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M D Manson

Publications and source records attributed to M D Manson.

34 records · Page 2Linked to original sources
PubMed1989

Evolution of chemotactic-signal transducers in enteric bacteria.

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.

Amino Acid Sequence↗
PubMed1988

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process.

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.

ATP-Binding Cassette Transporters↗
PubMed1988

Maltose chemoreceptor of Escherichia coli: interaction of maltose-binding protein and the tar signal transducer.

The maltose chemoreceptor in Escherichia coli consists of the periplasmic maltose-binding protein (MBP) and the Tar signal transducer, which is localized in the cytoplasmic membrane. We previously isolated strains containing malE mutations that cause specific defects in the chemotactic function of MBP. Four of these mutations have now been characterized by DNA sequence analysis. Two of them replace threonine at residue 53 of MBP with isoleucine (MBP-TI53), one replaces an aspartate at residue 55 with asparagine (MBP-DN55), and the fourth replaces threonine at residue 345 with isoleucine (MBP-TI345). The chemotactic defects of MBP-TI53 and MBP-DN55, but not of MBP-TI345, are suppressed by mutations in the tar gene. Of the tar mutations, the most effective suppressor (isolated independently three times) replaces Arg-73 of Tar with tryptophan. Two other tar mutations that disrupt the aspartate chemoreceptor function of Tar also suppress the maltose taxis defects associated with MBP-TI53 and MBP-DN55. One of these mutations introduces glutamine at residue 73 of Tar, the other replaces arginine at residue 69 of Tar with cysteine. These results suggest that regions of MBP that include residues 53 to 55 and residue 345 are important for the interaction with Tar. In turn, arginines at residues 69 and 73 of Tar must be involved in the recognition of maltose-bound MBP and/or in the production of the attractant signal generated by Tar in response to maltose-bound MBP.

ATP-Binding Cassette Transporters↗
PubMed1987

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors.

The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the beta-methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly74 of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.

Amino Acid Sequence↗
PubMed1986

Mutations in tar suppress defects in maltose chemotaxis caused by specific malE mutations.

Maltose-binding protein (MBP), which is encoded by the malE gene, is the maltose chemoreceptor of Escherichia coli, as well as an essential component of the maltose uptake system. Maltose-loaded MBP is thought to initiate a chemotactic response by binding to the tar gene product, the signal transducer Tar, which is also the aspartate chemoreceptor. To study the interaction of MBP with Tar, we selected 14 malE mutants which had specific defects in maltose taxis. Three of these mutants were fully active in maltose transport and produced MBP in normal amounts. The isoelectric points of the MBPs from these three mutants were identical to (malE461 and malE469) or only 0.1 pH unit more basic than (malE454) the isoelectric point of the wild-type protein (pH 5.0). Six of the mutations, including malE454, malE461, and malE469, were mapped in detail; they were located in two regions within malE. We also isolated second-site suppressor mutations in the tar gene that restored maltose taxis in combination with the closely linked malE454 and malE461 mutations but not with the malE469 mutation, which maps in a different part of the gene. This allele-specific suppression confirmed that MBP and Tar interact directly.

ATP-Binding Cassette Transporters↗
PubMed1985

Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein.

Maltose-binding protein (MBP) is essential for maltose transport and chemotaxis in Escherichia coli. To perform these functions it must interact with two sets of cytoplasmic membrane proteins, the MalFGK transport complex and the chemotactic signal transducer Tar. MBP is present at high concentrations, on the order of 1 mM, in the periplasm of maltose-induced or malTc constitutive cells. To determine how the amount of MBP affects transport and taxis, we utilized a series of malE signal-sequence mutations that interfere with export of MBP. The MBP content in shock fluid from cells carrying the various mutations ranged from 4 to 23% of the malE+ level. The apparent Km for maltose transport varied by less than a factor of 2 among malE+ and mutant strains. At a saturating maltose concentration 9% (approximately 90 microM) of the malE+ amount of MBP was required for half-maximal uptake rates. Transport exhibited a sigmoidal dependence on the amount of periplasmic MBP, indicating that MBP may be involved in a cooperative interaction at some stage of the transport process. The chemotactic response to a saturating maltose stimulus exhibited a first-order dependence on the amount of periplasmic MBP. Thus, interaction of a single substrate-bound MBP with Tar appears sufficient to initiate a chemotactic signal from the transducer. A half-maximal chemotactic response occurred at 25% of the malE+ MBP level, suggesting that in vivo the KD for binding of maltose-loaded MBP to Tar is quite high (approximately 250 microM).

ATP-Binding Cassette Transporters↗
PubMed1985

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria.

