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Biomedical subjects

I B Holland

Publications and source records attributed to I B Holland.

At least 19 recordsLinked to original sources
PubMed1995

Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway.

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.

Ampicillin↗
PubMed1995

Evidence for post-transcriptional regulation of the synthesis of the Escherichia coli HlyB haemolysin translocator and production of polyclonal anti-HlyB antibody.

Extensive attempts were made to overexpress the Escherichia coli haemolysin translocator protein HlyB, and HlyB fragments, utilising high copy number plasmids or hlyB expressed from strong promoters including lambda PR, ptrp and the T7 promoter. Analysis of both cytoplasmic and membrane fractions failed to detect any overexpression of the protein, although all the constructs showed biological activity and there was no evidence of HlyB-induced toxicity. In some constructs, the effect of removing a stem-loop structure, immediately upstream of the start codon and implicated in rho-independent termination of transcription, was tested but this did not lead to over-expression. Nevertheless, analysis of hlyB specific mRNA synthesis revealed that some constructs showed at least a 50-fold increase in mRNA levels, indicating that expression of HlyB may be limited at the translational level. When HlyB was expressed as a hybrid, downstream of LacZ, extremely high level overproduction was then detected in total cell extracts. When the expression of HlyB or HlyB fragments expressed from a T7 promoter was examined, the C-terminal ATPase domain was dramatically overexpressed but the production of fragments encompassing the N-terminal membrane domain, was reduced at least 1000-fold. These results indicate that mRNA structures corresponding to the membrane domain of HlyB greatly limit the post-transcriptional expression of HlyB. When such structures are deleted, or disrupted when part of a larger mRNA, HlyB or the HlyB ATPase domain can be overproduced in milligram quantities and this has facilitated the production of high titre antibodies to HlyB.

Antibodies↗
PubMed1995

Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus in Escherichia coli.

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30 degrees C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30 degrees C and was temperature sensitive (ts) at 42 degrees C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the ts defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca++ levels which affect the activity of the TFP target in some way.

Acyl-Carrier Protein S-Malonyltransferase↗
PubMed1994

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli.

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Gly10Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.

ATP-Binding Cassette Transporters↗
PubMed1994

Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator.

Heterologous proteins synthesized in the Gram-negative bacterium Escherichia coli in bioreactor culture may accumulate in one of three 'compartments':the cytoplasm, the periplasm, or the extracellular medium. Many overexpressed proteins from various origins have been purified from each of these locations. However, to date, each system has required specific tailoring to meet the stringent requirements for each protein product to ensure correct folding, activity and appropriate yield. The E. coli haemolysin secretion system appears to provide a flexible mechanism with which to secrete a wide variety of heterologous fusion proteins into the extracellular medium.

Bacterial Proteins↗
PubMed1994

Protein secretion pathway in Escherichia coli.

The export of proteins to the Escherichia coli periplasm is a well established system for heterologous protein production. With a better understanding of the protein export (SecA, Y-dependent) process and a greater awareness of the conditions necessary for correct folding of proteins in the periplasm, serious efforts are now being made to manipulate this system to achieve substantial increases in the yield of authentically folded proteins. Further advances in the development of methods for the recovery of recombinant proteins from the culture medium have made the use of fusion proteins secreted by the protein A or haemolysin pathways a more attractive option. Recent studies of the haemolysin system indicate its ability to secrete a wide range of polypeptides, including normally cytoplasmic proteins. As their features and potential applications become much clearer, a rapidly expanding number of protein-secretion mechanisms in Gram-negative bacteria are becoming available for heterologous protein expression. Most, if not all, of these systems can be successfully transplanted into E. coli, providing a wider choice of systems for the future.

Adenosine Triphosphatases↗
PubMed1992

Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein.

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.

Amino Acid Sequence↗
PubMed1992

Characterization of the hemolysin transporter, HlyB, using an epitope insertion.

The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.

Amino Acid Sequence↗
PubMed1992

Identification of individual amino acids required for secretion within the haemolysin (HlyA) C-terminal targeting region.

The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes. Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, -D and possibly TolC. HlyA is targeted to the medium via the membrane translocation complex, by a novel C-terminal secretion signal. Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20-60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function. In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C-terminus. The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue -46 and which contains at least four individual residues essential for maximal secretion levels. We propose that such residues act co-operatively, forming multiple contact points with the translocator proteins, with the 'best fit' promoting maximal levels of secretion.

Amino Acid Sequence↗
PubMed1991

Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins.

Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

Amino Acid Sequence↗
PubMed1991

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.

Base Sequence↗
PubMed1991

DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: genetic analysis.

Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied. Results show that a mutation, located at 57.2 min on the linkage map of E. coli, is responsible for the hyper-resistant phenotype. It is also responsible for the synthesis of a 55-kdal protein in high concentrations. In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C. There are indications that the mutation may have occurred in a regulatory gene, puvR, and as a result the operon, including a putative puvA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively. A model for the control of the PUV operon is proposed.

Bacterial Proteins↗
PubMed1991

Calcium in bacteria: a solution to which problem?

Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNA replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipid synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism which have yet to be discovered.

Bacteria↗
PubMed1991

Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain.

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.

Amino Acid Sequence↗
PubMed1991

Lung vascular injury after administration of viable hemolysin-forming Escherichia coli in isolated rabbit lungs.

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor. In the present study, the possible significance of hemolysin production was investigated in a model of septic lung failure through infusion of viable bacteria in isolated rabbit lungs; 10(4) to 10(7) E. coli/ml perfusate caused a dose- and time-dependent appearance of hemolysin, accompanied by release of potassium, thromboxane A2, and PGI2 into the perfusate. Concomitantly, marked pulmonary hypertension developed. Inhibitor studies suggested that the pressor response was predominantly mediated by pulmonary thromboxane generation. Administration of hemolysin-forming E. coli additionally caused a protracted, dose-dependent increase in the lung capillary filtration coefficient, followed by severe edema formation. The permeability increase was independent of lung prostanoid generation. An E. coli strain that releases an inactive form of hemolysin completely failed to provoke the described biophysical and biochemical responses. Preapplication of 2 x 10(8) human granulocytes was without effect in the present experimental model. We conclude that the hemolysin produced by low numbers of E. coli organisms can provoke thromboxane-mediated pulmonary hypertension and severe vascular leakage. E. coli hemolysin and, possibly, other related cytolysins may thus contribute directly to the pathogenesis of acute respiratory failure under conditions of sepsis or pneumonia.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

The mechanism of secretion of hemolysin and other polypeptides from gram-negative bacteria.

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is a two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of the E. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.

Amino Acid Sequence↗