Program review. Challenges and opportunities for training the next generation of biophysicists: perspectives of the directors of the Molecular Biophysics Training Program at Northwestern University

Molecular biophysics is a broad, diverse, and dynamic field that has presented a variety of unique challenges and opportunities for training future generations of investigators. Having been or currently being intimately associated with the Molecular Biophysics Training Program at Northwestern, we present our perspectives on various issues that we have encountered over the years. We propose no cookie-cutter solutions, as there is no consensus on what constitutes the "ideal" program. However, there is uniformity in opinion on some key issues that might be useful to those interested in establishing a biophysics training program.     

Temperature-Driven Adaptation of the Bacterial Community in Peat Measured by Using Thymidine and Leucine Incorporation

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, or 55°C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55°C (thermophilic activity) and 25°C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25°C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log₁₀ ratio of TdR incorporation at 55 and 25°C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55°C 3 or 11 days before incubation at 25°C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.     

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily.

The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (TC #2.A.66) consists of four previously recognized families: (a) the ubiquitous multi-drug and toxin extrusion (MATE) family; (b) the prokaryotic polysaccharide transporter (PST) family; (c) the eukaryotic oligosaccharidyl-lipid flippase (OLF) family and (d) the bacterial mouse virulence factor family (MVF). Of these four families, only members of the MATE family have been shown to function mechanistically as secondary carriers, and no member of the MVF family has been shown to function as a transporter. Establishment of a common origin for the MATE, PST, OLF and MVF families suggests a common mechanism of action as secondary carriers catalyzing substrate/cation antiport. Most protein members of these four families exhibit 12 putative transmembrane alpha-helical segments (TMSs), and several have been shown to have arisen by an internal gene duplication event; topological variation is observed for some members of the superfamily. The PST family is more closely related to the MATE, OLF and MVF families than any of these latter three families are related to each other. This fact leads to the suggestion that primordial proteins most closely related to the PST family were the evolutionary precursors of all members of the MOP superfamily. Here, phylogenetic trees and average hydropathy, similarity and amphipathicity plots for members of the four families are derived and provide detailed evolutionary and structural information about these proteins. We show that each family exhibits unique characteristics. For example, the MATE and PST families are characterized by numerous paralogues within a single organism (58 paralogues of the MATE family are present in Arabidopsis thaliana), while the OLF family consists exclusively of orthologues, and the MVF family consists primarily of orthologues. Only in the PST family has extensive lateral transfer of the encoding genes occurred, and in this family as well as the MVF family, topological variation is a characteristic feature. The results serve to define a large superfamily of transporters that we predict function to export substrates using a monovalent cation antiport mechanism.     

Ultrastructure of the Cuticle of Loricifera and Demonstration of Chitin using Gold-Labelled Wheat Germ Agglutinin

The pharyngeal and lorical cuticles of adult and larval Loricifera were investigated by transmission electron microscopy. LR White sections of larval and adult Loricifera were labelled with the lectin wheat germ agglutinin (WGA) conjugated to colloidal gold. The pharyngeal cuticle of Nanaloricus mysticus exhibits a multilaminate epicuticle and an amorphous basal layer with osmiophilic fibres. The lorical cuticle consists of an osmiophilic or trilaminate epicuticle, one to three amorphous layer(s), and a basal fibrous layer which is strongly labelled by the lectin-gold conjugate. Chitinase treatment or competitive inhibition with N-, N '-, N "-triacetylchitotriose exclude labelling almost completely, whereas competitive inhibition with N -acetyl-D-glucosamine does not affect labelling intensity. The binding of WGA in connection with competition experiments indicates the presence of chitin in the fibrous layer. In most areas of a section, three amorphous layers extend below the epicuticle of the Nanaloricidae. Only in favourably orientated sections can all three "amorphous" layers be seen to be formed by stacks of lamellae. Modified articulation sites with bundles of osmiophilic longitudinal fibres and an osmiophilic plate (Nanaloricidae only) occur in adult Loricifera, but not in the larval stages. The ultrastructure of the lorical cuticle of the Loricifera resembles that of other Nemathelminthes (= Aschelminthes). The morphology of the articulation sites and the number of lorical plates seem to differ between the Loricifera and Priapulida. Therefore, it is currently not possible to conclude whether the lorica of the Loricifera and Priapulida are homologous structures. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Steric effects on penicillin-sensitive peptidoglycan synthesis in a membrane-wall system Gaffkya homari.

Residues 4 and 5 of the pentapeptide moiety, R-Ala1-DGlu2-Lys3-DAla4-DAla5, of peptidoglycan play an important role in the donor phase of cross-linked glycan synthesis. To assess the role of these residues in this phase, a series of UDP-MurNAc-peptides were biosynthesized with residues 4 and 5 replaced singly by either D-alpha-amino-n-butyric acid, D-norvaline, or D-valine. The six nucleotides were compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla (reference) in nascent (penicillin-insensitive) peptidoglycan synthesis and in penicillin-sensitive peptidoglycan synthesis. The synthesis of penicillin-sensitive peptidoglycan is catalyzed by membrane-walls isolated from Gaffkya homari and would appear to require the concerted action of transglycosylase and transpeptidase. The membrane-wall system shows a high degree of discrimination for the steric substituents, -CH3 and -CH2CH3, in residue 4. For example, for UDP-MurNAc-Ala-DGly-Lys-DAbu-DAla and -Ala-DGlu-Lys-DAla-DAbu, Vmax/km is 0.19 and 0.95 and Vmax is 0.03 and 0.52, respectively, of the value for the reference nucleotide. In contrast, for the synthesis of nascent peptidoglycan with these nucleotides Vmax/Km is 0.75 and 0.80, and Vmax is 0.71 and 1.0, respectively, of the value for the reference nucleotide. This trend was also illustrated with the other nucleotides in the time course experiments. These results indicate that the penicillin-sensitive enzyme(s), presumably the transpeptidase, has a higher degree of specificity in the donor phase for D-alanine in residue 4 than for D-alanine in residue 5 in the cross-linking stage of peptidoglycan synthesis.     

Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.