Predicting and Analyzing Interactions between Mycobacterium tuberculosis and Its Human Host

The outcome of infection by Mycobacterium tuberculosis (Mtb) depends greatly on how the host responds to the bacteria and how the bacteria manipulates the host, which is facilitated by protein–protein interactions. Thus, to understand this process, there is a need for elucidating protein interactions between human and Mtb, which may enable us to characterize specific molecular mechanisms allowing the bacteria to persist and survive under different environmental conditions. In this work, we used the interologs method based on experimentally verified intra-species and inter-species interactions to predict human-Mtb functional interactions. These interactions were further filtered using known human-Mtb interactions and genes that are differentially expressed during infection, producing 190 interactions. Further analysis of the subcellular location of proteins involved in these human-Mtb interactions confirms feasibility of these interactions. We also conducted functional analysis of human and Mtb proteins involved in these interactions, checking whether these proteins play a role in infection and/or disease, and enriching Mtb proteins in a previously predicted list of drug targets. We found that the biological processes of the human interacting proteins suggested their involvement in apoptosis and production of nitric oxide, whereas those of the Mtb interacting proteins were relevant to the intracellular environment of Mtb in the host. Mapping these proteins onto KEGG pathways highlighted proteins belonging to the tuberculosis pathway and also suggested that Mtb proteins might use the host to acquire nutrients, which is in agreement with the intracellular lifestyle of Mtb. This indicates that these interactions can shed light on the interplay between Mtb and its human host and thus, contribute to the process of designing novel drugs with new biological mechanisms of action.     

Purification and Properties of a Thermophilic Bacteriophage Lytic Enzyme

A phage lytic enzyme was isolated from lysates of Bacillus stearothermophilus (NCA 1503-4R). The enzyme was purified 1,998-fold with a 27% recovery of enzyme activity. By use of polyacrylamide gel electrophoresis and sucrose gradient centrifugation the enzyme was judged free from protein contaminants. The lytic enzyme was active over a pH range of 6.0 to 7.0, with a maximum at 6.3, and it was stable between pH 7.0 and 8.0 and at 5.0 and unstable between pH 5.5 and 6.5. The temperature coefficient (Q(10)) was 2.27 between 35 and 45 C, 2.01 between 45 and 55 C, and 2.00 between 50 and 60 C. Lytic enzyme in 0.1 m sodium phosphate was not inactivated after a 1-hr exposure to temperatures below 65.5 C, whereas a 1% inactivation was observed at 70.6 C. A 2-hr exposure at 60.1, 65.5, and 70.6 C resulted in an inactivation of 1.2, 9.6, and 12.0%, respectively. A sodium phosphate concentration of at least 0.1 m was necessary for the prolonged exposure of lytic enzyme at 55 C (pH 6.3), whereas 0.005 m was required for maximal lytic activity. Lytic activity was stimulated 169, 165, and 160% by 10(-4)m Mg(++), Ca(++), and Mn(++), respectively. Lytic activity was inhibited 75% by 10(-4)m ethylenediaminetetraacetic acid (EDTA). The EDTA inhibition could be reversed by the addition of excess Mg(++), Ca(++), or Mn(++). Lytic activity was not affected by NaCl, KCl, or NH(4)Cl. Lytic activity was inhibited 100, 91, 25, 61, and 56% by 10(-4)m Hg(++), Cu(++), Zn(++), p-chloromercuribenzoate, and p-hydroxymercuribenzoate, respectively. Cysteine or 2-mercaptoethanol did not stimulate lytic activity, nor were these sulfhydryl compounds required for maintenance of enzyme activity during handling or storage. Cell walls were rapidly solubilized when incubated with lytic enzyme. Lytic action was complete after 1.5 min, with a 70% reduction in optical density (OD). Cell walls without lytic enzyme showed no reduction in OD during this period. The solubilization of N-terminal amino groups paralleled the reduction in OD and reached a level of 0.3 mumole/mg of cell wall after 4 min of incubation. Cell walls with and without lytic enzyme treatment showed a 3- and a 1.3-fold increase, respectively, in N-terminal amino groups after 3 hr of incubation. There was no release of reducing power in either the untreated cell wall suspensions or those treated with lytic enzyme. Electron micrographs of treated and untreated cell walls showed that the enzyme partially degrades the cell wall with the release of small wall fragments.     

