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Biomedical subjects

K T Hughes

Publications and source records attributed to K T Hughes.

At least 19 recordsLinked to original sources
PubMed2001

Detection of simple mutations and polymorphisms in large genomic regions.

We have developed a novel technology that makes it possible to detect simple nucleotide polymorphisms directly within a sample of total genomic DNA. It allows, in a single Southern blot experiment, the determination of sequence identity of genomic regions with a combined length of hundreds of kilobases. This technology does not require PCR amplification of the target DNA regions, but exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endonuclease CEL I to cleave heteroduplex DNA with a very high specificity and sensitivity. We have used this technique to detect various simple mutations directly in the genomic DNA of isogenic pairs of recombinant Pseudomonas aeruginosa, Escherichia coli and Salmonella isolates. Also, by using a cosmid DNA library and genomic fractions as hybridization probes, we have compared total genomic DNA of two clinical P.aeruginosa clones isolated from the same patient, but exhibiting divergent phenotypes. The mutation scan correctly detected a GA insertion in the quorum-sensing regulator gene rhlR and, in addition, identified a novel intragenomic polymorphism in rrn operons, indicating very high stability of the bacterial genomes under natural non-mutator conditions.

Bacterial Proteins↗
PubMed2001

A multipartite interaction between Salmonella transcription factor sigma28 and its anti-sigma factor FlgM: implications for sigma28 holoenzyme destabilization through stepwise binding.

Transcription of the late (Class 3) flagellar promoters in Salmonella typhimurium is dependent upon the flagellar specific sigma factor, sigma28, encoded by the fliA gene. sigma28-dependent transcription is inhibited by an anti-sigma factor, FlgM, through a direct interaction. FlgM can bind both to free sigma28 to prevent it from forming a complex with core RNA polymerase, and to sigma28 holoenzyme to destabilize the complex. A collection of fliA mutants defective for negative regulation by FlgM (fliA* mutants) were isolated. This collection included 27 substitution mutations that conferred insensitivity to FlgM in vivo. The distribution of mutations defined three potential FlgM binding domains in conserved sigma factor regions 2.1, 3.1 and 4 of sigma28. A subset of mutants from each region was assayed for FlgM binding and transcriptional activity in vitro. The results strongly support a multipartite interaction between sigma28 and FlgM. Region 4 mutations, but not region 2.1 or 3.1 mutations, interfered with the ability of FlgM to destabilize sigma28 from core RNA polymerase. We present refined models for FlgM inhibition of sigma28, and for FlgM destabilization of sigma28 holoenzyme.

Amino Acid Sequence↗
PubMed2001

How and when are substrates selected for type III secretion?

Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum. Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate. However, substrates of type III systems lack a single, defined secretion signal. There is evidence for the existence of three independent secretion signals - the 5' region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion - that direct substrates for secretion through the type III pathways. One or more of these signals might be used for a given substrate. A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism. We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors. The potential role of translation in regulating the timing of substrate secretion is also discussed.

5' Untranslated Regions↗
PubMed2001

Hin recombinase mutants functionally disrupted in interactions with Fis.

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

Carrier Proteins↗
PubMed2000

Translation/secretion coupling by type III secretion systems.

Type III secretion systems mediate export of virulence proteins and flagellar assembly subunits in Gram-negative bacteria. Chaperones specific to each class of secreted protein are believed to prevent degradation of the secreted substrates. We show that an additional role of chaperones may be to regulate translation of secreted proteins. We show that the chaperone FIgN is required for translation of the flgM gene transcribed from one mRNA transcript (a flagellar class 3 transcript), but not from another (a flagellar class 2 transcript). FIgM translated from the class 3 transcript is primarily secreted whereas FIgM translated from the class 2 transcript is primarily retained in the cytoplasm. These results suggest FIgM and other type III secretion substrates possess both mRNA and amino acid secretion signals, and supports a new role for type III chaperones in translation/secretion coupling.

Bacterial Proteins↗
PubMed2000

Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription.

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook-basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook-basal body structure.

Antiporters↗
PubMed2000

The flagellar hook protein, FlgE, of Salmonella enterica serovar typhimurium is posttranscriptionally regulated in response to the stage of flagellar assembly.

We investigated the posttranscriptional regulation of flgE, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the flgE gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook-basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly.

Bacterial Proteins↗
PubMed2000

Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli.

How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor sigma(28). FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of sigma(28)-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.

