First Report of Streptomyces galilaeus Associated with Common Scab in China.

During a survey of potato scab pathogens in China from 2003 to 2012, a new pathogen was found in Shanxi and Neimenggu provinces. The incidence was approximately 20% of all recovered strains. The lesions caused by the pathogen were slightly raised and similar to those caused by Streptomyces scabies (3). Lesions were excised (approximately 10 mm3) from 40 infected tubers, surface-disinfested with 0.3% NaOCl for 30 s, rinsed in sterile water three times, cut into 5 mm3, then sliced into 1-mm pieces, and plated on water agar amended with ampicillin (50 µg/ml). Plates were incubated at 28°C in the dark for 4 days. The spores of Streptomyces sp. strains growing from the tuber pieces were collected from single bacterial colonies and cultured on oatmeal agar. To fulfill Koch's postulates, one strain, CPS-2, was grown at 28°C for 10 days and the spores were washed from the plates as inoculum. One hundred milliliters of inoculum (1 × 105 CFU/ml) was mixed with autoclaved soil and vermiculite (1:1) in each pot (15 cm in diameter). Cut tubers were planted in the pots (potato cv. Favorita, one plant per pot, five replicates) and grown under greenhouse conditions (22 ± 5°C). Typical common scab symptoms consisting of small, brown, raised lesions developed on potato tubers 12 weeks after planting. The same strain was re-isolated from the lesions of the new scabby tubers. Non-inoculated plants, treated as described above, but without strain CPS-2, remained healthy. The CPS-2 strain was identified based on morphological and physiological characterization and 16S rDNA sequence. On yeast-malt extract agar, the test strain produced grayish-white aerial hypha, reddish brown substrate mycelium and pigments, and loose spiral spore chains. Spores were smooth and were 0.8 to 0.9 × 1.1 to 1.2 µm in size (diameter and length). The ability of the strain to use single sources of carbon and nitrogen was verified according to the International Streptomyces project (4). The strain grew in media supplemented with L-arabinose, D-fructose, D-glucose, rhamnose, raffinose, meso-inositol, sucrose, and D-xylose, but not D-mannitol. It used L-hydroxyproline, L-methionine, and L-histidine, and produced melanin on tyrosine and peptone yeast extract agar. The strain did not grow at a pH less than 5.0 and was sensitive to streptomycin (20 µg/ml), phenol (0.1%), and crystal violet (0.5 µg/mL), but not to penicillin (10 IU/ml). The strain also produced hydrogen sulfide. The biological characteristics of strain CPS-2 were in accord with Streptomyces galilaeus. CPS-2 produced thaxtomin A in oatmeal liquid medium and the txt AB gene fragment was successfully amplified using specific primers (2). The 16S rDNA sequence of CPS-2 was amplified by PCR with primers 16S1-F: 5'-CATTCACGGAGAGTTTGATCC-3' and 16S1-R: 5'-AGAAAGGAGGTGATCCAGCC-3' (1) and sequenced. A BLAST search of the 16S rDNA sequence for CPS-2 was conducted using the NCBI GenBank database, resulting in 99.8% similarity to S. galilaeus (NR_040857). The 16S rDNA sequence for CPS-2 (1,388 bp) was deposited in GenBank (AY621378). To our knowledge, this is the first report of S. galilaeus causing common scab of potato in China. References: (1) R. A. Bukhalid et al. Appl. Environ. Microbiol. 68:738, 2002. (2) R. Flores-González et al. Plant Pathol. 57:162, 2008. (3) D. H. Lambert and R. Loria. Int. J. Syst. Bacteriol. 39:387, 1989. (4) E. B. Shirling and D. Gottlieb. Int. J. Syst. Bacteriol. 16:313, 1966.

Environmental stresses induce the expression of putative glycine-rich insect cuticular protein genes in adult Leptinotarsa decemlineata (Say).

The deposition of cuticular proteins in insects usually occurs during the moulting process. Three putative glycine-rich insect cuticular proteins, Ld-GRP1 to 3, were identified and characterized from the Colorado potato beetle, Leptinotarsa decemlineata. The Ld-GRPs contained conserved GXGX and/or GGXG sequence repeats. Ld-GRP1 also contained a conserved AAPA/V motif commonly found in cuticular proteins. The transcripts of Ld-GRP1 and Ld-GRP2 were detected in the epidermal cell layer by in situ hybridization, making them putative insect cuticular proteins. The putative cuticular protein genes were highly induced by the insecticide azinphosmethyl (organophosphorous) 2-3 weeks after adult moulting. Putative cuticular protein gene expression level was higher in azinphosmethyl-resistant beetles than in susceptible beetles. Furthermore, two of the putative cuticular protein genes were highly induced by dry environmental conditions. These results suggest that the insect might increase cuticular component deposition in the adult stage in response to environmental stresses. This ability may allow the insect to adapt to new or changing environments.