High Temperature,High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially,but not Their Silencing Suppression Efficiencies

We compared infection of Nicotiana benthamiana plants by the positive-sense RNA viruses Cucumber mosaic virus (CMV), Potato virus Y (PVY), and by a Potato virus X (PVX) vector, the latter either unaltered or expressing the CMV 2b protein or the PVY HCPro suppressors of silencing, at 25°C vs. 30°C, or at standard (~401 parts per million, ppm) vs. elevated (970 ppm) CO₂ levels. We also assessed the activities of their suppressors of silencing under those conditions. We found that at 30°C, accumulation of the CMV isolate and infection symptoms remained comparable to those at 25°C, whereas accumulation of the PVY isolate and those of the three PVX constructs decreased markedly, even when expressing the heterologous suppressors 2b or HCPro, and plants had either very attenuated or no symptoms. Under elevated CO₂ plants grew larger, but contained less total protein/unit of leaf area. In contrast to temperature, infection symptoms remained unaltered for the five viruses at elevated CO₂ levels, but viral titers in leaf disks as a proportion of the total protein content increased in all cases, markedly for CMV, and less so for PVY and the PVX constructs. Despite these differences, we found that neither high temperature nor elevated CO₂ prevented efficient suppression of silencing by their viral suppressors in agropatch assays. Our results suggest that the strength of antiviral silencing at high temperature or CO₂ levels, or those of the viral suppressors that counteract it, may not be the main determinants of the observed infection outcomes.     

Network Analysis Highlights Complex Interactions between Pathogen,Host and Commensal Microbiota

Interactions between bacteria and their host represent a full continuum from pathogenicity to mutualism. From an evolutionary perspective, host-bacteria relationships are no longer considered a two-component system but rather a complex network. In this study, we focused on the relationship between brook charr (Salvelinus fontinalis) and bacterial communities developing on skin mucus. We hypothesized that stressful conditions such as those occurring in aquaculture production induce shifts in the bacterial community of healthy fish, thus allowing pathogens to cause infections. The results showed that fish skin mucus microbiota taxonomical structure is highly specific, its diversity being partly influenced by the surrounding water bacterial community. Two types of taxonomic co-variation patterns emerged across 121 contrasted communities’ samples: one encompassing four genera well known for their probiotic properties, the other harboring five genera mostly associated with pathogen species. The homeostasis of fish bacterial community was extensively disturbed by induction of physiological stress in that both: 1) the abundance of probiotic-like bacteria decreased after stress exposure; and 2) pathogenic bacteria increased following stress exposure. This study provides further insights regarding the role of mutualistic bacteria as a primary host protection barrier.     

Inhibition of Ribonucleic Acid Bacteriophage Release from Its Host by Rifampin

Rifampin, in addition to interfering with intracellular growth of the ribonucleic acid-containing phage MS2, also inhibits the release of mature phage particles from Escherichia coli cells.     

Production and Purification of the Thermophilic Bacteriophage TP-84

A new procedure for production and purification of the thermophilic bacteriophage TP-84 in high yields is described. Cultures of Bacillus stearothermophilus strain 10, enriched with nutrients to obtain heavy growth and to prevent sporulation and maintained at a pH of 6.5, were infected with the phage in a 100-liter fermentor. Addition of magnesium chloride (0.01 M) and a temperature of 58-C were essential for maximal phage production. Phage (5 times 1011 infective particles/ml) was precipitated with polyethylene glycol (molecular weight 6,000) in the presence of sodium chloride and was further purified by cesium chloride density centrifugation.     

