Microbial whole‐cell arrays

The coming of age of whole‐cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines – the whole‐cell array. In the present review, we highlight the state‐of‐the‐art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high‐performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis and – most importantly – enhanced long‐term maintenance of viability and activity on the fabricated biochips.     

The microbial community of Vetiver root and its involvement into essential oil biogenesis

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.     

The HrpB-HrpA two-partner secretion system is essential for intracellular survival of Neisseria meningitidis

In this study we used HeLa cells to investigate the role of the HrpB-HrpA two-partner secretion (TPS) system in the meningococcal infection cycle. Although there is evidence that several pathogenic microorganisms may use TPS systems to colonize epithelial surfaces, the meningococcal HrpB-HrpA TPS system was not primarily involved in adhesion to or invasion of HeLa cells. Instead, this system was essential for intracellular survival and escape from infected cells. Gentamicin protection assays, immunofluorescence and transmission electron microscopy analyses demonstrated that, in contrast to the wild-type strain, HrpB-HrpA-deficient mutants were primarily confined to late endocytic vacuoles and trapped in HeLa cells. Haemolytic tests using human erythrocytes suggested that the secreted HrpA proteins could act as manganese-dependent lysins directly involved in mediating vacuole escape. In addition, we demonstrated that escape of wild-type meningococci from infected cells required the use of an intact tubulin cytoskeleton and that the hrpB-hrpA genes, which are absent in other Neisseria spp., were upregulated during infection.     

A model-based approach for detecting coevolving positions in a molecule

We present a new method for detecting coevolving sites in molecules. The method relies on a set of aligned sequences (nucleic acid or protein) and uses Markov models of evolution to map the substitutions that occurred at each site onto the branches of the underlying phylogenetic tree. This mapping takes into account the uncertainty over ancestral states and among-site rate variation. We then build, for each site, a "substitution vector" containing the posterior estimates of the number of substitutions in each branch. The amount of coevolution for a pair of sites is then measured as the Pearson correlation coefficient between the two corresponding substitution vectors and compared to the expectation under the null hypothesis of independence. We applied the method to a 79-species bacterial ribosomal RNA data set, for which extensive structural characterization has been done over the last 30 years. More than 95% of the intramolecular predicted pairs of sites correspond to known interacting site pairs.     

The ConSurf-HSSP database: the mapping of evolutionary conservation among homologs onto PDB structures

The HSSP (Homology-Derived Secondary Structure of Proteins) database provides multiple sequence alignments (MSAs) for proteins of known three-dimensional (3D) structure in the Protein Data Bank (PDB). The database also contains an estimate of the degree of evolutionary conservation at each amino acid position. This estimate, which is based on the relative entropy, correlates with the functional importance of the position; evolutionarily conserved positions (i.e., positions with limited variability and low entropy) are occasionally important to maintain the 3D structure and biological function(s) of the protein. We recently developed the Rate4Site algorithm for scoring amino acid conservation based on their calculated evolutionary rate. This algorithm takes into account the phylogenetic relationships between the homologs and the stochastic nature of the evolutionary process. Here we present the ConSurf-HSSP database of Rate4Site estimates of the evolutionary rates of the amino acid positions, calculated using HSSP's MSAs. The database provides precalculated evolutionary rates for nearly all of the PDB. These rates are projected, using a color code, onto the protein structure, and can be viewed online using the ConSurf server interface. To exemplify the database, we analyzed in detail the conservation pattern obtained for pyruvate kinase and compared the results with those observed using the relative entropy scores of the HSSP database. It is reassuring to know that the main functional region of the enzyme is detectable using both conservation scores. Interestingly, the ConSurf-HSSP calculations mapped additional functionally important regions, which are moderately conserved and were overlooked by the original HSSP estimate. The ConSurf-HSSP database is available online (http://consurf-hssp.tau.ac.il).     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

Ratios of radical to conservative amino acid replacement are affected by mutational and compositional factors and may not be indicative of positive Darwinian selection

The ratio of radical to conservative amino acid replacements is frequently used to infer positive Darwinian selection. This method is based on the assumption that radical replacements are more likely than conservative replacements to improve the function of a protein. Therefore, if positive selection plays a major role in the evolution of a protein, one would expect the radical-conservative ratio to exceed the expectation under neutrality. Here, we investigate the possibility that factors unrelated to selection, i.e., transition-transversion ratio, codon usage, genetic code, and amino acid composition, influence the radical-conservative replacement ratio. All factors that have been studied were found to affect the radical-conservative replacement ratio. In particular, amino acid composition and transition-transversion ratio are shown to have the most profound effects. Because none of the studied factors had anything to do with selection (positive or otherwise) and also because all of them (singly or in combination) affected a measure that was supposed to be indicative of positive selection, we conclude that selectional inferences based on radical-conservative replacement ratios should be treated with suspicion.     

Association between the cell cycle and neural crest delamination through specific regulation of G1/S transition

Delamination of premigratory neural crest cells from the dorsal neural tube depends both upon environmental signals and cell-intrinsic mechanisms and is a prerequisite for cells to engage in migration. Here we show that avian neural crest cells synchronously emigrate from the neural tube in the S phase of the cell cycle. Furthermore, specific inhibition of the transition from G1 to S both in ovo and in explants blocks delamination, whereas arrest at the S or G2 phases has no immediate effect. Thus, the events taking place during G1 that control the transition from G1 to S are necessary for the epithelial to mesenchymal conversion of crest precursors.