Isolation and characterization of a novel filamentous phage from Stenotrophomonas maltophilia

In this study, a novel filamentous phage, φSHP1, of the environmental Stenotrophomonas maltophilia strain P2 was isolated and characterized. Electron microscopy showed that φSHP1 resembled members of the family Inoviridae and was about 2.1?μm long. The 6,867-nucleotide genome of φSHP1 was a circular single-stranded DNA and had a replication form designated pSH1. Ten putative open reading frames (ORFs) were found in the φSHP1 genome, and six predicted proteins showed similarity to proteins in databases. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis of φSHP1 displayed one major structural polypeptide of approximately 4.0?kDa. N-terminal sequencing showed that it was the mature product of ORF5 and that its N-terminal 27 amino acid residues had been cleaved off from the predicted nascent protein. Finally, phylogenetic trees were constructed to analyze the phylogenetic relationship of φSHP1 to other known filamentous phages. φSHP1 appears to be the first reported Stenotrophomonas filamentous phage.     

Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities. A novel thiosulfate:quinone oxidoreductase from strain SH (SH-TQO) has been purified from its solubilized membrane fraction. The gene for SH-TQO was determined from the draft genome sequence of the strain SH. Amino acid sequences of peptides generated by the in-gel trypsin digestion of SH-TQO were found in a protein encoded by locus tag B1757_09800 of the genome of the strain SH. The gene encoded 444 amino acids with a signal peptide of 29 amino acids and was annotated to encode a porin. The gene was located in a unique genomic region, not found in A. thiooxidans strains, suggesting that the strain SH acquired this region through a horizontal gene transfer. A protein–protein basic local alignment search revealed that sulfur-oxidizing bacteria, such as Acidithiobacillus species have proteins homologous to SH-TQO, though the degree of homologies was relatively low. The protein, DoxXA, which is homologous to TQO from Acidianus amvibalens, was also found in the genomic region.     

Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.     

Isolation and characterization of a novel bacteriophage against Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease, has a doubling time of 24 hours, making rapid detection very difficult. Mycobacteriophages can be used in the detection of disease-causing mycobacteria such as MAP. Isolation and sequencing the genomes of lytic MAP bacteriophages are important preliminary steps towards designing phage-based rapid detection assays for this bacterium. A simple optimized protocol was developed to allow reproducible production of confluent growth of MAP on plates within four to six weeks of incubation at 30 °C. This protocol was applied to the screening of environmental and fecal samples for bacteriophages inhibiting the growth of MAP. As a result, a lytic phage, vB_MapS_FF47, was isolated from bovine feces. FF47 contains a double-stranded DNA genome ~48 kb in length with 73 protein coding sequences. It does not carry temperate or known virulence genes. This phage was shown to be most closely related to Mycobacterium phage Muddy, isolated in South Africa, and Gordonia phage GTE2; however, it could not infect any of the tested Gordonia, Rhodococcus, or Nocardia spp. that GTE2 could. The protocols that were developed for growth and phage isolation have potential applications in a high-throughput screening for compounds inhibiting the growth of MAP. This work describes the first time that a phage was isolated against M. paratuberculosis.     

SNDV, a novel virus of the extremely thermophilic and acidophilic archaeon Sulfolobus

We describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand. SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends. The virion consists of a core and a coat. The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops. The genome is cccDNA of 20 kb, which is modified by dam-like methylation. It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences. The DNA-modifying system differentiates between virus and host. We postulate a virus-encoded methylase that is active on hemimethylated DNA. The host range of SNDV is confined to few Sulfolobus strains from New Zealand. The virus persists in an unstable carrier state rather than as a prophage. Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae.     