Vertical Migrations of a Deep-Sea Fish and Its Prey

It has been speculated that some deep-sea fishes can display large vertical migrations and likely doing so to explore the full suite of benthopelagic food resources, especially the pelagic organisms of the deep scattering layer (DSL). This would help explain the success of fishes residing at seamounts and the increased biodiversity found in these features of the open ocean. We combined active plus passive acoustic telemetry of blackspot seabream with in situ environmental and biological (backscattering) data collection at a seamount to verify if its behaviour is dominated by vertical movements as a response to temporal changes in environmental conditions and pelagic prey availability. We found that seabream extensively migrate up and down the water column, that these patterns are cyclic both in short-term (tidal, diel) as well as long-term (seasonal) scales, and that they partially match the availability of potential DSL prey components. Furthermore, the emerging pattern points to a more complex spatial behaviour than previously anticipated, suggesting a seasonal switch in the diel behaviour mode (benthic vs. pelagic) of seabream, which may reflect an adaptation to differences in prey availability. This study is the first to document the fine scale three-dimensional behaviour of a deep-sea fish residing at seamounts.     

Thermophilic Temperature Optimum for Crenarchaeol Synthesis and Its Implication for Archaeal Evolution

The isoprenoid lipid crenarchaeol is widespread in hot springs of California and Nevada. Terrestrial and marine data together suggest a maximum relative abundance of crenarchaeol at ~40°C. This warm temperature optimum may have facilitated colonization of the ocean by (hyper)thermophilic Archaea and the major marine radiation of Crenarchaeota.     

The Influence of Inorganic Ions on the Heat Stability of a Thermophilic Bacteriophage

A thermophilic bacteriophage was isolated from soil. Heat inactivation of this phage, suspended in tryptone starch broth at 65°C and 70°C, was found to be a monomolecular reaction. The phage was more heat stable in tryptone broth than in tris buffer. When the tris buffer was supplemented with calcium or magnesium ions, the survival percentage increased from 0.0 to 18.0 after two hours of heating at 65°C. The addition of sodium or potassium ions to the tris buffer had no significant effect. Equimolar solutions of calcium and magnesium chloride had the same effect on the heat stability of the phage. Maximum stability was attained in 2.5 mM solutions of these salts, and a further increase in the concentration up to 10.0 mM did not increase the percentage of surviving phages.     

Isolation and Characterization of Bacteriophage SBX-1 and Its Bacterial Host, Both Endemic in Soybeans

A phage, SBX-1, and its bacterial host, Xanthomonas sp. 1, were isolated consistently from roots, internal portions of stems, and leaves of soybean plants. Phage titer in leaves was highly variable. It was very low in seedlings, reached a maximum of 10(4) PFU/ml of sap after 11 weeks of plant growth and again dropped to very low levels. We isolated SBX-1 from plants of all 45 varieties studied, but not consistently from some. Plants of some varieties also carried Xanthomonas sp. 2, which was resistant to infection by SBX-1. The SBX-1 particle has a polyhedral head containing DNA of density 1.709. It has an edge-to-edge diameter of 80 nm and a tail length of 112 nm. The tail has a base plate and spikes. This is the first report of the extensive and continuous occurrence of a phage and its host bacterium in plants.     

Dissociation by Chelating Agents and Substructure of the Thermophilic Bacteriophage TP84

The thermophilic bacteriophage TP84 is dissociated into its head, tail, and released deoxyribonucleic acid (DNA) by chelating agents such as ethylenediaminetetraacetic acid (EDTA) and phosphate. The phage is more sensitive to EDTA than to phosphate, and dialysis against either agent causes more effective dissociation than standing in their presence. The tail possesses a knobbed structure which is inserted into the head of the intact phage and to which the DNA appears to be attached. The method of dissociating TP84 described in this paper provides a source of undamaged structural components and intact strands of DNA for subsequent investigations. A possible mechanism of chelate inactivation is discussed.     

