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Biomedical subjects

C Manoil

Publications and source records attributed to C Manoil.

At least 19 recordsLinked to original sources
PubMed2001

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa.

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR-LasI and RhlR-RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Amino Acid Sequence↗
PubMed2001

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning.

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.

Animals↗
PubMed2001

Drosophila as a model host for Pseudomonas aeruginosa infection.

Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

Animals↗
PubMed2000

Insertion of in-frame sequence tags into proteins using transposons.

Several methods based on the use of transposons allow the efficient generation of relatively short (e.g., <35 residues) in-frame insertions in proteins. The analysis of such insertions has provided a simple means to identify sites that tolerate dramatic sequence changes without loss of function ("permissive" sites) and to dissect protein structure-function relationships. In addition, epitope and protease cleavage site "tags" introduced in such insertions have made it possible to analyze the oligomerization state and transmembrane topologies of several proteins. Finally, the DNA inserted by these methods generally carries restriction sites which may facilitate the construction of in-frame deletions and gene fusions encoding a variety of chimeric proteins.

Amino Acid Sequence↗
PubMed1999

Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa.

Identification of host factors that interact with pathogens is crucial to an understanding of infectious disease, but direct screening for host mutations to aid in this task is not feasible in mammals. The nematode Caenorhabditis elegans is a genetically tractable alternative for investigating the pathogenic bacterium Pseudomonas aeruginosa. A P. aeruginosa toxin, produced at high cell density under control of the quorum-sensing regulators LasR and RhlR, rapidly and lethally paralyzes C. elegans. Loss-of-function mutations in C. elegans egl-9, a gene required for normal egg laying, confer strong resistance to the paralysis. Thus, activation of EGL-9 or of a pathway that includes it may lead to the paralysis. The molecular identity of egl-9 was determined by transformation rescue and DNA sequencing. A mammalian homologue of EGL-9 is expressed in tissues in which exposure to P. aeruginosa could have clinical effects.

Amino Acid Sequence↗
PubMed1999

Analysis of F factor TraD membrane topology by use of gene fusions and trypsin-sensitive insertions.

This report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in. Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion.

Alkaline Phosphatase↗
PubMed1998

Mutant membrane protein toxicity.

This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells. In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10(4)-fold. Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed. The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix. The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding. An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease.

Amino Acid Sequence↗
PubMed1998

Missense mutations that inactivate Escherichia coli lac permease.

Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poorly understood. To establish a model for investigating this issue, we identified 51 missense mutations arising in vivo that inactivate Escherichia coli lac permease, a well-characterized membrane transport protein. The mutants were isolated using a genetic screening procedure which eliminates mutations that block expression of the lac permease gene, such as nonsense and frameshift mutations. The majority of the 51 missense mutations caused highly non-conservative changes in membrane-spanning sequences, such as the introduction of charged residues. Nevertheless, the greatest clustering of substitutions occurred in the two regions of lac permease thought to be most important for transport function. The existence of this clustering indicates that even highly non-conservative substitutions may cause relatively localized structural defects. Conservative inactivating substitutions were scattered throughout lac permease and may affect residues that make contacts required for normal folding. Two unexpected phenotypes were observed in the collection of mutants: about 20% of the substitutions led to cold-sensitive lactose utilization, and one substitution made the mutant lac permease toxic to cells. This relatively unbiased collection of mutants should provide a resource for further studies of how missense mutations inactivate membrane proteins in vivo.

Base Sequence↗
PubMed1997

A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease.

Proteins can be remarkably tolerant of major mutational changes. Sites that accomodate large insertions without loss of function ("permissive" sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding. The identification of such sites can aid in the engineering of functional derivatives of a protein with novel properties. To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertions in cloned genes. In a first step, a beta-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon derivative into the target gene. Requiring beta-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the transposon relative to the target gene. In a second step, most of the transposon sequences are excised in vitro, leaving the in-frame insertion. Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety of proteins. As a test case, a set of 31-codon insertions in the Escherichia coli lac permease gene was generated. The lactose transport activities of the mutant proteins followed a simple pattern: most of the proteins (10/12) with insertions in sequences thought to face the cytoplasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive. The only exceptions were two inactive proteins with insertions in the third cytoplasmic region. Most of the inactive proteins were detected at reduced levels in cells, presumably due to proteolytic breakdown. These studies thus illustrate the use of the new method to identify permissive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease. In addition to screening for permissive sites, 31-codon insertion mutagenesis may be useful in epitope-tagging proteins at multiple internal positions, in analyzing membrane protein topology, and in dissecting structure-function relationships in proteins.

Amino Acid Sequence↗
PubMed1997

Engineering trypsin-sensitive sites in a membrane transport protein.

