Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.     

Fibrinogen structure in projection at 18 A resolution. Electron density by co-ordinated cryo-electron microscopy and X-ray crystallography

Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the beta domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the gamma domains interact. A possible interpretation of this finding is provided by assuming substructure in the gamma domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a starting point for obtaining the three-dimensional structure.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Cloning and expression of a pathway for benzene and toluene fromBacillus stearothermophilus

Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil. The chromosomally-located aromatic pathway from this isolate was cloned intoEscherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.     

Molecular analysis of tick-borne protozoan and rickettsial pathogens in small ruminants from two South African provinces

Tick-borne protozoan and rickettsial diseases are a major threat to livestock in tropical and sub-tropical regions of Africa. In this study we investigated the presence and distribution of Theileria spp., Babesia ovis, Anaplasma ovis, Anaplasma phagocytophilum, Ehrlichia ruminantium and SFG Rickettsia in sheep and goats from Free State and KwaZulu-Natal provinces. A total of 91 blood samples were screened in this study, 61 from goats and 30 from sheep. PCR assay was conducted using primers based on Theileria spp. 18S rRNA, Babesia ovis (BoSSU rRNA), Anaplasma ovis (AoMSP4), Anaplasma phagocytophilum epank1, Ehrlichia ruminantium pCS20 and SFG Rickettsia OmpA. Overall infection rates of Theileria spp., Anaplasma ovis and Ehrlichia ruminantium were 18 (19.8%), 33 (36.3%) and 13 (14.3%), respectively. The co-infection of two pathogens were detected in 17/91 (18.7%) of all samples, goats having higher rates of co-infection compared to sheep. Phylogenetic tree analysis sequence of pCS20 gene of E. ruminantium of this study was found to be in the same clade with Kumm2 and Riverside strains both from South Africa. The phylogram of SSU rRNA of Theileria ovis had longer branch length compared to all other sequences most of which were from Asia and Middle East. This study provides important data for understanding the tick-borne diseases occurrence in the study area and it is expected to improve the approach for the diagnosis and control of these diseases.     

Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites

A loop-mediated isothermal amplification (LAMP) technique has been used as a novel nucleic acid detection method, whereby the target DNA can be amplified with high specificity and sensitivity under an isothermal condition using a set of four specific primers. In this study, we designed two sets of the LAMP primers for rhoptry-associated protein-1 genes of Babesia bovis and B. bigemina, in which a restriction enzyme cleavage site was inserted into two pairs of species-specific primers to construct a multiplex LAMP (mLAMP) method by combining these two sets totaling eight primers. The mLAMP method was distinguishable between B. bovis and B. bigemina, simultaneously, due to the subsequent restriction enzyme analysis. The sensitivities of the mLAMP method were 10(3) and 10(5) times higher on the detection limits for B. bovis and B. bigemina, respectively, than those of the classical PCR methods. Of 40 blood samples collected from cattle living in Ghana, 12 and 27% were positively detected by the mLAMP for B. bovis and B. bigemina, respectively. Furthermore, 14 and 23% of 90 blood samples from cattle in Zambia showed mLAMP-positive reactions to B. bovis and B. bigemina, respectively. These findings indicate that this mLAMP method is a new convenient tool for simultaneous detection of the bovine Babesia parasites.