Academic literature on the topic 'Sporosarcina ureae'

Dormant spores of Sporosarcina halophila and Sporosarcina ureae contained no detectable ATP, significant levels of ADP, even higher levels of AMP, and a large pool of 3-phosphoglyceric acid, similar to what is found in dormant spores of Bacillus and Clostridium species. Sporosarcina halophila and S. ureae spores also contained significant pools of free amino acids, in particular glutamic acid, as in the case with spores of Bacillus but not Clostridium species. Levels of monovalent and divalent inorganic cations were comparable in spores of Sporosarcina, Clostridium, and Bacillus species, and cation levels in spores of the slight halophile S. halophila were similar to those in S. ureae spores. These data suggest that levels of small molecules are generally similar in spores of all Gram-positive organisms, and further suggest that these levels reflect fundamental and conserved features of the sporulation process and dormant spores in these organisms. The data are also consistent with the proposed close evolutionary relationship between Bacillus and Sporosarcina species.Key words: Bacillus, Clostridium, small molecules, spores, Sporosarcina.

Abstract. “Microbially induced carbonate precipitation” (MICP) is a biogeochemical process that can be applied to strengthen materials. The hydrolysis of urea by microbial catalysis to form carbonate is a commonly studied example of MICP. In this study, Sporosarcina ureae, a ureolytic organism, was compared to other ureolytic and non-ureolytic organisms of Bacillus and Sporosarcina genera in the assessment of its ability to produce carbonates by ureolytic MICP for ground reinforcement. It was found that S. ureae grew optimally in alkaline (pH ∼ 9.0) conditions which favoured MICP and could degrade urea (units U mL−1 represent µmol min−1 mL OD600) at levels (30.28 U mL−1) similar to S. pasteurii (32.76 U mL−1), the model ureolytic MICP organism. When cells of S. ureae were concentrated (OD600 ∼ 15–20) and mixed with cementation medium containing 0.5 M calcium chloride (CaCl2) and urea into a model sand, repeated treatments (3 × 24 h) were able to improve the confined direct shear strength of samples from 15.77 kPa to as much as 135.80 kPa. This was more than any other organism observed in the study. Imaging of the reinforced samples with scanning electron microscopy and energy-dispersive spectroscopy confirmed the successful precipitation of calcium carbonate (CaCO3) across sand particles by S. ureae. Treated samples were also tested experimentally according to model North American climatic conditions to understand the environmental durability of MICP. No statistically significant (p < 0.05, n= 3) difference in strength was observed for samples that underwent freeze–thaw cycling or flood-like simulations. However, shear strength of samples following acid rain simulations fell to 29.2 % of control MICP samples. Overall, the species S. ureae was found to be an excellent organism for MICP by ureolysis to achieve ground strengthening. However, the feasibility of MICP as a durable reinforcement technique is limited by specific climate conditions (i.e. acid rain).

A 3-D reconstruction of the regular surface layer of the bacterium Sporosarcina ureae was performed with negatively stained preparations. Particular attention was paid to two major sources of artifacts that may cause problems in the interpretion and comparison of 3-D reconstructions: effects created by missing information due to limited tilting ("missing cone"), and distortions of the protein due to adsorption to the carbon support.

Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.

Les sols qui ne rencontrent pas les normes d’ingénierie civile doivent êtres soumis à des améliorations géotechniques car les vibrations causées par les tremblements de terre ou par la surcharge sur des infrastructures en hauteur peuvent mener à la liquéfaction partielle ou totale des sols saturés en eau. Ceci peut donc entrainer des dommages importants aux structures construites sur ces sols. Certaines méthodes existent pour remédier à ce problème, mais elles demeurent couteuses et parfois toxiques car elles utilisent de l’acrylamide et des lignosulfates. La bio-précipitation in situ de calcite dans les sols représente une méthode alternative. Le tout se fait avec des bactéries qui démontrent une activité uréolytique. La présente étude s’est intéressée à l’activité uréolytique des souches Escherichia coli, Sporosarcina ureae, Bacillus pasteurii, Lysinibacillus sphaericus, Bacillus subtilis et Bacillus megaterium. Les résultats démontrent que l’urée est seulement dégradée par les souches S. ureae et S. pasteurii. L’incubation de S. ureae en présence de Ni2+ (0.1-1 ppm) et Fe2+ (1-10 ppm) a toutefois permis d’augmenter l’activité catalytique de la souche, ce qui démontre l’importance des éléments nutritifs lors de l’hydrolyse de l’urée. Afin de tester l’activité uréolytique des autres souches, nous avons introduit un système d’expression uréase dans la souche E. coli en substituant des amino-acides dans la structure primaire des protéines. Suite à cette modification, l’activité uréolytique de E. coli s’est améliorée et est devenue comparable à celle des souches S. ureae et S. pasteurii. L’injection de S. ureae et du mutant E. coli dans des sables non-consolidés a permis de cimenter de façon significative (p < 0.05) le matériel par rapport à des sables non inoculés, et ce après seulement 48 heures. Le transfert du système recombinant de E coli vers S. ureae est présentement en cours. Ces résultats prometteurs indiquent qu’il est possible de stimuler la précipitation in situ de calcite en utilisant des bactéries et de stabiliser les sols prônes à la liquéfaction. === Soils often do not satisfy functional requirements for civil engineering projects and as a result geotechnical improvements to soils are often made. Dynamic shaking during earthquakes or static overloading by overlying structures may still result in liquefaction in partially or fully water saturated soils. These have little bearing capacity for structures. Severe damages can result. Moreover, preventative soil grouting strategies are expensive, toxic, and permanent due to acrylamides, lignosulfonates, and otherwise harmful compounds present therein. Alternative methods of strength enhancement are advisable. Microbial induced calcite precipitation (MICP) was assessed in this investigation to consolidate loose, sandy soils. Ureolytic activty of Escherichia coli, Sporosarcina ureae, Bacillus pasteurii, Lysinibacillus sphaericus, Bacillus subtilis and Bacillus megaterium were assessed. Urea was readily degraded foremost by S. ureae and next by S. pasteurii with no significant (p <0.05) activity in other strains. Incubation of S. ureae with 0.1 - 1ppm Ni2+ and 1-10ppm Fe2+ was shown to improve catalytic activity, suggesting their importance as a dietary source for urea hydrolysis. A urease expression system was established in E. coli and particular amino acid substitutions in protein primary structure made. Enhanced ureolytic activity was observed in these E. coli mutants, comparable to native S. ureae activity. Application of wild type S. ureae and recombinant E. coli for MICP in a model sand showed significant (p < 0.05) improvements compared to controls after 48 hours. Transfer of the recombinant system in E. coli to S. ureae is currently underway. These results provide valuable insight affirming that a practical system for the application of MICP may be feasible in the field for the strength enhancement of native and construction-laid loose, sandy soils.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

