苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

The α Subunit of Toluene Dioxygenase from Pseudomonas putida F1 Can Accept Electrons from Reduced FerredoxinTOL but Is Catalytically Inactive in the Absence of the β Subunit

The oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP_TOL) consisting of α (TodC1) and β (TodC2) subunits. Purified TodC1 gave absorbance and electron paramagnetic resonance spectra identical to those given by purified ISP_TOL. TodC1 was reduced by NADH and catalytic amounts of Reductase_TOL and Ferredoxin_TOL. Reduced TodC1 did not oxidize toluene, and catalysis was strictly dependent on the presence of purified TodC2.     

The amphioxus (Branchiostoma floridae) genome contains a highly diversified set of G protein-coupled receptors

Background

When natural hybridization occurs at sites where the hybridizing species differ in abundance, the pollen load delivered to the rare species should be predominantly from the common species. Previous authors have therefore proposed a hypothesis on the direction of hybridization: interspecific hybrids are more likely to have the female parent from the rare species and the male parent from the common species. We wish to test this hypothesis using data of plant hybridizations both from our own experimentation and from the literature.

Results

By examining the maternally inherited chloroplast DNA of 6 cases of F1 hybridization from four genera of plants, we infer unidirectional hybridization in most cases. In all 5 cases where the relative abundance of the parental species deviates from parity, however, the direction is predominantly in the direction opposite of the prediction based strictly on numerical abundance.

Conclusion

Our results show that the observed direction of hybridization is almost always opposite of the predicted direction based on the relative abundance of the hybridizing species. Several alternative hypotheses, including unidirectional postmating isolation and reinforcement of premating isolation, were discussed.     

Three types of taxis used in the response of Acidovorax sp. strain JS42 to 2-nitrotoluene

Acidovorax sp. strain JS42 is able to utilize 2-nitrotoluene (2NT) as its sole carbon, nitrogen, and energy source. We report here that strain JS42 is chemotactic to 2NT and that the response is increased when cells are grown on compounds such as 2NT that are known to induce the first step of 2NT degradation. Assays with JS42 mutants unable to oxidize 2NT showed that the first step of 2NT metabolism was required for the induced response, but not for a portion of the constitutive response, indicating that 2NT itself is an attractant. The 2NT metabolite nitrite was shown to be a strong attractant for strain JS42, and sufficient nitrite was produced during the taxis assay to account for a large part of the induced response. A mutant with an inactivated ntdY gene, which is located adjacent to the 2NT degradation genes and codes for a putative methyl-accepting chemotaxis protein, showed a defect in taxis toward 2NT that may involve a reduced response to nitrite. Responses of a mutant defective for the energy-taxis receptor, Aer, indicated that a functional aer gene is required for a substantial part of the wild-type induced response to 2NT. In summary, strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive 2NT-specific chemotaxis to directly sense 2NT, metabolism-dependent nitrite-specific chemotaxis that may be mediated by NtdY, and energy taxis mediated by Aer.     

Chemotaxis to Pyrimidines and Identification of a Cytosine Chemoreceptor in Pseudomonas putida