Detection,identification and typing of Acidithiobacillus species and strains: a review

The genus Acidithiobacillus comprises several species of Gram-negative acidophilic bacteria that thrive in natural and man-made low pH environments in a variety of geo-climatic contexts. Beyond their fundamental interest as model extreme acidophiles, these bacteria are involved in the processing of minerals and the desulfurization of coal and natural gas, and are also sources of environmental pollution due to their generation of acid mine drainage and corrosion of cement and concrete structures. Acidithiobacillus spp. are therefore considered a biotechnologically relevant group of bacteria, and their identification and screening in natural and industrial environments is of great concern.Several molecular typing methodologies have been instrumental in improving knowledge of the inherent diversity of acidithiobacilli by providing information on the genetic subtypes sampled in public and private culture collections; more recently, they have provided specific insight into the diversity of acidithiobacilli present in industrial and natural environments. The aim of this review is to provide an overview of techniques used in molecular detection, identification and typing of Acidithiobacillus spp. These methods will be discussed in the context of their contribution to the general and specific understanding of the role of the acidithiobacilli in microbial ecology and industrial biotechnology. Emerging opportunities for industrial and environmental surveillance of acidithiobacilli using next-generation molecular typing methodologies are also reviewed.     

Isolation and characterization of a novel elastase inhibitor, AFLEI from Aspergillus flavus.

A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac1-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by alpha2-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.     

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.     

Isolation and characterization of a novel Francisella sp. from human cerebrospinal fluid and blood

We describe the isolation of a Francisella sp. from normally sterile sites in acutely ill patients in two different states within 2 years. Microbiologic and molecular analyses indicate that this organism represents a novel Francisella sp. Clinicians and microbiologists should be aware of this new potential pathogen, as infection may be more common than recognized.     

Isolation and characterization of novel rhesus monkey embryonic stem cell lines

ESCs are important as research subjects since the mechanisms underlying cellular differentiation, expansion, and self-renewal can be studied along with differentiated tissue development and regeneration in vitro. Furthermore, human ESCs hold promise for cell and tissue replacement approaches to treating human diseases. The rhesus monkey is a clinically relevant primate model that will likely be required to bring these clinical applications to fruition. Monkey ESCs share a number of properties with human ESCs, and their derivation and use are not affected by bioethical concerns. Here, we summarize our experience in the establishment of 18 ESC lines from rhesus monkey preimplantation embryos generated by the application of the assisted reproductive technologies. The newly derived monkey ESC lines were maintained in vitro without losing their chromosomal integrity, and they expressed markers previously reported present in human and monkey ESCs. We also describe initial efforts to compare the pluripotency of ESC lines by expression profiling, chimeric embryo formation, and in vitro-directed differentiation into endodermal, mesodermal, and ectodermal lineages.     

Isolation and characterization of a novel indigenous intestinal N4-related coliphage vB_EcoP_G7C

Lytic coliphage vB_EcoP_G7C and several other highly related isolates were obtained repeatedly from the samples of horse feces held in the same stable thus representing a component of the normal indigenous intestinal communities in this population of animals. The genome of G7C consists of 71,759 bp with terminal repeats of about 1160 bp, yielding approximately 73 kbp packed DNA size. Seventy-eight potential open reading frames, most of them unique to N4-like viruses, were identified and annotated. The overall layout of functional gene groups was close to that of the original N4 phage, with some important changes in late gene area including new tail fiber proteins containing hydrolytic domains. Structural proteome analysis confirmed all the predicted subunits of the viral particle. Unlike N4 itself, phage G7C did not exhibit a lysis-inhibited phenotype.     

Isolation and partial characterization of a novel subset of human T lymphocytes defined by monoclonal antibodies

Delayed-type hypersensitivity (DTH) responses to alloantigens were found to correlate with both skin and tumour allograft rejection in 224 reconstituted ATXBM-CBA mice. Furthermore, DTH responses and allograft rejection were observed only in mice that had received Ly-1 cells. Depletion of Thy-1+ or Ly-1+ cells led to indefinite graft survival and the absence of DTH responses, whereas depletion of Ly-2+ cells led to rapid graft rejection and strong DTH responses. The same result was obtained with CBA mice responding to grafts of either C57BL/6 skin, the B16 melanoma, or the EL4 lymphoma; and for (CBA X A)F1 mice responding to H-2K region alloantigens of AQR skin grafts. Thus, DTH and allograft rejection are both mediated by a Ly-1 T cell and it is considered that these are two different manifestations of the same transplantation response.     

Isolation and characterization of a canine rotavirus

Summary Canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was chracterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.With 4 Figures