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T Ferenci

Publications and source records attributed to T Ferenci.

At least 37 records · Page 2Linked to original sources
PubMed1996

The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA.

The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined. surA was composed of 1338 bp and encoded 445 amino acid residues. The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose. The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA. SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability. The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer. SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B. subtilis enzyme, had no detectable sucrose phosphate hydrolase activity.

Amino Acid Sequence↗
PubMed1996

Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP.

Free-living bacteria are expert in adapting to variations in nutrient availability, often using an array of transport systems of different affinities to scavenge for particular substrates (multiport). This review concentrates on the regulation of expression of different transporters contributing to multiport in response to varying nutrient levels. A novel mechanism of controlling bacterial transport affinity under sugar limitation is described. In particular, switching from glucose-rich to glucose-limited conditions results in Escherichia coli orchestrating outer membrane changes as well as the induction of a periplasmic binding protein-dependent (ABC-type) transport system. The changes leading to the high affinity transport pathway are directed towards uptake of rapidly utilisable concentrations and are optimal close to 10-6 M medium glucose. High affinity transport is absent under both glucose-rich 'feast' and glucose-starved 'famine' conditions hence high affinity transporters are not simply repressed by excess nutrient. Rather, the improvement in glucose scavenging involves induction of genes in 2 distinct regulons (mgl/gal and mal/lamB) through synthesis of 2 different endogenous inducer molecules (galactose, maltotriose). Endoinducer levels are tightly controlled by extracellular glucose concentration at different glucose-limited growth rates. Aside from endoinducers, the elevated intracellular level of cAMP plays a role in induction of the high-affinity pathway but cAMP-mediated relief from catabolite repression is not itself sufficient for high affinity transport. In contrast to the repressive role of glucose when present at millimolar concentrations, micromolar glucose also leads to the induction of transport systems for other sugars, further broadening the scavenging potential of nutrient-limited bacteria for other substrates.

Adaptation, Physiological↗
PubMed1996

Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli?

treA and osmY expression and RpoS protein levels were investigated in glucose-limited continuous culture. The level of induction of these stationary-phase markers became as high during growth at a D of 0.1 to 0.2 h(-1) as in carbon-starved batch cultures but only in rpoS+ bacteria. The stress protectant trehalose was actually produced at higher levels at low growth rates than in stationary-phase cultures. The pattern of induction of RpoS-dependent activities could be separated from those regulated by cyclic AMP (cAMP) or endoinduction, and the induction occurred at extreme glucose limitation. Escherichia coli turns to a protective stationary-phase response when nutrient levels fall below approximately 10(-7) M glucose, which is insufficient to saturate scavenger transporters regulated by cAMP plus endoinducers, and this response is optimally expressed at 10(-6) M glucose. The high-level induction of protective functions also explains the maintenance energy requirement of bacterial growth at low dilution rates.

Bacterial Proteins↗
PubMed1995

Sequence alignment and structural modelling of the LamB glycoporin family.

lamB gene segments were obtained from Yersinia enterocolitica and Vibrio parahaemolyticus by the PCR and the DNA sequence determined. The deduced polypeptide sequences showed high similarity to six other LamB-related proteins and all contained typical signature sequences present in all members of the family but not other proteins. The aligned amino acid sequences permitted derivation of a model of LamB folding across the bacterial outer membrane using an approach successfully applied in the identification of structural features in other porins (Ferenci,T. (1994) Mol. Microbiol. 14:188-189). The alignment-based model differs from previous LamB structure predictions and is also more complex than that found for OmpF-related porins; more than 16 conserved stretches of amino acid sequence potentially corresponded to membrane-spanning segments.

Amino Acid Sequence↗
PubMed1995

Differential expression of mal genes under cAMP and endogenous inducer control in nutrient-stressed Escherichia coli.

LamB glycoporin has an important general role in carbohydrate uptake during growth at low extracellular sugar concentrations. lamB and mal regulon induction during glucose starvation and glucose-limited continuous culture was investigated using lacZ fusions. A low-level burst of lamB induction occurred upon entry into glucose starvation-induced stationary phase but returned to basal levels during continued nutrient deprivation. Glucose-limited continuous culture elicited much higher expression of transporter genes in the mal regulon, as well as [14C]-maltose-transport activity; malEFG and malKlamB operons in glucose-limited chemostats were expressed to close to half of the level of maltose-induced batch cultures. Limitation-induced expression was dependent on both Crp-cAMP and MalT activation but was independent of RpoS function. As expected for a gene with a Crp-controlled promoter, malT expression was maximal under conditions which elicited the highest cAMP levels, but lamB induction did not behave in a corresponding fashion. Rather, maximal lamB induction occurred at rapid but suboptimal growth rates with micromolar or submicromolar medium glucose. Maximal transport and lamB induction coincided with increased endogenous maltotriose (inducer) concentrations during growth on glucose. Hence regulation of glycoporin and the maltose-transport system is not a starvation- or stationary-phase response but facilitates the adaptation of Escherichia coli to low-nutrient environments through endoinduction.

Bacterial Proteins↗
PubMed1994

Epitope mapping by cysteine mutagenesis: identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli.

Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329-342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370-412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328-338 regions. The lamB mutants were also tested for phage lambda receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12.

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MalG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.