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 ΔmcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.Motile bacteria are capable of detecting chemical gradients in the environment and swim toward or away from them, a behavior known as chemotaxis. Historically, the enteric bacterium Escherichia coli has been the model organism for chemotaxis studies. E. coli has four transmembrane chemoreceptors called methyl-accepting chemotaxis proteins (MCPs), each of which binds a set of chemicals directly or in complex with specific periplasmic binding proteins. MCPs send signals to the flagellar motor via a complex signal transduction system that is composed of six soluble chemotaxis proteins, through which the bacterium modifies its swimming behavior based on the signal(s) received (for reviews, see references 5 and 15). The MCPs of E. coli sense a variety of stimuli, including amino acids, sugars, and dipeptides (30, 44). We recently reported that E. coli also responds to the pyrimidines thymine and uracil and demonstrated that Tap, the MCP known to mediate chemotaxis to dipeptides, is required for pyrimidine taxis (29).Pseudomonads are environmental bacteria that are widespread in nature, and all Pseudomonas species are motile. They have conserved chemotaxis proteins that are homologous to those present in E. coli, but their chemosensory systems appear to be more complex (6, 39, 55). Unlike E. coli, which has only one set of chemotaxis (che) genes in a single gene cluster, Pseudomonas species have multiple che gene homologs organized in several unlinked gene clusters (39). In addition, genome sequence analyses have revealed that Pseudomonas strains have numerous putative MCP genes. For example, the genome of Pseudomonas aeruginosa PAO1 (46) encodes 26 MCP-like proteins, Pseudomonas putida KT2440 (34) has 27, and Pseudomonas syringae DC3000 (9) has 49 (39).The best-studied chemotaxis system in Pseudomonas is that of the opportunistic pathogen P. aeruginosa. More than 75 different chemoattractants have been identified for P. aeruginosa (39), and 13 of its 26 MCP-like proteins have been functionally characterized. Eight MCPs have been shown to mediate positive responses to amino acids (PctABC), inorganic phosphate (CtpH and CtpL), malate (PA2652), ethylene (TlpQ), and chloroethylenes (McpA) (3, 25-27, 42, 47, 54). Two MCPs (McpA and McpB) were shown to be required for general optimal chemotaxis (16), and one MCP-like protein (Aer) was found to mediate energy taxis (22). The MCP-like proteins BdlA and PilJ were shown to be involved in biofilm formation and biosynthesis of type IV pili, respectively (10, 12, 32).P. putida is a common soil bacterium and, unlike P. aeruginosa, is not known to be pathogenic. Although P. putida and P. aeruginosa each have approximately the same number of MCP-like genes in their genomes, most of the protein products show relatively low amino acid sequence similarity. Based on our BLAST searches, three putative P. putida F1 MCPs have no obvious counterparts in P. aeruginosa PAO1. Most of the others share between 30 and 70% amino acid sequence identity, with the highest sequence conservation in the C-terminal signaling domains. The most highly conserved MCP-like proteins in the two species are Aer and PilJ (both are 77% identical to the corresponding homologs). These observations suggest that the two organisms respond to different subsets of attractants, which most likely reflects their different lifestyles and environmental niches. P. putida is known for its catabolic versatility (45), and we expect that members of the species are capable of responding to a correspondingly wide range of organic attractants. We are interested in defining the range of attractant and repellent responses and the functions of the MCPs present in P. putida compared to those of P. aeruginosa. In this study, we used P. putida strains F1 and PRS2000 and P. aeruginosa strain PAO1 to investigate the chemotactic responses to pyrimidines.     

Aerobic fitness and performance in elite female futsal players

Despite its growing popularity, few studies have investigated specific physiological demands for elite female futsal. The aim of this study was to determine aerobic fitness in elite female futsal players using laboratory and field testing. Fourteen female futsal players from the Venezuelan National team (age =21.2±4.0 years; body mass =58.6±5.6 kg; height =161±5.0 cm) performed a progressive maximal treadmill test under laboratory conditions. Players also performed a progressive intermittent futsal-specific field test for endurance, the Futsal Intermittent Endurance Test (FIET), until volitional fatigue. Outcome variables were exercise heart rate (HR), VO₂, post-exercise blood lactate concentrations ([La]b) and running speeds (km · h^-1). During the treadmill test, VO₂max, maximal aerobic speed (MAS), HR and peak [La]b were 45.3±5.6 ml · kg^-1 · min^-1, 12.5±1.77 km · h^-1, 197±8 beats · min^-1 and 11.3±1.4 mmol · l^-1, respectively. The FIET total distance, peak running velocity, peak HR and [La]b were 1125.0±121.0 m, 15.2±0.5 km · h^-1, 199±8 beats · min^-1 and 12.5±2.2 mmol · l^-1, respectively. The FIET distance and peak speed were strongly associated (r= 0.85-87, p < 0.0001) with VO₂max and MAS, respectively. Peak HR and [La]b were not significantly different between tests. Elite female futsal players possess moderate aerobic fitness. Furthermore, the FIET can be considered as a valid field test to determine aerobic fitness in elite level female futsal players.     

Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Background  

The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-F_B1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-O_B1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene.     

Biocatalytic degradation of pollutants

Microbial reactions play key roles in biocatalysis and biodegradation. The recent genome sequencing of environmentally relevant bacteria has revealed previously unsuspected metabolic potential that could be exploited for useful purposes. For example, oxygenases and other biodegradative enzymes are benign catalysts that can be used for the production of industrially useful compounds. In conjunction with their biodegradative capacities, bacterial chemotaxis towards pollutants might contribute to the ability of bacteria to compete with other organisms in the environment and to be efficient agents for bioremediation. In addition to the bacterial biomineralization of organic pollutants, certain bacteria are also capable of immobilizing toxic heavy metals in contaminated aquifers, further illustrating the potential of microorganisms for the removal of pollutants.     

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.