In Escherichia coli, the periplasmic maltose-binding protein (MBP), the product of the malE gene, is the primary recognition component of the transport system for maltose and maltodextrins. It is also the maltose chemoreceptor, in which capacity it interacts with the signal transducer Tar (taxis to aspartate and some repellents). In studies of the maltose system in other members of the family Enterobacteriaceae, we found that MBP is produced by Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens. MBP from all of these species cross-reacted with antibody against the E. coli protein and had a similar molecular weight (about 40,000). The Shigella flexneri and Proteus mirabilis strains we examined did not synthesize MBP. The isoelectric points of MBP from different species varied from the acid extreme of E. coli (4.8) to the basic extreme of E. aerogenes (8.9). All species with MBP transported maltose with high affinity, although the Vmax for K. pneumoniae was severalfold lower than that for the other species. Maltose chemotaxis was observed only in E. coli and E. aerogenes. In S. typhimurium LT2, Tar was completely inactive in maltose taxis, although it signaled normally in response to aspartate. MBP isolated from all five species could be used to reconstitute maltose transport and taxis in a delta malE strain of E. coli after permeabilization of the outer membrane with calcium.

ATP-Binding Cassette Transporters↗
PubMed1984

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants.

Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C. After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay. About 20% of tethered cells responded to 10(-4) M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s). In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells. The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells. Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.

ATP-Binding Cassette Transporters↗
PubMed1984

Transposon Tn10-dependent expression of the lamB gene in Escherichia coli.

Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains. The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain. Transport of maltose at concentrations of below 10(-6) M was reduced about threefold. When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored. We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.

Bacterial Outer Membrane Proteins↗
PubMed1977

A protonmotive force drives bacterial flagella.

Streptococcus strain V4051 is motile in the presence of glucose. The cells move steadily along smooth paths (run), jump about briefly with little net displacement (twiddle), and then run in new directions. They stop swimming when deprived of glucose. These cells become motile when an electrical potential or a pH gradient is imposed across the membrane. Starved cells suspended in a potassium-free medium respond to the addition of valinomycin by a brief period of vigorous twiddling. They also twiddle, although less vigorously, when the external pH is lowered. Valinomycin-induced twiddling occurs in the absence of external alkali or alkaline earth cations and without significant net synthesis of ATP. When a chemoattractant is added to cells swimming in the presence of glucose, twiddles are transiently suppressed, and the cells run for a time. Similarly, when starved cells are suspended in a potassium-free medium containing both valinomycin and an attractant, many cells initially run rather than twiddle. We conclude that the flagella are driven by a protonmotive force.

Adenosine Triphosphate↗
PubMed1976

Naturally occurring sites within the Shigella dysenteriae tryptophan operon severely limit tryptophan biosynthesis.

We investigated the structural, functional, and regulatory properties of the Shigella dysenteriae tryptophan (trp.) operon in transduction hybrids in which the cysB-trp-region of Escherichia coli is replaced by the corresponding region from S. dysenteriae. Tryptophan biosynthesis was largely blocked in the hybrids, although the order of the structural genes was identical with that of E. coli. Nutritional tests and enzyme assays revealed that the hybrids produced a defective anthranilate synthetase (ASase). Deletion mapping identified two distinct sites in trpE, each of which was partially responsible for the instability and low activity of ASase. We also discovered a pleiotropic site trpP (S) that maps outside the structural gene region and is closely linked to the S. dysenteriae trp operator. trpP (S) reduced the rate of trp messenger ribonucleic acid synthesis, and consequently trp enzyme levels, 10-fold relative to wild-type E. coli. In recombinants in which the structural genes of E coli were under the control of the S. dysenteriae promoter, enzyme levels were also reduced 10-fold. In some fast-growing revertants of the original hybrids, the rates of trp messenger ribonucleic acid synthesis and levels of tryptophan synthetase were restored to values characteristic of wild-type E.coli. Thus, the Trp auxotrophy associated with the S dysenteriae trp operon derives from the combination of a defective ASase and decreased expression of the entire operon imposed by trpP (S).

Anthranilate Phosphoribosyltransferase↗
PubMed1976

Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae.

Anthranilate Phosphoribosyltransferase↗
PubMedDate not supplied

Peptide chemotaxis in E. coli involves the Tap signal transducer and the dipeptide permease.

Bacterial chemotaxis provides a simple model system for the more complex sensory responses of multicellular eukaryotic organisms. In Escherichia coli, methylation and demethylation of four related membrane proteins, the methyl-accepting chemotaxis proteins (or MCPs), is central to chemotactic sensing and signal transduction. Three of these proteins, Tar, Tsr and Trg, have been assigned specific roles in chemotaxis. However, the role of the fourth MCP, Tap, has remained obscure. We demonstrate here that Tap functions as a conventional signal transducer, enabling the cell to respond chemotactically to dipeptides. This provides the first evidence of specific bacterial chemotaxis towards peptides. Peptide taxis requires the function of a periplasmic component of the dipeptide permease. This protein represents the first example of a periplasmic chemoreceptor that does not have a sugar substrate.

Bacterial Proteins↗