Oxidative Processes in Photosystem I of Thermophilic Cyanobacteria at High Temperatures

We studied the mechanisms of the relationships between the generation of millisecond delayed fluorescence in photosystem I (DF) and the oxidative destruction of chlorophyll in the membranes of a thermophilic cyanobacteria Synechococcus elongatusin the temperature range 60–80°C at various irradiation levels and in the presence of substances affecting the intensity of DF. Light and temperature dependencies of the chlorophyll oxidation rates were similar to those of the DF of PSI. Anions Cl^–, Br^–, and NO^– ₃, which quench the triplet states of chlorophyll, almost completely inhibited the chlorophyll oxidation and reduced the intensity of the DF maximum by 70%. Under anaerobic conditions and in the presence of sodium ascorbate, the rate of chlorophyll oxidation also markedly decreased. We found that the long-wavelength chlorophyll forms were the most susceptible to oxidation and related the temperature-dependent changes in the DF of PSI and in the oxidative processes in the membranes of thermophilic cyanobacteria to an increase in the concentration of the triplet states of P₇₀₀and other chlorophyll forms. The latter result from the temperature-dependent inactivation of carotenoids and the inhibition of electron transfer to ferredoxin in PSI.     

一株产高温蛋白酶嗜热菌A-2的筛选鉴定及酶学特性分析

本研究为从云南腾冲热泉中分离纯化得到一株产高温蛋白酶的菌株并对其进行驯化培养,用以探究该菌株的生长条件及酶学特性,通过选择培养基筛选能够分解脱脂奶粉产蛋白酶的菌株,应用常规方法液体培养菌体,探究温度、pH、碳源、氮源对菌株生长情况的影响,并采用福林酚法测蛋白酶活性。并提取蛋白酶液对酶的最适pH、温度以及热稳定性、pH稳定性进行研究。结果发现通过含脱脂奶粉的固体培养基筛选得到一株产蛋白酶菌株A-2,经过生理生化试验和16S rDNA鉴定知该菌种属于Aneurinibacillus属。酵母粉、葡萄糖、55℃、pH值7.5分别为菌株生长的最适氮源、碳源、温度和pH。此外该菌株所产的蛋白酶最适温度为60℃,在pH值7~9具有较好的酶活性。因此,该菌株为嗜热芽孢杆菌,所产的碱性蛋白酶具有较高的耐受温度和pH稳定性,为进一步开发利用提供参考的价值。     

A Novel Filtering Mutualism between a Sponge Host and Its Endosymbiotic Bivalves

Sponges, porous filter-feeding organisms consisting of vast canal systems, provide unique substrates for diverse symbiotic organisms. The Spongia (Spongia) sp. massive sponge is obligately inhabited by the host-specific endosymbiotic bivalve Vulsella vulsella, which benefits from this symbiosis by receiving protection from predators. However, whether the host sponge gains any benefit from this association is unclear. Considering that the bivalves exhale filtered water into the sponge body rather than the ambient environment, the sponge is hypothesized to utilize water exhaled by the bivalves to circulate water around its body more efficiently. We tested this hypothesis by observing the sponge aquiferous structure and comparing the pumping rates of sponges and bivalves. Observations of water currents and the sponge aquiferous structure revealed that the sponge had a unique canal system enabling it to inhale water exhaled from bivalves, indicating that the host sponge adapted morphologically to receive water from the bivalves. In addition, the volume of water circulating in the sponge body was dramatically increased by the water exhaled from bivalves. Therefore, this sponge-bivalve association can be regarded as a novel mutualism in which two filter-feeding symbionts promote mutual filtering rates. This symbiotic association should be called a “filtering mutualism”.     

Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase

The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (Wzc_CD). Wzc_CD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from Wzc_CD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine.