Mark S. Thomas

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.

Iron acquisition mechanisms of the Burkholderia cepacia complex

The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members of the Bcc appear to possess efficient mechanisms for iron capture. These bacteria specify up to four different types of siderophore (ornibactin, pyochelin, cepabactin and cepaciachelin) that employ the full repertoire of iron-binding groups present in most naturally occurring siderophores. Members of the Bcc are also capable of utilising some exogenous siderophores that they are not able to synthesise. In addition to siderophore-mediated mechanisms of iron uptake, the Bcc possess mechanisms for acquiring iron from haem and from ferritin. The Bcc therefore appear to be well-equipped for life in an iron-poor environment.

Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

The Ornibactin Biosynthesis and Transport Genes of Burkholderia cenocepacia Are Regulated by an Extracytoplasmic Function σ Factor Which Is a Part of the Fur Regulon

Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) sigma factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated sigma(70)-dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF sigma factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin.

Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein.

MelR is a melibiose‐triggered transcription activator that belongs to the AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR‐dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR‐and CRP‐binding sites at the melAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position −100.5 (Site 1) and position −62.5 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1′, centred at position −120.5, located immediately upstream of Site 1, and Site R, at position −238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1′, Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2′, centred at position −42.5) located immediately downstream of Site 2, and overlapping the −35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding Site 1 and Site 2. Thus, tandem‐bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2′. Optimal expression of the melAB promoter requires Sites 1′, Site 1, Site 2 and Site 2′; CRP acts as a ‘bridge’ between MelR bound at Sites 1′ and 1 and at Sites 2 and 2′, increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2′ removes the requirement for Site 1′ and Site 1, and short circuits the effects of CRP.

Immunization with an Interferon-γ–gp120 Fusion Protein Induces Enhanced Immune Responses to Human Immunodeficiency Virus gp120

Cytokines, including interferon (IFN)-gamma, can be effective immunologic adjuvants but often lack the potency of other, more reactogenic compounds. On the basis of the observation that attachment of IFN-gamma to antigen could further enhance its adjuvanticity, a chimeric protein involving IFN-gamma and gp120 of human immunodeficiency virus was produced, using varying lengths of amino acid linkers between the two moieties. All resultant fusion proteins appeared to be dimerized, but full IFN-gamma biological activity was present only with the longest, 34-aa linker. Immunization with the fusion protein gave rise to enhanced primary antibody responses to gp120, particularly of the IgG2a subclass. In addition, both T cell proliferation and IFN-gamma production in response to antigen were strongly enhanced by primary immunization with the fusion protein. IFN-gamma fused to antigen is a more potent adjuvant for Th1-like responses than is IFN-gamma mixed with antigen.

Role of the Stationary Growth Phase Sigma Factor RpoS of Burkholderia pseudomallei in Response to Physiological Stress Conditions

The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.

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.

The Burkholderia cepacia fur gene: co-localization with omlA and absence of regulation by iron

The ferric uptake regulator (Fur) functions as a transcription repressor of many genes in bacteria in response to iron, but the presence of a functional equivalent of this protein has not been demonstrated in Burkholderia cepacia. A segment of the Burkholderia pseudomallei fur gene was amplified using degenerate primers and used to identify chromosomal restriction fragments containing the entire fur genes of B. cepacia and B. pseudomallei. These fragments were cloned and sequenced, revealing the Fur protein of both species to be a polypeptide of 142 amino acids possessing a high degree of amino acid sequence identity to Fur of other members of the beta subclass of the Proteobacteria. Primer extension analysis demonstrated that transcription of B. cepacia fur originated from a single promoter located 36 bp upstream from the fur translation initiation codon. The Fur polypeptide of B. cepacia was shown to functionally substitute for Fur in an Escherichia coli fur mutant. Single copy fur-lacZ fusions were constructed and used to examine the regulation of B. cepacia fur. The B. cepacia fur promoter was not responsive to iron availability, the presence of hydrogen peroxide or the superoxide generator methyl viologen. In addition, fur expression was not significantly influenced by carbon source. Interestingly, the presence of the divergently transcribed omlA/smpA gene upstream of fur in some members of the gamma subclass of the Proteobacteria is retained in several genera within the beta taxon, including Burkholderia.

The Escherichia coli RNA polymerase α subunit linker: length requirements for transcription activation at CRP‐dependent promoters

The C‐terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD) plays a key role in transcription initiation at many activator‐dependent promoters. This domain is connected to the N‐terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on αCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the α linker decrease CRP‐dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP‐dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker‐modified α subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP‐dependent promoters.