Marc J. A. Bailey

QUANTITATION OF PROTEIN

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Methods are described to provide information on how to analyze protein concentration using UV protein spectroscopy measurements, traditional dye-based absorbance measurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The observation that no single assay dominates the market is due to specific limitations of certain methods that investigators need to consider before selecting the most appropriate assay for their sample. Many of the dye-based assays have unique chemical mechanisms that are prone to interference from chemicals prevalent in many biological buffer preparations. A discussion of which assays are prone to interference and the selection of alternative methods is included.

RfaH and the ops element, components of a novel system controlling bacterial transcription elongation

The RfaH protein controls the transcription of a specialized group of Escherichia coli and Salmonella operons that direct the synthesis, assembly and export of the lipopolysaccharide core, exopolysaccharide, F conjugation pilus and haemolysin toxin. RfaH is a specific regulator of transcript elongation; its loss increases transcription polarity in these operons without affecting initiation from the operon promoters. The operons of the RfaH‐dependent regulon contain a short conserved 5′ sequence, the ops element, deletion of which increases operon polarity to an extent similar to that caused by loss of RfaH. The ops element is also present upstream of polysaccharide gene clusters of Shigella flexneri, Yersinia enterocolitica, Vibrio cholerae and Klebsiella pneumoniae and the RP4 fertility operon of Pseudomonas aeruginosa, suggesting that this is a widely spread control system. The mechanistic coupling of RfaH and the ops element has been demonstrated in vitro and in vivo, and we suggest that the ops element recruits RfaH and potentially other factors to the RNA polymerase complex, modifying the complex to increase its processivity and allowing transcription to proceed over long distances.

Escherichia coli HIyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes

Synthesis and secretion of the 110kDa haemolysin toxin of Escherichia coli and other pathogenic Gram‐negative bacteria are governed by the four genes of the hly operon. We have identified, by transposon mutagenesis, an E. coli cellular locus, hlyT, required for the synthesis and secretion of haemolysin encoded in trans by intact hly operons carrying the hly upstream regulatory region. Mutation of the hlyT locus specifically reduced the level of hlyA structural gene transcript 20‐100‐fold and thus markedly lowered both intracellular and extracellular levels of the HlyA protein. Genetic and structural analysis of the hlyT locus mapped it at co‐ordinate 3680 kbp (minute 87) on the chromosome adjacent to the fadBA operon, and identified it specifically as the rfaH (sfrB) locus which is required for transcription of the genes encoding synthesis of the sex pilus and also the lipopolysaccharide core for attachment of the O‐antigen of E. coli and Salmonella. Expression of the hly operon in the E. coli hlyT mutant was restored in trans by both the hlyT and rfaH genes, suggesting that the rfaH gene is an important activator of regulon structures that are central to the fertility and virulence of these pathogenic bacteria. DNA sequencing of the hlyT locus identifies the HlyT/RfaH transcriptional activator as a protein of 162 amino acids (Mr 18325) which shows no identity to characterized transcription factors.

SUPPRESSION OF TRANSCRIPTION POLARITY IN THE ESCHERICHIA COLI HAEMOLYSIN OPERON BY A SHORT UPSTREAM ELEMENT SHARED BY POLYSACCHARIDE AND DNA TRANSFER DETERMINANTS

Expression of the Escherichia coli hlyCABD operon encoding synthesis, maturation and export of haemolysin toxin was strongly dependent upon a 35 bp DNA sequence, spanning the element GGCGGTAG, located 2 kbp upstream. When the hly operon was placed under the control of the inducible tac promoter, expression remained dependent upon this element, when transcribed in its native orientation 3′ of the promoter. The increase in ptac‐directed transcription was strongest for the distal, export genes of the hly operon, and was particularly striking when ptac and the element were placed far upstream. The element did not influence transcript stability, and we suggest that it is a key component of a novel regulatory mechanism may suppresses transcription polarity within operons. The mechanism that be of widespread importance in bacterial gene expression because the 8 bp element is present in many Gram‐negative species as an upstream component of operons encoding the production of toxins and the surface assembly of polysaccharides and components required for the conjugal transfer of DNA. We name it the ops element for operon polarity suppressor.

REQUIREMENT FOR FLHA IN FLAGELLA ASSEMBLY AND SWARM-CELL DIFFERENTIATION BY PROTEUS MIRABILIS

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin. A non‐swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane trans‐location and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation‐specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild‐type strain. Transcription of flhA was strongly induced during differentiation, from its own putative σ28 promoter. The results suggest a mechanistic coupling of flagella assembly and swarm‐cell differentiation.

INCREASED DISTAL GENE TRANSCRIPTION BY THE ELONGATION FACTOR RFAH, A SPECIALIZED HOMOLOGUE OF NUSG

The Escherichia coli RfaH protein is required for the expression of operons directing synthesis and export of the toxin haemolysin, the lipopolysaccharide core, and the F‐factor sex pilus. Mutation of rfaH increases transcriptional polarity along all three operons. By demonstrating strong RfaH‐dependent suppression of transcription polarity in vitro, we have established RfaH as a novel transcriptional activator, and we reveal that RfaH is a homologue of the essential protein NusG that modulates general transcriptional pausing and termination in prokaryotes. Full transcription of the distal genes from an upstream promoter required RfaH and the 5′ cis‐acting ops element, both in vivo and in vitro. In vivo the requirement for the ops element was suppressed by overexpressing RfaH, and in vitro the presence of ops lowered the concentration of RfaH required to stimulate transcript elongation. We suggest that RfaH directs transcript elongation in an analogous way to NusG, but does so in a subset of bacterial operons primarily engaged in the production of extracellular components required for virulence and fertility.

Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly

A transposon mutant of Escherichia coli 5K was isolated which reduced 10‐ to 50‐fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate‐containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC‐directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate‐zonal centrifugation the mutant‐secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide‐independent secretion process.

In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element.

Abstract An unusual regulatory mechanism that controls transcription elongation in long fertility and virulence operons in bacteria is effected by two specialised components, the RfaH protein and the nucleic acid ops element. Without direct interaction, ops acts to reduce the concentration of RfaH required to stimulate distal gene transcription, and we have proposed that ops recruits RfaH to the transcription machinery. To provide direct experimental evidence for this view, we used gel fitration to identify potential RfaH complexes assembled in Escherichia coli cell extracts that carry out RfaH-dependent transcription. This novel molecular weight shift assay revealed that RfaH-dependent transcription elongation occurs concomitantly with recruitment of RfaH into a high molecular weight transcription complex, and that this recruitment is specifically directed by the ops element. Assembly of this complex required RNA polymerase and nucleotide hydrolysis, but not processive transcription. Neither assembly of the complex nor RfaH-dependent transcription was observed in in vitro reactions containing only ops, RfaH and purified core (αββ′ ) RNA polymerase; both processes required the combination of subcellular fractions containing the RNA polymerase complex, the cytoplasmic membrane and ribosomes. The data confirm that the ops element directs recruitment of RfaH into a multi-component RNA polymerase complex that resists transcription termination.