Streptococcal collagen-like protein 1, Scl1, modulates group a Streptococcus adhesion, biofilm formation and virulence

Background: The collagens comprise a large family of versatile proteins found in all three domains of life. The streptococcal collagen-like protein 1, scl1, of group A Streptococcus (GAS) binds extracellular matrix components (ECM), cellular fibronectin and laminin, via the surface-exposed globular domain. GAS strains express scl1 and form biofilm in vitro, except for M3-type strains that are particularly invasive to humans. Hypothesis: Lack of scl1 adhesin in M3 GAS results in decreased adherence and biofilm formation, and increased virulence. Results and Discussion : First crystal structure of the globular domain revealed a unique six-helical bundle fold, consisting of three pairs of alpha helices connected by variable loops. ECM binding by Scl1 promotes the formation of stable tissue microcolonies, which was demonstrated in vitro during infection of wounded human skin equivalents. A conserved nonsense mutation was identified in the scl1 allele of the M3-type strains (scl1.3) that truncates the coding sequence, presumably resulting in a secreted Scl1 variant. Absence of Scl1 on the surface of M3-type GAS was demonstrated experimentally, as well as diminished expression of the scl1 transcript in M3 strains relative to other M-types. Therefore, M3-type strains have reduced biofilm capacity on ECM coatings relative to other M-types. Constructed full-length recombinant Scl1.3 protein displayed binding capacity to cellular fibronectin and laminin, and M3 strains complemented with functional Scl1.3 adhesin displayed increased biofilm formation. The isoallelic M3 strain, carrying a rare carrier allele encoding cell-associated Scl1.3 variant, showed decreased pathology in mice, compared to the invasive M3 strain. Similarly, scl1 inactivation in biofilm-capable M28- and M41-type GAS led to increased lesion size during subcutaneous infection. Conclusions: The studies presented here demonstrate the importance of surface Scl1 in modulating biofilm formation and virulence of GAS, and provide insight into the structure and function of Scl proteins

An examination of novel archaeal stress response genes

The regulation of gene expression is the fundamental system employed by cells and organisms to respond to changes in their surroundings. To further understand the origins and nature of the processes underlying gene regulatory systems, the stress response in the thermophilic archaeon, Thermococcus zilligii, was examined. Microorganisms respond to stress by triggering physiological and morphological changes, which are largely brought about by adaptive changes in gene expression. The use of a thermophilic archaeon in this research is significant, since this group of organisms is widely regarded to resemble ancient life more closely than other extant life on Earth. Moreover, since the informational processing systems in archaea are thought to be closely related to those of the eukaryal nucleus, the study of the archaeal stress-response may further the understanding of the evolutionary relationship between the Archaea and the Eucarya. The stress response of T. zilligii was examined by analysing the total cytoplasmic protein complements from cultures ranging from mid-log to late stationary phase. Using N-terminal sequencing and BLASTP searches, homologues of four low molecular weight proteins that varied in level were identified in the genome sequences of members of the closely related genus, Pyrococcus. The genes originated from four distinct families, which were named srb, src, srd and sre. The srb family represents an essentially undescribed group of genes (COGI433), that occurs primarily amongst members of the Euryarchaeota. Due to the sporadic distribution of srb genes amongst extant life, the srb genes are suggested to have undergone horizontal transfer across large phylogenetic distances on several occasions. A sequence homologous to srb family members was identified as part of a hypothetical ORF in Clostridium perfringens, which encodes a protein that consists of a fusion of a transcriptional regulator belonging to the ArsR family and an SRB homologue. Several srb genes were found to occur as part of putative operons, the majority of which included genes homologous to the MinD family of P-loop ATPases. The src family encodes the highly conserved SaclOb family of DNA-binding proteins (COG1581). src genes occur exclusively amongst archaeal genomes, but were absent in Halobacterium sp. NRC-1, Methanopyrus kandleri, Methanosarcina mazei and M. acetivorans. The consensus secondary structure of the SRC proteins consists of a relatively long α-helix flanked by two small β-strands, followed by a second α-helix, and then three further β-strands. The srd family encodes proteins that are homologous to the C-terminal half of members of the Lrp/AsnC family of transcriptional regulators (COG1522). It is suggested here that the SRD family forms a logical subgroup that is structurally, and presumably functionally, distinct to the other members of the Lrp/AsnC family. srd genes occur in all nonmethanogenic archaea and in some bacteria. It is suggested that the srd family originated in the archaeal lineage and was later transferred to members of the Bacteria. The srd genes often appear as part of operon-like arrangements, which seem to be unrelated to one another, and consequently it is suggested that the SRD proteins are involved in a relatively generic function, such as transcriptional regulation. The sre family represents an undescribed group of highly conserved genes (COG1698) that occur exclusively amongst archaeal genomes. One sre gene was found in every archaeal genome sequenced to date, except for Pyrobaculum aerophilum and Halobacterium sp. NRC-1. Comparison of the SRE and 16S rRNA distance trees suggests that the sre genes were not prone to horizontal transfer. The secondary structure predicted from the SRE sequences consists of four large helical regions separated by short coiled regions. Recombinant proteins encoded by the srbPab1, srbPab2, srdPab2 and srePab genes cloned from Pyrococcus abyssi were produced in Escherichia coli and purified. An examination of the predicted quaternary associations of the recombinant proteins revealed that each of the proteins, with the exception rSRBPab2, formed multimeric structures. rSRBPabl formed dimer-sized multimers that occurred at 80°C, but not at room temperature. rSRDPab2 formed dimer-sized molecules that occurred preferentially at 80°C and in the presence of DNA. rSREPab formed structures that were consistent in size with those predicted for dimers and tetramers. Neither rSRBPabl, rSRDPab2 or rSREPab bound to DNA in the conditions examined