Urease is a nickel-dependent enzyme that catalyzes hydrolysis of urea in the last step of organic nitrogen mineralization. Its active site contains a dinuclear center for Ni(II) ions that must be inserted into the apo-enzyme through the action of four accessory proteins (UreD, UreE, UreF, UreG) leading to activation of urease. UreE, acting as a metallo-chaperone, delivers Ni(II) to the preformed complex of apo-urease-UreDFG and has the capability to enhance the GTPase activity of UreG. This study, focused on characterization of UreE from Sporosarcina pasteurii (SpUreE), represents a piece of information on the structure/mobility-function relationships that control nickel binding by SpUreE and its interaction with SpUreG. A calorimetric analysis revealed the occurrence of a binding event between these proteins with positive cooperativity and a stoichiometry consistent with the formation of the (UreE)2-(UreG)2 hetero-oligomer complex. Chemical Shift Perturbations induced by the protein-protein interaction were analyzed using high-resolution NMR spectroscopy, which allowed to characterize the molecular details of the protein surface of SpUreE involved in the complex formation with SpUreG. Moreover, backbone dynamic properties of SpUreE, determined using 15N relaxation analysis, revealed a general mobility in the nanoseconds time-scale, with the fastest motions observed at the C-termini. The latter analysis made it possible for the first time to characterize of the C-terminal portions, known to contain key residues for metal ion binding, that were not observed in the crystal structure of UreE because of disorder. The residues belonging to this portion of SpUreE feature large CSPs upon addition of SpUreG, showing that their chemical environment is directly affected by protein-protein interaction. Metal ion selectivity and affinity of SpUreE for cognate Ni(II) and non cognate Zn(II) metal ions were determined, and the ability of the protein to select Ni(II) over Zn(II), in consistency with the proposed role in Ni(II) cations transport, was established.

The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.

Increasing the integration density of electron device components will necessitate the use of new nanofabrication paradigms that complement and extend existing technologies. One potential approach to overcome the current limitations of electron-beam lithography may involve the use of hybrid systems, in which existing lithographic techniques are coupled with “bottom up” approaches such as supramolecular self-assembly. In this respect, biological systems offer some unique possibilities as they combine both self-organization and spatial patterning at the nanometer length scale. In particular, Surface Layer Proteins (S-layers) can facilitate high order organization and specific orientation of inorganic structures as they are two-dimensional porous crystalline membranes with regular structure at the nanometer scale. In this framework, the aim of the present work was the characterization of the S-layer of Sporosarcina ureae ATCC 13881 (SslA) with respect to its self-assembling properties and modification that would allow it to be employed as a patterning element and a new building block for nanobiotechnology. In vitro recrystallization experiments have shown that wild type SslA self-assembles into monolayers, multilayers or tubes. Factors such as initial monomer concentration, Ca2+ ions, pH of the recrystallization buffer and the presence of a silicon substrate have a strong influence on the recrystallization process. SslA monolayers proved to be an excellent biotemplate for ordered assembly of gold nanoparticle arrays. The recombinant SslA after expression and purification formed micrometer sized, crystalline monolayers exhibiting the same lattice structure as the wild type protein (p4 symmetry). This remarkable property of self-assembling has been preserved even when SslA was truncated. The deletion of both, N- and C-terminal SslA domains does not hinder self-assembly; the resulting protein is able to form extended monolayers that exhibit the p4 lattice symmetry. The central SslA-domain is self sufficient for the self-assembly. The possibility to change the natural properties of S-layers by genetic engineering techniques opens a new horizon for the tuning of their structural and functional features. The SslA-streptavidin fusion protein combines the remarkable property of self-assembling with the ligand i.e. biotin binding function. On silicon wafers, this chimeric protein recrystallized into coherent protein layers and exposes streptavidin, fact demonstrated by binding studies using biotinylated quantum dots. In this way, it can serve as a functional surface for controlled immobilization of biologically active molecules but also as a platform for the synthesis of planar arrays of quantum dots. Furthermore, the results open up exciting possibilities for construction of hybrid S-layers, structures that may ultimately promote the fabrication of miniaturized, nanosized electronic devices.