Cinzia Calvio

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors — the spliceosome — is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Swarming Differentiation and Swimming Motility in Bacillus subtilis Are Controlled by swrA, a Newly Identified Dicistronic Operon

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA, that controls both swimming motility and swarming differentiation in B. subtilis. Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52:357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

Autoregulation of swrAA and Motility in Bacillus subtilis

We demonstrate that transcription of the gene swrAA, required for swarming migration in Bacillus subtilis, is driven by two promoters: a sigD-dependent promoter and a putative sigA-dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis.

Knockout of pgdS and ggt genes improves γ-PGA yield in B. subtilis.

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012.

pH-Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase

γ‐Glutamyltransferases (γ‐GTs) are heterodimeric enzymes that catalyze the transfer of a γ‐glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate γ‐glutamyl enzyme. In our search for a γ‐GT from a generally recognized as safe microorganism suitable for the production of γ‐glutamyl derivatives with flavor‐enhancing properties intended for human use, we cloned and overexpressed the γ‐GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis γ‐GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis γ‐GT on pH, also in relation to the pKa of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four γ‐glutamyl moieties are linked to a single glutamine. Moreover, we found that d‐glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis γ‐GT‐catalyzed transpeptidation reaction is feasible, and the observed activities of γ‐GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material γ‐polyglutamic acid.

The Role of SwrA, DegU and PD3 in fla/che Expression in B. subtilis

In B. subtilis swarming and robust swimming motility require the positive trigger of SwrA on fla/che operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σA-dependent fla/che promoter PA(fla/che) through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at PA(fla/che) while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses PA(fla/che) in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of PA(fla/che) when combined with SwrA. Finally, we demonstrate that the σD-dependent PD3(fla/che) promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when swrA is switched to the non-functional swrA - status.

DNA extraction from soil: comparison of different methods using spore-forming bacteria and theswrAA gene as indicators

Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of theswrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodium-dodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L) expressing theStSy gene for resveratrol production and thebar gene for Basta® tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genusBacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Metal Leaching and Reductive Dissolution of Iron from Contaminated Soil and Sediment Samples by Indigenous Bacteria and Bacillus Isolates

ABSTRACT The purpose of this study was to leach Cu, Zn, As, and Fe from contaminated soil and sediment samples with indigenous heterotrophic bacteria isolated from the study sites. The sediment contained Fe in the form of goethite and low concentrations of other metals. The soil contained hematite and high concentrations of other metals. The environmental conditions affected the bacterial activity in the metals dissolution. As and Fe were the major metals leached from the sediment sample while a minor fraction of Cu was solubilized. Cu and Zn were the major metals leached from the soil sample while only a minor fraction of Fe was dissolved. As a control, a disinfectant was used for partial inactivation of indigenous bacteria. This treatment had a negative effect on the leaching of Fe, Zn and As from soil and sediment samples, but it increased Cu dissolution from the sediment. Bacterial different dissolution of Fe during soil and sediment bioleaching was also investigated with ferrihydrite. The iron concentration was much higher during ferrihydrite dissolution when indigenous bacteria from sediment were used compared to indigenous bacteria isolated from soil. The indigenous bacterial inoculum provided more biological and metabolic diversity which may account for the difference in reductive iron reduction from ferrihydrite. The Bacillus cultures isolated from soil and sediment samples showed similar efficiencies in reductive dissolution of ferrihydrite. The synergetic bacterial inhibition effect created by the environmental conditions can influence bioremediation effect.

A BioBrick™-Compatible Vector for Allelic Replacement Using the XylE Gene as Selection Marker

BackgroundCircular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids.FindingsWe illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering.ConclusionsCompared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.