Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

Investigating the Structure of FtsZ to Understand its Functional Role in Bacterial Cell Division

FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?

One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.

The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.

Electrostatic Contributions to the Thermodynamics of Ribonuclease P Protein Folding

Electrostatic interactions are of fundamental importance in determining the structure and stability of macromolecules. For example, charge-charge interactions modulate the folding and binding of proteins and influence protein solubility. Electrostatic interactions are highly variable and can be both favorable and unfavorable. The ability to quantify these interactions is challenging but vital to understanding the detailed balance and major roles that they have in different proteins and biological processes. Measuring pKa values of ionizable groups provides a sensitive method for experimentally probing the electrostatic properties of a protein.

pKa values report the free energy of site-specific proton binding and provide a direct means of studying protein folding and pH-dependent stability. Using a combination of NMR, circular dichroism, and fluorescence spectroscopy along with singular value decomposition, we investigated the contributions of electrostatic interactions to the thermodynamic stability and folding of the protein subunit of Bacillus subtilis ribonuclease P, P protein. Taken together, the results suggest that unfavorable electrostatics alone do not account for the fact that P protein is intrinsically unfolded in the absence of ligand because the pKa differences observed between the folded and unfolded state are small. Presumably, multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the beta- and gamma-proteobacteria. Many fliC genes were deduced to be under the control of sigma(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (<= 1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.

Biogenic opal and diatoms in surface sediments of the South Western Atlantic

Based on 66 surface sediment samples collected in the SW Atlantic Ocean between 27 and 50°S, this study presents an overview of the spatial distribution of biogenic opal and diatom concentrations, and diatom assemblages. Biogenic opal has highest values in the deepest, pelagic stations and decreases toward the slope. Diatoms closely follow the spatial trend of opal. Diatom assemblages reflect the present-day dominant hydrographical features. Antarctic diatoms are the main contributors to the preserved diatom community in core top sediments, with coastal planktonic and tropical/subtropical diatoms as secondary components. Dominance of Antarctic diatoms between 35 and 50°S in the pelagic realm mirrors the northward displacement of Antarctic-source water masses, characterized by high nutrient content and low salinity. Northward of ca. 35°S, the highest contribution of tropical/subtropical, pelagic diatoms, typical for nutrient-poor and high salinity waters, matches the main southward path of the Brazil Current. Mixing of Antarctic and tropical waters down up to 45°S is clearly illustrated by the diatom assemblage. Concentrations of biogenic opal and diatoms rather reflect the path of predominant water masses, but are less correlated with surface water productivity in the SW Atlantic.

Distribution of diatom species as abundance estimates from vertical multinet hauls from 150 m water depth in the Indian Ocean

During the Indian Ocean Expedition of R/V METEOR phytoplankton samples were taken with a multiple closing net (Multinet) at 103 stations. In this material the diatoms were investigated. In all 247 taxa could be identified which belong to 242 species and 5 varieties of formae of 80 genera. Of these 1 variety, 15 pecies, and 3 genera are newly described. New combinations were made for 18 species, and a number of old combinations was reinstated.

Distribution of diatoms, coccolithophores and planktic foraminifera in the Arabian Sea

The distribution of diatoms, coccolithophores and planktic foraminifers mirrored the hydrographic and trophic conditions of the surface ocean (0-100 m) across the upwelling area off the Oman coast to the central Arabian Sea during May/June 1997 and July/August 1995. The number of diatoms was increased in waters with local temperature minimum and enhanced nutrient concentration (nitrate, phosphate, silicate) caused by upwelling. Vegetative cells of Chaetoceros dominated the diatom assemblage in the coastal upwelling area. Towards the more nutrient depleted and stratified surface waters to the southeast, the number of diatoms decreased, coccolithophore and planktic foraminiferal numbers increased, and floral and faunal composition changed. In particular, the transition between the eutrophic upwelling region off Oman and the oligotrophic central Arabian Sea was marked by moderate nutrient concentration, and high coccolithophore and foraminifer numbers. Florisphaera profunda, previously often referred as a 'lower-photic-zone-species', was frequent in water depths as shallow as 20 m, and at high nutrient concentration up to 14 µmol NO3/l and 1.2 µmol PO4/. To the oligotrophic southeast of the divergence, cell densities of coccolithophores declined and Umbellosphaera irregularis prevailed throughout the water column down to 100 m depth. In general, total coccolithophore numbers were limited by nutrient threshold concentration, with low numbers (<10*10**3 cells/l) at high [NO3] and [PO4], and high numbers (>70*10**3 cells/l) at low [NO3] and [PO4]. The components of the complex microplankton succession, diatoms, coccoliths and planktic foraminifers (and possibly others), should ideally be used as a combined paleoceanographic proxy. Consequently, models on plankton ecology should be resolved at least for the seasonality, to account for the bias of paleoceanographic transfer calculations.