Identifying Novel Lignocellulosic Processing Enzymes from Cellulomonas fimi using Transcriptomic, Proteomic and Evolution Adaptive Studies

The declining reserves of fossil fuel twined with an increasing concern about the environmental consequences of burning these fuels and rising carbon dioxide levels, means that a more sustainable replacement is required. Lignocellulosic biomass is an attractive candidate that has been shown to be the best sustainable alternative source to produce bioethanol for liquid transportation fuels. It has enormous availability, is renewable and cost-effective. As an agricultural residue, it does not compete with food production. However, lignocellulosic biomass of plant cell walls is composed mainly of cellulose, hemicellulose and lignin, which are extremely resistant to digestion. Converting this biomass to useful products of fermentable sugars for bioethanol production has met with little success as harsh pretreatment and costly enzyme applications are required. An arsenal of enzymes and a synergistic mechanism are required to deconstruct recalcitrant lignocellulosic biomass for an efficient production of lignocellulosic bioethanol. To achieve this goal, this study used transcriptomic and proteomic approaches with the objective of identifying new genes and enzymes involved in lignocellulose degradation. This revealed that the only one AA10 of Cellulomonas fimi was among the highest enzymes identified during the degradation of cellulose. Another other 20 hypothetical proteins co-expressed with CAZymes have been identified including a potentially exclusively new C. fimi β-glucosidase (PKDP1) that contains a PKD-domain and oxidoreductase predicted function of PQQ-domain. A naturally mutagenized C. fimi population also was screened from an adaptive evolution experiment involving exposure to a wheat straw environment. One of the strains in the adaptive population (Strain-6) showed a higher association with wheat straw biomass, which may be an indication of the strategy that being used by the adapted strain to tackle obstinate substrates to sustain growth. These results show many new enzymes would be revealed from the C. fimi repertoire in order to have a better enzymatic cocktails for lignocellulose breakdown. For the future, this encourages a deeper understanding of lignocellulose deconstruction mechanisms by an orchestra of multiple enzymes in a bacterial system

National synchrotron light source. Activity report, October 1, 1995--September 30, 1996

The hard work done by the synchrotron radiation community, in collaboration with all those using large-scale central facilities during 1995, paid off in FY 1996 through the DOE`s Presidential Scientific Facilities Initiative. In comparison with the other DOE synchrotron radiation facilities, the National Synchrotron Light Source benefited least in operating budgets because it was unable to increase running time beyond 100%-nevertheless, the number of station hours was maintained. The major thrust at Brookhaven came from a 15% increase in budget which allowed the recruitment of seven staff in the beamlines support group and permitted a step increment in the funding of the extremely long list of upgrades; both to the sources and to the beamlines. During the December 1995 shutdown, the VUV Ring quadrant around U10-U12 was totally reconstructed. New front ends, enabling apertures up to 90 mrad on U10 and U12, were installed. During the year new PRTs were in formation for the infrared beamlines, encouraged by the investment the lab was able to commit from the initiative funds and by awards from the Scientific Facilities Initiative. A new PRT, specifically for small and wide angle x-ray scattering from polymers, will start work on X27C in FY 1997 and existing PRTs on X26C and X9B working on macromolecular crystallography will be joined by new members. Plans to replace aging radio frequency cavities by an improved design, originally a painfully slow six or eight year project, were brought forward so that the first pair of cavities (half of the project for the X-Ray Ring) will now be installed in FY 1997. Current upgrades to 350 mA initially and to 438 mA later in the X-Ray Ring were set aside due to lack of funds for the necessary thermally robust beryllium windows. The Scientific Facilities Initiative allowed purchase of all 34 windows in FY 1996 so that the power upgrade will be achieved in FY 1997

Modelling an Ammonium Transporter with SCLS

The Stochastic Calculus of Looping Sequences (SCLS) is a recently proposed modelling language for the representation and simulation of biological systems behaviour. It has been designed with the aim of combining the simplicity of notation of rewrite systems with the advantage of compositionality. It also allows a rather simple and accurate description of biological membranes and their interactions with the environment.<P>In this work we apply SCLS to model a newly discovered ammonium transporter. This transporter is believed to play a fundamental role for plant mineral acquisition, which takes place in the arbuscular mycorrhiza, the most wide-spread plant-fungus symbiosis on earth. Due to its potential application in agriculture this kind of symbiosis is one of the main focuses of the BioBITs project. <p>In our experiments the passage of NH3 / NH4+ from the fungus to the plant has been dissected in known and hypothetical mechanisms; with the model so far we have been able to simulate the behaviour of the system under different conditions. Our simulations confirmed some of the latest experimental results about the LjAMT2;2 transporter. The initial simulation results of the modelling of the symbiosis process are promising and indicate new directions for biological investigations