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C F Higgins

Publications and source records attributed to C F Higgins.

At least 127 records · Page 7Linked to original sources
PubMed1991

Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein.

The dipeptide permease (Dpp) is one of three genetically distinct peptide-transport systems in enteric bacteria. Dpp also plays a role in chemotaxis towards peptides. We have devised three selections for dpp mutations based on resistance to toxic peptides (bacilysin, valine-containing peptides, and bialaphos). All dpp mutations mapped to a single chromosomal locus between 77 and 78 min in Salmonella typhimurium and at 79.2 min in Escherichia coli. Expression of dpp was constitutive in both species but the absolute level of expression varied widely between strains. At least in part this difference in expression levels is determined by cis-acting sequences. The dpp locus of E. coli was cloned. The first gene in the operon, dppA, encodes a periplasmic dipeptide-binding protein (DBP) required for dipeptide transport and chemotaxis. Downstream of dppA are other genes required for transport but not for chemotaxis. The dipeptide-binding protein was found to share 26.5% sequence identity with the periplasmic oligopeptide-binding protein OppA.

Amino Acid Sequence↗
PubMed1990

Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli.

The TonB protein is required for several outer membrane transport processes in bacteria. A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy. The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration. This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors. Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein. As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm. These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane.

Amino Acid Sequence↗
PubMed1990

TonB protein of Salmonella typhimurium. A model for signal transduction between membranes.

The tonB gene product is required for several outer membrane transport processes in bacteria. The tonB gene from Salmonella typhimurium was sequenced and found to be similar to that of Escherichia coli. The TonB protein is highly proline-rich and includes an unusual segment consisting of multiple X-Pro dipeptide repeats. A synthetic peptide corresponding to this segment has been used to raise anti-TonB antibodies. TonB was shown to be associated with the cytoplasmic membrane, apparently anchored via a single hydrophobic N-terminal segment. Protease accessibility studies, and the use of a series of TonB-beta-lactamase fusions, showed that the rest of the TonB protein is periplasmic. Unusually, export of TonB is not accompanied by cleavage of the N-terminal signal peptide. In the accompanying paper, we show that TonB interacts directly with the outer membrane FhuA (TonA) receptor. Thus, TonB must span the periplasm, providing a link between the cytoplasmic membrane and receptors in the outer membrane. On the basis of these data, and those published by other laboratories, we propose a model whereby TonB serves as a "mechanical" linkage that, by transmitting protein conformational changes from the cytoplasmic membrane across the periplasm, acts as a means of coupling energy to outer membrane transport processes. Such a mechanism has general implications for signal transduction within and between proteins.

Amino Acid Sequence↗
PubMed1990

Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria.

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the "histone-like" protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.

Bacterial Proteins↗
PubMed1990

Chromosome rearrangements induced by recombinant coliphage lambda placMu.

Operon fusions to lacZ, commonly used to study bacterial gene expression in vivo, are normally constructed using phage derivatives such as lambda placMu53 or Mud-1. These derivatives contain a part of trp operon, and we have found that, when integrated into the chromosome, recombination can occur at high frequency between this trp DNA and the chromosomal trp operon leading to chromosomal inversions which fuse lacZ to the trp promoter. Large segments of the chromosome can be inverted by such rearrangements and their occurrence can seriously complicate the isolation of regulatory mutations and other studies unless appropriate precautions are taken. This phenomenon provides a simple means of isolating inversions of defined chromosomal segments and determining the direction of transcription of some lacZ operon fusions.

Bacteriophage lambda↗
PubMed1990

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport.

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri.

Bacterial pathogens undergo profound physiological changes when they infect their hosts, requiring coordinated regulation of gene expression in response to the stresses encountered during infection. A number of environmental stresses (such as osmotic shock and anaerobiosis) have been shown to induce changes in DNA supercoiling that can directly affect the transcription of a specific subset of bacterial genes, at least some of which (the outer-membrane porins and type 1 fimbriae) play a part in bacterial virulence. Here, we demonstrate that the virR gene of S. flexneri, implicated in the temperature regulation of plasmid-encoded virulence genes, is equivalent to the osmZ gene of Escherichia coli, which has previously been shown to mediate its regulatory effects through changes in DNA supercoiling. Our results imply that environmentally induced changes in DNA supercoiling are important in the coordinated control of virulence gene expression in S. flexneri and have general implications for the control of bacterial virulence.

DNA, Bacterial↗
PubMed1990

Periplasmic binding protein-dependent transport systems: the membrane-associated components.

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.

Adenosine Triphosphate↗
PubMed1990

Binding protein-dependent transport systems.

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.

Adenosine Triphosphate↗
PubMed1990

Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence?

There has been a recent revival of interest in one of the most abundant Escherichia coli proteins, H1 (also called H-NS). This protein was first identified many years ago as a major component of the bacterial nucleoid, and has been characterized biochemically by several groups. However, no clear function for the protein emerged from these studies. Our thinking has been transformed by recent findings which complement the biochemistry with genetic data. Several mutations, selected over many years by virtue of their diverse effects on gene expression, have turned out to be allelic and to fall within the structural gene for H1. Bringing together the genetics and the biochemistry has demonstrated that the whole is worth more than the sum of the parts! These findings have far-reaching implications for the mechanisms by which gene expression is regulated and also, perhaps, for the control of bacterial virulence.

Amino Acid Sequence↗
PubMed1990

RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus.

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7 kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease identified as having a general role in the chemical decay of E. coli mRNA.

Cloning, Molecular↗
PubMed1990

Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress?

Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain a relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine (N-trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.

Biological Transport↗
PubMed1989

Identification and localization of the membrane-associated, ATP-binding subunit of the oligopeptide permease of Salmonella typhimurium.

The OppF protein, a component of the oligopeptide permease of Salmonella typhimurium, is an ATP-binding protein and is believed to couple ATP hydrolysis to the transport process. This protein is an example of a large family of closely related proteins which couple ATP to a variety of different biological processes. The oppF gene has been cloned and sequenced. In order to identify and characterize its protein product we overproduced the protein from the cloned gene. Anti-OppF antibodies were raised against a synthetic peptide. Using these antibodies as a probe we identified OppF in wild-type and overproducing strains. Protease accessibility studies showed the protein to be a peripheral membrane protein located on the cytoplasmic side of the inner membrane. These findings have general implications for the organization and function of this class of prokaryotic and eukaryotic transport system.

Bacterial Proteins↗
PubMed1989

Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo.

Periplasmic binding protein-dependent transport systems mediate the accumulation of many diverse substrates in prokaryotic cells. Similar transport systems, including the P-glycoprotein responsible for multidrug resistance in human tumors, are also found in eukaryotes. The mechanism by which energy is coupled to the accumulation of substrate by these transport systems has been controversial. In this paper we demonstrate that ATP hydrolysis occurs in vivo concomitantly with transport. These data strongly suggest that ATP hydrolysis directly energizes substrate accumulation by these transport systems. The apparent stoichiometry is one to two molecules of ATP hydrolyzed per molecule of substrate transported.

ATP-Binding Cassette Transporters↗
PubMed1989

Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments.

Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions. The implications of this finding for cell division and other periplasmic processes are discussed.

Biological Transport↗
PubMed1989

DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus.

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.

Blotting, Southern↗