(Appendix A) Radiolarian abundance in ODP Hole 202-1241A

A quantitative radiolarian study at Ocean Drilling Program Site 1241 in the eastern tropical Pacific enables us to reconstruct paleoceanographic changes that occurred since the latest middle Miocene. Today, this site is located just under the Eastern Pacific Warm Pool (EPWP). Based on the abundance variations of radiolarian characteristic species which are indicators of upwelling and thermocline changes, it is suggested that three notable changes occurred at 10.6, 9.8, and 4.2 Ma in the region. Four distinct periods of oceanographic conditions bounded by these notable changes were characterized on the basis of the following: (1) stratified seawater (12.0 to 10.6 Ma); (2) a shallowing of the thermocline and an increasing of upwelling (10.6 to 9.8 Ma); (3) significant inflow of warm water to the eastern tropical Pacific caused by an intensified Northern Equatorial Countercurrent (NECC), resulting in the formation of EPWP (9.8 to 4.2 Ma); and (4) the reduction of the EPWP and the NECC, and an increase in upwelling (4.2 to 0 Ma). The timing of these paleoceanographic events indicated the strong relations with the opening and closing of the Indonesian and Central American (Panama) Seaways. The reduction of the EPWP (this study) and the deepening of the thermocline in western Pacific at about 4.2 Ma (Cannariato and Ravelo, 1997; Chaisson and Ravelo, 2000) indicated a change from a state resembling El Niño in the late Miocene and the early Pliocene time to a state resembling La Niña by the late Pliocene

The Appendicularia and Chaetognatha from the Great Meteor Bank, Northeast Atlantic

At a longtime station near the "Grosse Meteor Bank" in the North Atlantic 41 subsequent hauls were made in April 1967 with the Helgoland larva net with changing bucket device. In addition 9 hauls were made during July 1967. The catches from the depth ranges of 900-700 m, 700-500 m, 500-300 m, 300-200 m, 200-100 m, and 100-0 m were collected in separate buckets during each catch series. Contamination, though possible on principle, does not seem to be of much consequence in appendicularia. After some comments on certain species caught it is shown that at this station in the open ocean the density of appendicularia not only varies with the season, but that clouds of plankton may pass by it within a few hours, in which the density may vary at a ratio of ten or more to one. In the composition of species as many as four species may in turn be the most abundant. For one species the composition as to size and stage of maturity may change in the same way. Regarding the depth distribution there are no species restricted to deeper layers. Below 100 m the number falls to about 1 % of the uppermost layer. Oikopkura longicauda, O. cophocerca, O.parva and Althoffia tumida as well as Fritillaria species are found between 900 and 100 m in comparatively higher numbers than Stegosoma magnum, Oikopleura albicans and O. intermedia. The Chaetognaths were collected in the depth of 900-0 m in vertical hauls with the Helgoland larva net with changing bucket device; buckets had been changed in the depth of 700, 500, 300, 200,1 00 m. In the course of the investigation it appeared that for Chaetognaths the sampling method with changing bucket device is insufficient. Many specimens remained in the net and entered the bucket at a higher level than that in which they had lived, mostly during flushing the net (sample 100-0 m); this means considerable contamination. In spite of this difficulty deep layers of higher abundance could be traced for Sagitta lyra and some other species. For some species large local variations in the number of specimens within a short time were found. Moreover notes have been made of foodorganisms, parasits and anatornic metamorphoses during maturing.

Late Holocene diatom abundaces in sediment core HC-11

The study of diatoms in core HC11 collected from the southwestern part of Chukchi Sea, allowed to distinguish 3 diatoms ecological zones, reflecting paleoenvironmental changes during the last 2300 years. The sediment age was based on the sedimentation rates, determined by 210Pb and radiocarbon dating of mollusk shells. The environmental changes of Chukchi Sea revealed by examination of diatoms correlates with global climate changes - the warming of the early and middle Subatlantic and cooling of the late Subatlantic (Little Ice Age). Warming early and middle Subatlantic in the Chukchi Sea was probably stronger than the warming of the late 20th century and was not accompanied by significant changes in sea level.