Amino Acid Sequence↗
PubMed1999

Measurement of calcium flux through ionotropic glutamate receptors using Cytostar-T scintillating microplates.

Human embryonic kidney cells (HEK293), expressing the human GluR4 receptor sub-unit of 2-amino-3-hydroxy-methylisoxazol-4-ylpropionic acid (AMPA) type non-NMDA receptors were used, in combination with Cytostar-T scintillating microplates, to develop an assay system for the screening of novel compounds with potential AMPA antagonistic characteristics. Agonist dose responses were measured using the agonists: AMPA; quisqualic acid; L-glutamic acid and kainic acid (KA), and EC50 values of 40, 10, 100 and 100 microM were estimated for each of the agonists, respectively. The AMPA receptor antagonists LY293558 and GYK152466 were tested and shown to inhibit agonist induced [45Ca] influx into the cells. An IC50 value of 600 microM was estimated for the competitive antagonist LY293558 and a value of 100 microM estimated for the non-competitive antagonist GYK152466. The developed assay system is homogeneous, allowing increased assay precision and speed. This allows the potential for automation of the assay and it may be used for screening large numbers of novel compounds.

Calcium↗
PubMed1998

The flagellar anti-sigma factor FlgM actively dissociates Salmonella typhimurium sigma28 RNA polymerase holoenzyme.

The anti-sigma factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific sigma factor, sigma28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free sigma28. We have analyzed FlgM-mediated inhibition of sigma28 activity in vitro. FlgM is able to inhibit sigma28 activity even when sigma28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both sigma28 and sigma28 holoenzyme (Esigma28). The Kd of the sigma28-FlgM complex is approximately 2 x 10(-10) M; missense mutations in FlgM that cause a defect in sigma28 inhibition in vivo increase the Kd of this interaction by 4- to 10-fold. SPR measurements of Esigma28 dissociation in the presence of FlgM indicate that FlgM destabilizes Esigma28, presumably via an interaction with the sigma subunit. Our data provide the first direct evidence of an interaction between FlgM and Esigma28. We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.

Bacterial Proteins↗
PubMed1998

The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-sigma28 domain.

The flagellar-specific anti-sigma factor, FIgM, inhibits the expression of late flagellar genes until the hook-basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FIgM monitors this assembly checkpoint by being a substrate for export via the hook-basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FIgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FIgM. Deletion of this region (S3-P11) resulted in lower intracellular FIgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FIgM spanning residues K27 to A65 that exhibited increased anti-sigma28 activity. These FIgM 'hyperinhibitor' mutants were secreted less than wild-type FIgM. Mutations that interfere with the secretion of FIgM without abolishing anti-sigma28 activity have a negative effect upon the secretion of a His-tagged FIgM mutant that lacks anti-sigma28 activity. Models are proposed to explain the dominant negative phenotype of the FIgM secretion mutants reported in this study.

Alanine↗
PubMed1998

In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase.

The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

Alleles↗
PubMed1998

Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium.

The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium. The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389-2400, 1997). In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains. The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected. This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains. No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.

Alleles↗
PubMed1998

The anti-sigma factors.

A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-sigma) factor. The cytoplasmic class of anti-sigma factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli sigma 70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor sigma 28; SpoIIAB, which inhibits the sporulation-specific sigma factor, sigma F and sigma G, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor sigma B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor sigma 32. In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane-bound anti-sigma factors has recently been discovered in a variety of eubacteria. This new class of anti-sigma factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors. The range of cell processes regulated by anti-sigma factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence.

Bacterial Outer Membrane Proteins↗
PubMed1997

The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, sigma 28.

The interaction between the flagellum specific sigma factor, sigma 28, and its inhibitor, FlgM, was examined using multidimensional heteronuclear NMR. Here we observe that free FlgM is mostly unfolded, but about 50% of the residues become structured when bound to sigma 28. Our analysis suggests that the sigma 28 binding domain of FlgM is contained within the last 57 amino acids of the protein while the first 40 amino acids are unstructured in both the free and bound states. Genetic analysis of flgM mutants that fail to inhibit sigma 28 activity reveal amino acid changes that are also isolated to the C-terminal 57 residues of FlgM. We postulate that the lack of structure in free and bound FlgM is important to its role as an exported protein.

Bacterial Proteins↗
PubMed1997

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites.

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.

Arginine↗