ATP-Binding Cassette Transporters↗
PubMed1994

Crystallization of monodisperse maltoporin from wild-type and mutant strains of various Enterobacteriaceae.

Maltoporin has been purified by affinity chromatography on starch gel columns. This single-step procedure affords the rapid purification of active protein from wild-type and mutants of E. coli, and from other Gram-negative bacteria. The monodisperse protein was crystallized under various conditions. Several preparations have yielded crystals amendable to X-ray analysis, notably a single cysteine substitution, S57C.

Bacterial Outer Membrane Proteins↗
PubMed1994

Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates.

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.

Bacterial Proteins↗
PubMed1993

The importance of the binding-protein-dependent Mgl system to the transport of glucose in Escherichia coli growing on low sugar concentrations.

Glucose limitation in chemostats derepressed the binding-protein-dependent Mgl transport system, which is strongly repressed during growth in batch culture with high glucose levels. The limitation-induced Mgl activity was higher than that of batch cultures "fully induced" for the Mgl system after growth on glycerol plus fucose. Mgl- strains were impaired compared to Mgl+ bacteria in removing glucose from sugar-limited chemostats and were outcompeted in mixed continuous culture on limiting glucose. The influence of Mgl was not observed on growth with limiting maltose or non-carbohydrates, and thus was specific for glucose, a known substrate of the Mgl system. In the absence of the two glucose-specific membrane components of the phosphoenolpyruvate:sugar phosphotransferase system, non-PTS-dependent growth on glucose was observed in continuous culture, but only under sugar-limited conditions derepressing the Mgl system and not in glucose-rich batches or continuous culture. Hence growth of Escherichia coli on glucose at micromolar concentrations involves a significant contribution of a binding-protein-dependent transport system. The participation of multiple transporters in glucose transport can account for the complex non-hyperbolic dependence of growth-rate on glucose concentration and for discrepancies in studies attempting to describe growth on glucose purely in terms of phosphotransferase kinetics.

Biological Transport, Active↗
PubMed1993

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function.

A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function. The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region. In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region. The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position. Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard. Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43. Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane. These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.

Amino Acid Sequence↗
PubMed1993

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.

Bacterial Outer Membrane Proteins↗
PubMed1992

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.

Amino Acid Sequence↗
PubMed1991

Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli.

Wild-type and 16 variant maltoporins with site-directed cysteine substitutions at 14 sites were purified by a novel one-step affinity-chromatographic procedure. The trimer stability of purified proteins with C22S, C38S and G103C substitutions was reduced compared to wild-type maltoporin. Quantitative labelling with N-ethyl[14C]maleimide, cross-linking with bifunctional bismaleimides and disulphide formation was used to test the reactivity of cysteines in the folded protein. The maleimide reactivity of the residues was in the order: 152 approximately equal to 153 greater than 265 greater than 30 approximately equal to 103 approximately equal to 120 approximately equal to 154 approximately equal to 382 greater than 57 approximately equal to 146, with the other sites (22, 38, 97, 184) poorly labelled. Only cysteines at 152 or 153 permitted the formation of inter-subunit disulphide bonds suggesting these residues are located within 0.5-0.9 nm of each other in homotrimers of maltoporin. S152C and S153C as well as S154C permitted the formation of inter-subunit cross-links using bifunctional bismaleimides. The cross-linkability and the high reactivity to N-ethylmaleimide of the 150 region was consistent with the current model of the structure of maltoporin in the outer membrane; the reactivity of the other sites is also discussed within the context of this model.

Amino Acid Sequence↗
PubMed1991

A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli.

Results presented in this study demonstrate that a mutation which inserts an additional tyrosine between the 2 tyrosines at residues 118 and 119 of mature LamB protein results in a temperature-dependent assembly defect. This defect leads to the accumulation of an intermediate at the restrictive temperature that is most likely an assembly-defective monomer. These monomers are rapidly degraded in the wild type (htrA+) strain, and the biphasic kinetics of this degradation indicate that the mutation affects the assembly process and not the final product, i.e. stable trimers. In addition, our data show that the temperature-dependent assembly defect in the mutant strain is reversible, and therefore the accumulated monomers represent a true assembly intermediate. Fractionation studies show that the monomers, which can be accumulated in htrA (degP) mutants at the restrictive temperature, are associated with the outer membrane, indicating that trimerization of LamB is not a prerequisite for localization.

ATP-Binding Cassette Transporters↗
PubMed1991

Genetic mapping of starch- and lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis.

Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.

Bacterial Outer Membrane Proteins↗
PubMed1989

Cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (LamB protein).

Maltoporin in the outer membrane of Escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence. The role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage lambda, was investigated. Site-directed mutagenesis was used to alter each of these residues to a serine. A double mutant lacking both cysteines was also isolated. None of the substitutions affected maltodextrin binding or the binding of phage lambda, suggesting the variant proteins retain a native binding-site conformation. The mutants were assembled at wild-type levels into the outer membrane as maltoporin trimers but the temperature-stability of the trimer greater than monomer dissociation was slightly reduced in the presence of the Cys 38 substitution. However, it is unlikely that the stability of trimers was due to disulfide linkages between subunits since the native trimers are stable under highly reducing conditions in the presence of SDS; more likely the Cys greater than Ser substitutions slightly perturb intra- or inter-subunit hydrophobic interactions in regions predicted to span across the outer membrane.

Bacterial Outer Membrane Proteins↗