Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.     

Characterisation of members of the <Emphasis Type="Italic">Fusarium incarnatum</Emphasis>-<Emphasis Type="Italic">equiseti</Emphasis> species complex from undisturbed soils in South Africa

The genus Fusarium hosts a large number of economically significant phytopathogens with a global distribution. Surprisingly, only a limited number of studies have tried to identify the natural distribution of members of this genus in undisturbed soils. Members of the Fusarium incarnatum-equiseti species complex (FIESC) are increasingly associated with plant disease, and human and animal health problems. Recently, an outbreak of kikuyu poisoning of cattle was attributed to the F. incarnatum-equiseti species complex. Thus, it is of importance to identify the natural distribution of members of the FIESC from the environment. The aim of this study was to use the phylogenetic signal within the TEF 1α gene region to characterise 54 F. incarnatum-equiseti isolates obtained from undisturbed soils from the grassland biome of South Africa. These isolates were further compared with members of the FIESC previously associated with kikuyu poisoning of cattle. The phylogenetic analysis indicated a high level of variation within this species complex. Several members were closely related to isolates implicated in the death of cattle from infected kikuyu grass.     

Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents

3-Methoxybenzamide (3-MBA) derivatives have been identified as novel class of potent antibacterial agents targeting the bacterial cell division protein FtsZ. As one of isosteres for the amide group, 1,2,3-triazole can mimic the topological and electronic features of the amide, which has gained increasing attention in drug discovery. Based on these considerations, we prepared a series of 1H-1,2,3-triazole-containing 3-MBA analogues via isosteric replacement of the terminal amide with triazole, which had increased antibacterial activity. This study demonstrated the possibility of developing the 1H-1,2,3-triazole group as a terminal amide-mimetic element which was capable of both keeping and modulating amide-related bioactivity. Surprisingly, a different action mode of these new 1H-1,2,3-triazole-containing analogues was observed, which could open new opportunities for the development of antibacterial agents.     

Assembly of eukaryotic algal chromosomes in yeast

Background

Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (>?~?150 kb), high G?+?C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G?+?C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (> 150 kb) eukaryotic DNA with moderate G?+?C content. The model diatom Phaeodactylum tricornutum has an average G?+?C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast.

Results

We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency.

Conclusions

Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G?+?C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.

     

Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline

Gram-negative, facultatively anaerobic bacterial strains were consistently isolated from oak trees displaying symptoms of extensive stem bleeding. In Britain, this disorder is called Acute Oak Decline (AOD). A similar condition has been noted on species of Mediterranean oak in Spain. The identity of bacterial isolates from symptomatic trees in both countries was investigated using molecular techniques and phenotypic assays. 16S rRNA gene sequencing indicated that the strains were most closely related to the genera Serratia, Kluyvera, Klebsiella and Raoultella (all>97%). Phylogenetic analysis revealed that the strains formed a distinct lineage within the family Enterobacteriaceae, which was confirmed by both gyrB- and rpoB-gene sequencing. DNA-DNA hybridization confirmed that the strains belonged to a single taxon which could also be differentiated phenotypically from its closest phylogenetic neighbours. The phylogenetic and phenotypic data both demonstrated that the strains isolated from oak represented a novel genus and species within the family Enterobacteriaceae for which the name Gibbsiella quercinecans gen. nov., sp. nov. (type strain=FRB 97(T)=LMG 25500(T)=NCPPB 4470(T)) is proposed.     

Thermal sensitivity of a Neotropical amphibian (Engystomops pustulosus) and its vulnerability to climate change

A species’ thermal sensitivity and its exposure to climate variation are key components in the prediction of its vulnerability to climate change. We tested the thermal sensitivity of a tropical amphibian that lives in a mild constant climate in which the thermal tolerance range is expected to closely match the experienced environmental temperature. The air temperature that this species is exposed to varies between 21.9 and 31.6°C with an annual mean of 27.2°C. We estimated the microhabitat water temperature variation under vegetation shade, which buffers the temperature by 1.8°C in relation to that of the air, and with open canopy, where the water was 1.9°C warmer than the air temperature. With broods of tadpoles split into five treatments (15°C, 21°C, 28°C, 31°C, and 33°C), we estimated the critical thermal maximum (CTMax) and critical thermal minimum (CTMin) after at least 7 days of acclimation. Both CTMax (42.3°C) and CTMin (11.8°C) were more extreme than the temperature range estimated for the field. We estimated the optimum temperature (T_o = 28.8°C) and the thermal performance breadth (range: 23.3–34.1°C) based on growth rate (g/day). The animals were able to acclimate more extensively to cold than to warm temperatures. These performance curve traits closely matched the air temperature. The estimated vulnerability varied according to the microhabitat prediction model used. The combination of tadpole data on thermal sensitivity and macro‐ and microhabitat variation provides a necessary framework to understand the effects of climate change on tropical amphibians.     

Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O₂-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.Hydrogen is a promising energy carrier for the future (10). Photosynthetic microbes such as cyanobacteria have attracted considerable attention, because they can split water photolytically to produce H₂. However, one major drawback of the processes is that their H₂-evolving hydrogenases are extremely sensitive to O₂, which is an inherent by-product of oxygenic photosynthesis. Thus, transfer of O₂-tolerant [NiFe] hydrogenases into cyanobacteria might be one approach to overcome this O₂ sensitivity issue. A small number of O₂-tolerant hydrogenases has been identified (9, 21, 47). However, they tend to favor H₂ uptake over evolution. Searching for novel O₂-tolerant [NiFe] hydrogenases from environmental microbes therefore becomes an important part of the effort to construct such biophotolytic systems.The oceans harbor an abundance of microorganisms with H₂ production capability. Traditionally, new hydrogenases have been screened only from culturable organisms. However, since only a few microbes can be cultured (14), many of them have not been identified, and their functions remain unknown. Metagenomics is a rapidly growing field, which allows us to obtain information about uncultured microbes and to understand the true diversity of microbes in their natural environments. Metagenomics analysis provides a completely new approach for identifying novel [NiFe] hydrogenases from the oceans in a culture-independent manner. The Global Ocean Sampling (GOS) expedition has produced the largest metagenomic data set to date, providing a rich catalog of proteins and protein families, including those enzymes involved in hydrogen metabolism (45, 52, 56-58). Putative novel [NiFe] hydrogenase enzymes that were identified from marine microbial metagenomic data in these expeditions can be examined to find potentially important new hydrogenases. Because source organisms for metagenomic sequences are not typically known, these hydrogenases have to be heterologously expressed in culturable foreign hosts for protein and functional analyses.Unlike most proteins, hydrogenases have a complex architecture and must be assembled and matured through a multiple-step process (7, 11). Hydrogenases are divided into three distinct groups based on their metal contents (54): Fe-S cluster-free hydrogenases (22, 23, 48), [FeFe] hydrogenases (1, 12, 25), and [NiFe] hydrogenases (2, 3, 55). [NiFe] hydrogenases are heterodimers composed of a large subunit and a small subunit, and their NiFe catalytic centers are located in the large subunits (2, 15, 19, 40). A whole set of accessory proteins are required to properly assemble the catalytic centers (7). The accessory protein HypE first interacts with HypF to form a HypF-HypE complex, and the carbamyl group linked to HypF is then dehydrated by HypE in the presence of ATP to release the CN group that is transferred to iron through a HypC-HypD-HypE complex (6). The origin of the CO ligand that is also bound to the iron is not clear, and possibly it comes from formate, formyl-tetrahydrofolate, or acetate. The liganded Fe atom is inserted into the immature large subunit, in which HypC proteins function as chaperones to facilitate the metal insertion (5, 34, 36). Ni is delivered to the catalytic center by the zinc-metalloenzyme HypA that interacts with HypB, a nickel-binding and GTP-hydrolyzing protein. The final step in the maturation process is endoproteolytic cleavage. Once the nickel is transferred to the active site, the endopeptidase, such as HyaD or HynD, cleaves the C-terminal end of the large subunit (33, 43), which triggers a conformational change of the protein so that the Ni-Fe catalytic center can be internalized.Heterologous expression of functional [NiFe] hydrogenases has been demonstrated in several studies (4, 18, 31, 39, 44, 50), suggesting that it could be a feasible approach to express novel hydrogenases from the environment for functional analysis. In this study, we sought to prove the concept that metagenomically derived environmental DNA can give rise to a functional [NiFe] hydrogenase through expression in a foreign host and that novel [NiFe] hydrogenases from environmental microbes can be studied in a culture-independent manner. We cloned environmental DNA that harbors the genes of a putative novel hydrogenase that shows strong homology to a known O₂-tolerant hydrogenase, HynSL, from the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (21, 28, 41, 59). We heterologously expressed the two structural genes (hyaA and hyaB) and two accessory genes (hupH and hyaD) of this novel environmental hydrogenase in T. roseopersicina, a foreign host that may already have the necessary machinery required to process the environmental hydrogenase since it carries the homologous hydrogenase HynSL. We analyzed the new hydrogenase protein and its functions. In addition, we compared the maturation mechanisms between the two homolog hydrogenases by performing complementation experiments.