Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects.

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.

Substitution of aspartate and glutamate for active center histidines in the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system maintain phosphotransfer potential.

The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIA(Glc) were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation. The mutants [H189E]enzyme I, [H15D]HPr, and [H90E]enzyme IIA(Glc) retained ability for phosphorylation as indicated by [(32)P]phosphoenolpyruvate labeling. As the active center histidines of both enzyme I and enzyme IIA(Glc) undergo phosphorylation of the N(epsilon2) atom, while HPr is phosphorylated at the N(delta1) atom, a pattern of successful substitution of glutamates for N(epsilon2) phosphorylations and aspartates for N(delta1) phosphorylations emerges. Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes.

Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization.

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.

Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis.

The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.

Determination of the binding constants for three HPr-specific monoclonal antibodies and their Fab fragments.

Jel42, Jel44 and Jel323 are mouse monoclonal antibodies specific for HPr, the histidine-containing phosphocarrier protein, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. The binding constants, Kd, of the three antibodies and their Fab fragments have been determined: Jel42, 2.8+/-1.6 nM; Jel42Fab, 3. 7+/-0.3 nM; Jel44, 5.1+/-0.4 nM; Jel44Fab, 6.3+/-1.1 nM; Jel323, 5. 7+/-0.5 nM; Jel323Fab, 5.1+/-0.9 nM. The binding constants were determined by a fluorescence polarization assay that used the mutants Arg17Cys HPr and Phe2Cys HPr specifically labeled with fluorescein-5-maleimide. The latter was used for Jel323 as interaction with fluorescein-5-maleimide-labeled Arg17Cys HPr gave quenching of the fluorescence intensity. The specificity of each antibody and the Fab fragments for binding to many HPr mutants was determined by this solution assay. The Fab fragments had the same specificity or cross-reactivity as the antibodies. Comparison of relative binding specificity determined by a solid phase assay showed that the results from both types of assay are comparable. Neither Jel42 nor Jel323 binding was affected by ionic strength (approximately 45 to 245 mM salt), but Jel44 varied about two- to threefold. Charged residues are prominent in the Jel44 epitope and paratope. Initial thermodynamic characterization was investigated by temperature-dependent determinations of the Kd. The binding of Jel42 and Jel323 to HPr was entropic at low temperatures and enthalpic at physiological temperatures. Jel44 showed no change in the contributions of entropy and enthalpy over the temperature range 3 to 37 degreesC. The 2.5 A resolution structure of the complex of Jel42 Fab fragment bound to HPr described in the accompanying paper provides some structural intepretation for the mutational effects.

Cloning of a cDNA encoding the 21.2 kDa oleosin isoform from Arabidopsis thaliana and a study of its expression in a mutant defective in diacylglycerol acyltransferase activity.

A full-length cDNA clone (pA23) of 832 bp encoding an oleosin from Arabidopsis thaliana was isolated by differential screening of a silique-specific cDNA library with probes prepared from poly(A)+ RNA isolated from developing seeds of wild-type (WT) Arabidopsis and from mutant AS11 with a lesion affecting diacylglycerol acyltransferase (DGAT) activity during embryo development. The encoded protein has a calculated molecular mass of 21.2 kDa, and its amino acid sequence shows strong sequence homology and structural similarity to other known oleosins. Transcription of the oleosin gene during seed development was both reduced and delayed in AS11 compared to WT. However, the level of oleosin protein did not appear to be down-regulated during seed development, and at maturity, the overall level of oleosin protein was similar in both WT and AS11. These findings indicate that regulation of oleosin gene expression is part of a highly complex, and co-ordinated expression of storage lipid biosynthesis and related (oleosin) genes during oilseed development.