Naheed Kaderbhai

Rapid identification of urinary tract infection bacteria using hyperspectral whole-organism fingerprinting and artificial neural networks

Three rapid spectroscopic approaches for whole-organism fingerprinting-pyrolysis mass spectrometry (PyMS), Fourier transform infra-red spectroscopy (FT-IR) and dispersive Raman microscopy--were used to analyse a group of 59 clinical bacterial isolates associated with urinary tract infection. Direct visual analysis of these spectra was not possible, highlighting the need to use methods to reduce the dimensionality of these hyperspectral data. The unsupervised methods of discriminant function and hierarchical cluster analyses were employed to group these organisms based on their spectral fingerprints, but none produced wholly satisfactory groupings which were characteristic for each of the five bacterial types. In contrast, for PyMS and FT-IR, the artificial neural network (ANN) approaches exploiting multi-layer perceptrons or radial basis functions could be trained with representative spectra of the five bacterial groups so that isolates from clinical bacteriuria in an independent unseen test set could be correctly identified. Comparable ANNs trained with Raman spectra correctly identified some 80% of the same test set. PyMS and FT-IR have often been exploited within microbial systematics, but these are believed to be the first published data showing the ability of dispersive Raman microscopy to discriminate clinically significant intact bacterial species. These results demonstrate that modern analytical spectroscopies of high intrinsic dimensionality can provide rapid accurate microbial characterization techniques, but only when combined with appropriate chemometrics.

Functional Genomics via Metabolic Footprinting: Monitoring Metabolite Secretion by Escherichia coli Tryptophan Metabolism Mutants Using FT–IR and Direct Injection Electrospray Mass Spectrometry

We sought to test the hypothesis that mutant bacterial strains could be discriminated from each other on the basis of the metabolites they secrete into the medium (their ‘metabolic footprint’), using two methods of ‘global’ metabolite analysis (FT–IR and direct injection electrospray mass spectrometry). The biological system used was based on a published study of Escherichia coli tryptophan mutants that had been analysed and discriminated by Yanofsky and colleagues using transcriptome analysis. Wild-type strains supplemented with tryptophan or analogues could be discriminated from controls using FT–IR of 24 h broths, as could each of the mutant strains in both minimal and supplemented media. Direct injection electrospray mass spectrometry with unit mass resolution could also be used to discriminate the strains from each other, and had the advantage that the discrimination required the use of just two or three masses in each case. These were determined via a genetic algorithm. Both methods are rapid, reagentless, reproducible and cheap, and might beneficially be extended to the analysis of gene knockout libraries.

Noninvasive, On-Line Monitoring of the Biotransformation by Yeast of Glucose to Ethanol Using Dispersive Raman Spectroscopy and Chemometrics

We describe the first application of dispersive Raman spectroscopy using a diode laser exciting at 780 nm and a charge-coupled device (CCD) detector to the noninvasive, on-line determination of the biotransformation by yeast of glucose to ethanol. Software was developed which automatically removed the effects of cosmic rays and other noise, normalized the spectra to an invariant peak, then removed the “baseline” arising from interference by fluorescent impurities, to obtain the “true” Raman spectra. Variable selection was automatically performed on the parameters of relevant Raman peaks (height, width, position of top and center, area and skewness), and a small subset used as the input to cross-validated models based on partial least-squares (PLS) regression. The multivariate calibration models so formed were sufficiently robust to be able to predict the concentration of glucose and ethanol in a completely different fermentation with a precision better than 5%. Dispersive Raman spectroscopy, when coupled with the appropriate chemometrics, is a very useful approach to the noninvasive, on-line determination of the progress of microbial fermentations.

A photoactivatable synthetic transit peptide labels 30 kDa and 52 kDa polypeptides of the chloroplast inner envelope membrane

A 24‐residue transit peptide based on the sequence of a precursor of the small subunit of wheat ribulose‐1,5‐bisphosphate carboxylase (Rubisco) was synthesized. The transit peptide was converted into a radioactive azido derivatised analogue. Photoactivation of the radiolabelled transit peptide analogue with isolated inner and outer membranes of the chloroplast envelope intensely labelled two proteins of 30 kDa and 52 kDa. In the outer membrane only the 52 kDa polypeptide was labelled. These findings are consistent with a recent report on the identification of the 30 kDa receptor protein for protein import in the chloroplast envelope contact zones [(1988) Nature 331, 232–237].

Rapid Analysis of High-Dimensional Bioprocesses Using Multivariate Spectroscopies and Advanced Chemometrics

There are an increasing number of instrumental methods for obtaining data from biochemical processes, many of which now provide information on many (indeed many hundreds) of variables simultaneously. The wealth of data that these methods provide, however, is useless without the means to extract the required information. As instruments advance, and the quantity of data produced increases, the fields of bioinformatics and chemometrics have consequently grown greatly in importance. The chemometric methods nowadays available are both powerful and dangerous, and there are many issues to be considered when using statistical analyses on data for which there are numerous measurements (which often exceed the number of samples). It is not difficult to carry out statistical analysis on multivariate data in such a way that the results appear much more impressive than they really are. The authors present some of the methods that we have developed and exploited in Aberystwyth for gathering highly multivariate data from bioprocesses, and some techniques of sound multivariate statistical analyses (and of related methods based on neural and evolutionary computing) which can ensure that the results will stand up to the most rigorous scrutiny.

Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).

Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli

SummaryA synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n=1) to six (n=6) gene copies. The gene, placed in pλ-ncyt under the control of the λPL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5. The expressed levels of cytochrome b5 in Escherichia coli pλ-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing pλ-6cyt produced 75 μg cytochrome b5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the halocytochrome b5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E. coli.

A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge.

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With‐in “sequence spaces” ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane‐associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of −1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore‐dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.

The extraction and reconstitution of the α-cyanocinnamate-sensitive pyruvate transporter from castor bean mitochondria

The pyruvate carrier from castor bean mitochondria has been solubilized with Triton X-114 and partially purified using hydroxyapatite column chromatography. SDS-polyacrylamide gel electrophoresis of the hydroxyapatite-eluate showed that there were 6 major protein bands of Mr, 74kDa, 66kDa, 34kDa, 32kDa, 30kDa 12kDa. When the eluate was reconstituted into liposomes it was shown to catalyze a pyruvate exchange reaction which was sensitive to N-ethyl maleimide and a series of analogues of alpha-cyanocinnamate. The characteristics of this pyruvate exchange activity are similar to that found in intact mitochondria, and it is concluded that one or more proteins in the hydroxyapatite-eluate correspond to the pyruvate carrier.

Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.

A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.