Potenzialità applicative delle alte pressioni di omogeneizzazione nel settore alimentare

Le alte pressioni di omogeneizzazione sono considerate una tecnologia non termica basata sull’applicazione di pressioni comprese tra 60 e 400 MPa ad alimenti fluidi o fluidificabili, con un tempo di trattamento di pochi millisecondi. Questa tecnologia permette di ottenere una serie di effetti sull’alimento che variano in rapporto all’entità del trattamento applicato e alla matrice fluida considerata. Pertanto, le alte pressioni di omogeneizzazione rappresentano una delle tecnologie maggiormente studiate in ragione delle loro buone opportunità applicative a livello industriale. Tale tecnologia viene comunemente applicata per modificare le proprietà funzionali di alcune macromolecole caratteristiche degli alimenti ed ha permesso l’ottenimento di prodotti di origine lattiero-casearia ed anche succhi di frutta caratterizzati da migliore texture, gusto, flavour e aumentata shelf-life. L’omogeneizzazione ad alta pressione, considerata come trattamento di sanitizzazione a freddo, è in grado di disattivare sia microrganismi patogeni che degradativi presenti in un determinato sistema, contribuendo a ridurre o contenere quindi lo sviluppo microbico nei prodotti alimentari. L’effetto di tale tecnologia, quando applicata a livelli compresi tra 60-200 MPa bar è stato valutato nei confronti di diversi patogeni quali Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, Salmonella typhimurium microrganismi degradativi, come Bacillus subtilis e lieviti, deliberatamente inoculati in prodotti diversi.

Effect of soil structure on temporal and spatial dynamics of bacteria

Soil is a complex heterogeneous system comprising of highly variable and dynamic micro-habitats that have significant impacts on the growth and activity of resident microbiota. A question addressed in this research is how soil structure affects the temporal dynamics and spatial distribution of bacteria. Using repacked microcosms, the effect of bulk-density, aggregate sizes and water content on growth and distribution of introduced Pseudomonas fluorescens and Bacillus subtilis bacteria was determined. Soil bulk-density and aggregate sizes were altered to manipulate the characteristics of the pore volume where bacteria reside and through which distribution of solutes and nutrients is controlled. X-ray CT was used to characterise the pore geometry of repacked soil microcosms. Soil porosity, connectivity and soil-pore interface area declined with increasing bulk-density. In samples that differ in pore geometry, its effect on growth and extent of spread of introduced bacteria was investigated. The growth rate of bacteria reduced with increasing bulk-density, consistent with a significant difference in pore geometry. To measure the ability of bacteria to spread thorough soil, placement experiments were developed. Bacteria were capable of spreading several cm’s through soil. The extent of spread of bacteria was faster and further in soil with larger and better connected pore volumes. To study the spatial distribution in detail, a methodology was developed where a combination of X-ray microtopography, to characterize the soil structure, and fluorescence microscopy, to visualize and quantify bacteria in soil sections was used. The influence of pore characteristics on distribution of bacteria was analysed at macro- and microscales. Soil porosity, connectivity and soil-pore interface influenced bacterial distribution only at the macroscale. The method developed was applied to investigate the effect of soil pore characteristics on the extent of spread of bacteria introduced locally towards a C source in soil. Soil-pore interface influenced spread of bacteria and colonization, therefore higher bacterial densities were found in soil with higher pore volumes. Therefore the results in this showed that pore geometry affects the growth and spread of bacteria in soil. The method developed showed showed how thin sectioning technique can be combined with 3D X-ray CT to visualize bacterial colonization of a 3D pore volume. This novel combination of methods is a significant step towards a full mechanistic understanding of microbial dynamics in structured soils.

Intercepting Cyclic Dinucleotide Signaling with Small Molecules

Bacterial infections, especially the ones that are caused by multidrug-resistant strains, are becoming increasingly difficult to treat and put enormous stress on healthcare systems. Recently President Obama announced a new initiative to combat the growing problem of antibiotic resistance. New types of antibiotic drugs are always in need to catch up with the rapid speed of bacterial drug-resistance acquisition. Bacterial second messengers, cyclic dinucleotides, play important roles in signal transduction and therefore are currently generating great buzz in the microbiology community because it is believed that small molecules that inhibit cyclic dinucleotide signaling could become next-generation antibacterial agents. The first identified cyclic dinucleotide, c-di-GMP, has now been shown to regulate a large number of processes, such as virulence, biofilm formation, cell cycle, quorum sensing, etc. Recently, another cyclic dinucleotide, c-di-AMP, has emerged as a regulator of key processes in Gram-positive and mycobacteria. C-di-AMP is now known to regulate DNA damage sensing, fatty acid synthesis, potassium ion transport, cell wall homeostasis and host type I interferon response induction. Due to the central roles that cyclic dinucleotides play in bacteria, we are interested in small molecules that intercept cyclic dinucleotide signaling with the hope that these molecules would help us learn more details about cyclic dinucleotide signaling or could be used to inhibit bacterial viability or virulence. This dissertation documents the development of several small molecule inhibitors of a cyclic dinucleotide synthase (DisA from B. subtilis) and phosphodiesterases (RocR from P. aeruginosa and CdnP from M. tuberculosis). We also demonstrate that an inhibitor of RocR PDE can inhibit bacterial swarming motility, which is a virulence factor.