The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA.

The eukaryotic mRNA 5' cap structure m7GpppX (where X is any nucleotide) interacts with a number of cellular proteins. Several of these proteins were studied in mammalian, yeast, and drosophila cells and found to be involved in translation initiation. Here we describe a novel cap-binding protein, the coat protein of L-A, a double-stranded RNA virus that is persistently maintained in many Saccharomyces cerevisiae strains. The results also suggest that the coat protein of a related double-stranded RNA virus (L-BC) is likewise a cap-binding protein. Strikingly, in contrast to the cellular cap-binding proteins, the interaction between the L-A virus coat protein and the cap structure is through a covalent bond.

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems.

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.

Controlled surface density of RGD ligands for cell adhesion: evidence for ligand specificity by using QCM-D.

RGD peptides (Arg-Gly-Asp) are known to promote cell adhesion. As a consequence, numerous materials have been functionalized using these peptides for several medical applications. We report herein the controlled functionalization of surfaces to study the influence of RGD density on cell selectivity. For this purpose, we selected a quartz crystal microbalance QCM-D as this technique allows real-time monitoring of cell adhesion to RGD surfaces. We observed that a critical spacing of nearly 40 nm between RGD ligands is required to observe selective cell adhesion whereas a higher density is not specific.

A wireless patch for sleep respiratory disorders applications.

This paper presents a conformable wireless patch and its mobile application for physical activity, spO2 and pCO2 recording associated to digital biomarkers that aim at providing the clinicians with a reliable computer-aided diagnosis tool for rapid and continuous monitoring of sleep respiratory disorders. Each part of the system is described and results are presented and discussed. The reflectance sp02 sensor has been tested in vivo on several body sites and several subjects then compared to a reference device. The electrochemical tcpO2 sensor has been validated in vitro. Based on these physiological parameters, the proposed algorithms to automatically identifying sleep respiratory events are compared to a reference index.

Isolation of a yeast gene encoding a protein homologous to the human Tat-binding protein TBP-1.

We have cloned a putative yeast homolog of the gene encoding the human Tat-binding protein, TBP-1. The gene termed TBPY encodes a 45,243-dalton protein displaying a heptad repeat of hydrophobic amino acids reminiscent of a leucine zipper. Secondary structure predictions suggest the possibility of formation of an amphipathic helix that could further be organized into a coiled-coil. Additionally, the protein product of TBPY shows amino acid signatures characteristic of a large family of RNA and DNA helicases. We propose that the hydrophobic region of yTBP-1 participates in self-dimerization or heterodimerization.

Genetic and family studies in Friedreich's ataxia.

This study consists of two parts: 1. A detailed genetic analysis of 35 sibships in which 58 individuals were affected with Friedreich's ataxia; and 2. Clinical and laboratory examinations of parents and siblings, in an attempt at carrier detection and diagnosis of the pre-clinical state. The increased parental consanguinity, the lack of affected individuals in other generations, and the lack of significance of extrinsic etiological variables, all suggested an autosomal recessive mode of inheritance, and this was confirmed by formal genetic analyses, employing several different methods. Associated abnormalities in our series of 58 patients included cardiomyopathy (51.7%), diabetes mellitus (19.0%), optic atrophy (5.2%), nerve deafness (5.2%) and congenital malformations (6.9%). The incidence of diabetes mellitus, congenital malformations, and epilepsy and/or febrile convulsions was elevated in first degree relatives of patients with Friedreich's ataxia.

Pathogenicity of Streptomyces scabies Mutants Altered in Thaxtomin A Production.

ABSTRACT To investigate the role of thaxtomin A in the pathogenicity of Streptomyces scabies, mutants altered in thaxtomin A production were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Mutants of S. scabies EF-35 could be differentiated according to levels of thaxtomin production. Mutants M1, M8, and M19 produced 2 to 20 times less thaxtomin A in oat bran medium than did EF-35. M1 and M19 were deficient in tryptophan catabolism. Thaxtomin production was reduced by about 300 times in mutant M16, which was a glutamic acid auxotroph. No thaxtomin A was detected in M13 culture supernatant. This mutant had a normal growth rate, was prototrophic, and catabolized tryptophan. Pathogenicity of mutants was tested on radish and potato. Mutants M1, M8, and M19 were pathogenic but, in most cases, less virulent than EF-35. M13 and M16 were nonpathogenic. These results suggest that thaxtomin A is an important pathogenicity determinant in S. scabies.

Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil.

The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorB(P) [Pseudomonas mandelii and closely related strains] and cnorB(B) [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB(P) population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB(B) guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB(P) populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB(P) population densities increased significantly with increasing rates of glucose addition. cnorB(B) guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.

Identification and characterization of cap-binding proteins from yeast.

Photochemical cross-linking of Saccharomyces cerevisiae ribosomal salt wash preparations to cap-labeled mRNA reveals, in addition to the previously characterized 24-kDa cap-binding protein (eIF-4E), the presence of two novel cap-binding proteins (CBPs) of apparent molecular masses of 96 and 150 kDa. Cross-linking of the 96-kDa CBP was found to occur spontaneously without UV light induction. Based on the ATP/Mg2+ requirements, the three CBPs can be subdivided into two classes: 1) ATP/Mg2+ independent (24- and 150 kDa) and 2) Mg2+ dependent (96 kDa). The co-purification of the 24- and 150-kDa CBPs through several different chromatographic steps is consistent with the existence of a yeast CBP complex, possibly analogous to mammalian eIF-4F.

A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.

Diastolic dysfunction is predictive of difficult weaning from cardiopulmonary bypass.

Diastolic function is receiving more attention since echocardiographic measurements were developed and have become widely available. The importance and significance of diastolic dysfunction (DD) observed before cardiac surgery and its relationship with adverse outcomes, such as difficult separation from cardiopulmonary bypass (CPB), have not been fully explored. In this study, we hypothesize that DD can be a predictor for the need of inotropic support to successfully separate from CPB. Ninety-two consecutive patients underwent surgery during the study period. Twenty-six patients were excluded. From the remaining 66 patients, 52 had coronary artery bypass grafting alone and 14 combined procedures, valvular surgery, and reoperations (redo). Systolic and diastolic function was evaluated by two experts blinded as to the clinical data except for the age. The evaluation of diastolic function was done according to published guidelines. The demographic, echocardiographic, and hemodynamic variables were entered in a logistic regression analysis to determine which variables were independent predictors of difficult separation from CPB and the need for postoperative vasoactive support. DD was present in 20 patients (30%). Patients with DD had lower weight (P = 0.046), less frequent coronary artery bypass grafting alone (P = 0.0004), more myocardial infarction before surgery (P = 0.02), higher regional wall motion score index (P = 0.0002), and larger left ventricle (P = 0.03). Total CPB time (P = 0.004) and ischemic time (P = 0.007) were longer in the DD group. Patients with DD required more frequent inotropic support at the end of surgery (P = 0.006) and up to 12 h after surgery (P = 0.003). Multivariate logistic regression identified female sex, DD, and total CPB time as predictive of difficult weaning and inotropic requirements up to 12 h after surgery.

Molecular cloning and sequencing of the hemD gene of Escherichia coli K-12 and preliminary data on the Uro operon.

DNA of plasmid pSAS1002TH (F' ilv+ hemD+ hemC+ cya+) was used to clone the hemD gene of Escherichia coli K-12. Due to poor transformability of the heme-deficient mutants, the restriction fragments of the F' plasmid were first cloned into a mobilizable derivative of pBR322, pSAS1211LP, which was then mobilized into a hemD recA mutant (E. coli SASX419AN). One recombinant plasmid, carrying a HindIII fragment of about 5 kilobases (kb), was shown to complement the hemD mutant and also a cya mutant of E. coli K-12, as well as a hemC mutant of Salmonella typhimurium LT2. Further subcloning of the insert enabled us to locate the hemD gene to a BamHI-PstI fragment (approximately 2.3 kb) which also carried the hemC gene. The hemD gene occupies a region close to the PstI end, since the deletion of a 0.6-kb fragment from this end resulted in loss of the ability to complement the hemD mutation. The use of the promoter-probe vector pK01 and the results of complementation showed that the hemD gene was transcribed under physiological conditions from the same promoter as the hemC gene, the direction of transcription being hemC-hemD. This allows us to define a new polycistronic operon of E. coli K-12, for which we propose the designation Uro operon. Sequencing of the hemD gene showed the presence of an open reading frame (ORF) of 738 nucleotides which could code for a protein with a molecular weight of 27,766, which should correspond to the hemD protein; the ORF starts with the last nucleotide of the hemC gene, the two genes having different reading frames. An ORF of at least 480 base pairs follows the hemD gene after a few nucleotides. The corresponding gene X, the function of which is unknown, might represent a third member of the Uro operon.

Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity.

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins