Rapid quantitative analysis of binary mixtures of Escherichia coli strains using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Pyrolysis mass spectrometry (PyMS) and multivariate calibration were used to show the high degree of relatedness between Escherichia coli HB101 and E. coli UB5201. Next, binary mixtures of these two phenotypically closely related E. coli strains were prepared and subjected to PyMS. Fully interconnected feedforward artificial neural networks (ANNs) were used to analyse the pyrolysis mass spectra to obtain quantitative information representative of the level of E. coli UB5201 in E. coli HB101. The ANNs exploited were trained using the standard back propagation algorithm, and the nodes used sigmoidal squashing functions. Accurate quantitative information was obtained for mixtures with >3% E. coli UB5201 in E. coli HB101. To remove noise from the pyrolysis mass spectra and so lower the limit of detection, the spectra were reduced using principal components analysis (PCA) and the first 13 principal components used to train ANNs. These PCA-ANNs allowed accurate estimates at levels as low as 1% E. coli UB5201 in E. coli HB101 to be predicted. In terms of bacterial numbers, it was shown that the limit of detection for PyMS in conjunction with ANNs was 3 × 10⁴ E. coli UB5201 cells in 1·6 × 10⁷ E. coli HB101 cells. It may be concluded that PyMS with ANNs provides a powerful and rapid method for the quantification of mixtures of closely related bacterial strains.     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.     

Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling

Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens.     

Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

A screen for bacterial endosymbionts in the model organisms Tribolium castaneum,T. confusum,Callosobruchus maculatus,and related species

Reproductive parasites such as Wolbachia are extremely widespread amongst the arthropods and can have a large influence over the reproduction and fitness of their hosts. Undetected infections could thus confound the results of a wide range of studies that focus on aspects of host behavior, reproduction, fitness, and degrees of reproductive isolation. This potential problem has already been underlined by work investigating the incidence of Wolbachia infections in stocks of the model system Drosophila melanogaster. Here we survey a range of lab stocks of further commonly used model arthropods, focusing especially on the flour beetles Tribolium castaneum and Tribolium confusum, the cowpea weevil Callosobruchus maculatus and related species (Coleoptera: Tenebrionidae and Bruchidae). These species are widespread stored product pests so knowledge of infections with symbionts further has potential use in informing biocontrol measures. Beetles were assessed for infection with 3 known microbial reproductive parasites: Wolbachia, Rickettsia, Spiroplasma. Infections with some of these microbes were found in some of the lab stocks studied, although overall infections were relatively rare. The consequences of finding infections in these or other species and the type of previous studies likely to be affected most are discussed.