Isolation of bacterial type IV machine subassemblies.

The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through isolation of machine subassemblies for further biochemical and ultrastructural analysis. Here, we describe a protocol for isolation and characterization of VirB protein complexes from the paradigmatic VirB/VirD4 T4SS of Agrobacterium tumefaciens. This protocol can be adapted for isolation of T4SS subassemblies from other gram-negative bacteria as well as gram-positive bacteria. The biological importance of isolated T4SS subcomplexes can be assessed by assaying for copurification of trapped or cross-linked substrates. This can be achieved with a modified form of the chromatin immunoprecipitation (ChIP) assay termed transfer DNA immunoprecipitation (TrIP). Here, a TrIP protocol is described for recovery of formaldehyde-cross-linked DNA substrate-channel subunit complexes from cells employing T4SSs for conjugative DNA transfer.

A novel method for the production of in vivo-assembled, recombinant Escherichia coli RNA polymerase lacking the α C-terminal domain.

The biochemical characterization of the bacterial transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric ß or ß' subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric α-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the α C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any α-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.

Downregulation of the Escherichia coli guaB promoter by upstream-bound cyclic AMP receptor protein.

The Escherichia coli guaB promoter (P(guaB)) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P(guaB) is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P(guaB) contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P(guaB). The CRP-mediated repression of P(guaB) activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P(guaB). Thus, GRDC of P(guaB) involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P(guaB) and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.

Downregulation of the Escherichia coli guaB promoter by FIS.

The Escherichia coli guaB promoter (P(guaB)) regulates transcription of two genes, guaB and guaA, that are required for the synthesis of guanosine 5'-monophosphate (GMP), a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from P(guaB) increases as a function of increasing cellular growth rate, and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions -11, +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of P(guaB) in vitro but not in vivo. Deletion of the fis gene results in increased P(guaB) activity in vivo, but GRDC of P(guaB) is maintained.

The UP element is necessary but not sufficient for growth rate-dependent control of the Escherichia coli guaB promoter.

The Escherichia coli guaB promoter (P(guaB)) regulates the transcription of two genes, guaB and guaA, that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P(guaB) is subject to growth rate-dependent control (GRDC). Here we show that the A+T-rich sequence located between positions -59 and -38 relative to the guaB transcription start site stimulates transcription from P(guaB) approximately 8- to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase alpha subunit for activity. Like the rrnB P1 UP element, the P(guaB) UP element contains two independently acting subsites located at positions -59 to -47 and -46 to -38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P(guaB) UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P(guaB) activity at lower growth rates.

The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma 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.

Escherichia coli can tolerate insertions of up to 16 amino acids in the RNA polymerase alpha subunit inter-domain linker.

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator-dependent promoters and at UP element-dependent promoters. This domain is connected to the alpha N-terminal domain (alphaNTD) by an unstructured linker. To investigate the requirements of the alpha inter-domain linker to support growth of E. coli, we utilised a recently described technique for the substitution of the chromosomal rpoA gene, encoding alpha, by mutant rpoA alleles. We found that it was possible to replace wild-type rpoA by mutant alleles encoding alpha subunits containing inter-domain linkers that were longer by as many as 16 amino acids. However, using this method, it was not possible to transfer to the chromosome rpoA alleles encoding alpha subunits that contained an insertion of 32 amino acids or short deletions within the inter-domain linker. The effect of lengthening the alpha linker on activator-dependent and UP element-dependent transcription in the "haploid" rpoA system was shown to be qualitatively the same as observed previously in the diploid system. The ability of E. coli to tolerate insertions within the alpha inter-domain linker suggests that lengthening the alpha linker does not severely impair transcription of essential genes.