of rpoA and was subsequently transferred to a plasmid by recombination in vivo. DNA sequence analysis revealed that the RpoA341 phenotype was the result of the substitution of lysine 271 by glutamate within the α polypeptide. We discuss this result in relation to our current understanding of the functional organization of the α subunit.

Involvement of the Escherichia coli RNA polymerase α subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins

Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.

Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli

SummaryWe have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12. The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme. Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively. The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes. Negative complementation suggests that the MetH enzyme exists as an oligomer. Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine. For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively. The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess. Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.

Structure‐function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression

We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5‐like bacteriophage BF23, and vitamin B12 (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non‐functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz, Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two‐dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic β‐strand analysis and sequence conservation amongst the TonB‐dependent receptors). In addition, we report that the extreme C‐terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane‐spanning β‐sheet with residue L588 situated on its hydrophobic surface. Two of the C‐terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.

Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase.

Transcription of genetic material is catalysed by the enzyme DNA‐dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed β and β′, are conserved throughout nature. The β subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the β subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the β subunit of RNA polymerase. To this end, the β structural gene rpoB was separated into four near‐equal, non‐overlapping segments (as well as ‘half’ genes) on the basis of ‘split’ genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence‐specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac‐controlled high‐copy number vector.

The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus

Spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37 degrees C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45-83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.

A Structure-Function Analysis of BtuB, the E.Coli. Vitamin B12 Outer Membrane Transport Protein

Vitamin B12 or cobalamin is a member of a group of compounds known as the corrinoids which are the most complex, nonpolymeric molecules so far found in nature. They exist in trace amounts throughout the natural world, usually within the concentration range lxl0-9 to 1x10-15 (Bradbeer, 1982). Vitamin B12 is found in virtually all animal tissues to a greater or lesser degree, its source being ingested tissue or synthesis by gut flora.

Investigation of the regulation of the Escherichia coli btuB gene using operon fusions

Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor. Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated. Mutations in metH, metE and ompR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.

In vivo cloning of a carboxy-terminalrpoB allele which confers altered transcriptional properties

A 3′-terminal mutation of the gene encoding the β subunit ofEscherichia coli RNA polymerase was isolated using anin vivo polA(Ts) technique. Cloning of the allele was monitored by virtue of the fact that the deletion Δ(rpoB) 1570-1 resulted in an altered-size restriction fragment. DNA sequencing confirmed the predicted nature and location of the mutation: Δ(rpoB) 1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967–1028. The phenotype conferred by Δ(rpoB) 1570-1 is discussed with respect to conserved domains within the β polypeptide.

Control of Bacterial Gene Expression

A myriad of gene products are involved in cellular metabolism, in the replicative and expressive machineries and in structural components. However, during the (normal) bacterial life cycle, certain products are required only at particular stages. Moreover, adjustment in cellular metabolism may be necessary when the bacterium is confronted with an unusual growth substance or altered external conditions. Thus, while many, if not the majority, of bacterial genes are expressed constitutively, others are actively controlled. Even when active, not all genes are expressed at the same rate, as suggested by the wide variation in the absolute levels of bacterial proteins (from 10 to 105 copies per cell). Control of gene expression protects against wasteful energy consumption since both RNA and protein production expend ATP at a high rate. (Indeed, control mutants that unnecessarily expend energy may be outgrown by their wild-type counterparts in liquid culture.) Gene control also, clearly, prevents monopolisation of the transcription-translation apparatus.