This paper describes a systematic procedure for introducing protease-sensitive sites into bacterial integral membrane proteins. Such sites should make it possible to monitor the subcellular localization of individual domains of a topologically complex protein. Escherichia coli lac permease was used as a model. Site-directed mutagenesis, targeted to a particular periplasmic domain, was used to generate insertion derivatives containing a lysine residue in different sequence contexts. Individual mutants were then screened for lactose transport activity and efficient cleavage by trypsin. To facilitate this screen, the mutagenesis was carried out using a gene fusion encoding an easily detected, bifunctional lac permease-galactosidase hybrid. Insertions were identified in the fourth and sixth periplasmic domains (P4 and P6) that were efficiently cleaved in both the hybrid protein and in unfused lac permease. One of the P6 insertion mutants exhibited lactose transport specific activity near that of the wild-type and was shown by sequence analysis to be cleaved at the expected site in the inserted sequence. As part of this analysis, we determined the range of cellular concentrations of lac permease over which lactose uptake was linear. The activity showed a plateau at a relatively low concentration corresponding to approximately five times the wild-type level.

Bacterial Proteins↗
PubMed1997

Insertion mutagenesis of the lac repressor and its implications for structure-function analysis.

We recently developed a simple technique for the generation of relatively large (31-codon) insertion mutations in cloned genes. To test whether the analysis of such mutations could provide insight into structure-function relationships in proteins, we examined a set of insertion mutants of the Escherichia coli lac repressor (LacI). Representatives of several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative phenotypes. The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein. In particular, the results support the prior designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain. These findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function relationships in proteins for which high-resolution structures are not available.

Bacterial Proteins↗
PubMed1995

Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy.

The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.

Alkaline Phosphatase↗
PubMed1995

Sequences determining the cytoplasmic localization of a chemoreceptor domain.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.

Alkaline Phosphatase↗
PubMed1995

Membrane protein assembly: genetic, evolutionary and medical perspectives.

Lipid bilayers are delicate structures that are easily disrupted by a variety of amphipathic molecules. Yet the viability of a cell requires the continued assembly of large amphipathic proteins within its membranes without damage. The need to minimize bilayer disruption may account for a number of fundamental features of membrane protein assembly. These include the use of redundant sequence information to establish the topologies and folded structures of membrane proteins, and the existence of efficient mechanisms to rid cells of misassembled proteins. Most missense mutations that inactivate a membrane protein probably do so by altering the folding of the membrane-inserted structure rather than by rearranging the topology or by changing key residues involved directly in function. Such misfolded membrane proteins may be toxic to cells if they escape cellular safeguards. This toxicity may underlie some human degenerative diseases due to mutant membrane proteins.

Biological Evolution↗
PubMed1994

Mutations eliminating the protein export function of a membrane-spanning sequence.

Individual membrane protein spanning sequences can promote protein export. To help define the sequence features necessary for this action, we identified mutations disrupting export mediated by the first spanning sequence (TM1) of the Escherichia coli serine chemoreceptor. Mutant spanning sequences were generated and characterized using beta-galactosidase and alkaline phosphatase gene fusions. The protein export function of TM1 was remarkably tolerant of single charged residues, and the introduction of pairs of charged amino acids was necessary to eliminate export. The results are accommodated by a model in which export requires a stretch of uncharged residues whose summed hydrophobicity exceeds a particular threshold value. This threshold approximates the minimum hydrophobicity required for cleavable signal sequence function. In addition, the threshold was near the minimum hydrophobicity observed for wild-type spanning sequences in a collection of topologically characterized membrane proteins.

Amino Acid Sequence↗
PubMed1994

An amphipathic sequence determinant of membrane protein topology.

We developed a screen involving alkaline phosphatase gene fusions to identify mutations altering the membrane topology of a bacterial chemoreceptor (Escherichia coli Tsr). We identified three informative classes of mutations causing increased export of the protein's normally cytoplasmic carboxyl-terminal domain. The first class consisted of deletions eliminating all or most of the membrane-spanning sequence (TM2) immediately amino-terminal to the cytoplasmic domain. The second class consisted of mutations altering a highly amphipathic sequence at the beginning of the domain. The third class of mutation was a deletion of an upstream spanning sequence (TM1). The amphipathic sequence appears to be a novel determinant of membrane topology whose function is not due to its positive residue density. The amphipathic character of the sequence is relatively well-conserved in chemoreceptors and their relatives. Although deletions removing the amphipathic sequence or TM1 alone caused only partial carboxyl-terminal domain export, a double mutation removing both caused efficient export. This result suggests that the two sequences function independently to promote normal membrane insertion. The independent functioning of the two sequences may help ensure that Tsr insertion is normally a high fidelity process.

Amino Acid Sequence↗
PubMed1994

Role of a small cytoplasmic domain in the establishment of serine chemoreceptor membrane topology.

The Escherichia coli serine chemoreceptor takes on a simple membrane topology with two transmembrane segments separating cytoplasmically disposed N and C termini from a central periplasmic domain. We investigated the role of the small N-terminal cytoplasmic domain in membrane insertion using alkaline phosphatase gene fusions. Mutations eliminating the positive charge of the domain altered insertion dramatically, with reciprocal effects on hybrids with periplasmic and C-terminal cytoplasmic fusion junctions. Efficient export of the normally cytoplasmic C-terminal domain required that, in addition to the N-terminal changes, a short amphiphatic sequence at the beginning of the C-terminal domain be also absent. These findings document the importance of the positive character of the N-terminal domain in chemoreceptor membrane insertion and imply that partially redundant sequence information controls the orientation of the second transmembrane segment.

Amino Acid Sequence↗