bluuebunny/crossref_metadata_2025 huggingface dataset:
PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells
Authors: Sun Shishinn , Steinberg Bettie M.
Published: August 2015

Laryngeal papillomas are caused by infection of the laryngeal epithelium by human papillomavirus type 6 or type 11 (HPV-6/-11). Previous studies in our laboratory have demonstrated an increase in PI3 kinase levels in papilloma tissue. However, activation of the downstream effector of PI3 kinase, protein kinase B (PKB/Akt), was reduced. This observation was explained by the elevated expression of the phosphatase and tensin homologue (PTEN), a recently characterized tumour suppressor, in papilloma tissue. Recent investigation of the possible functional roles of PTEN during papilloma development has now indicated that the level of tyrosine(705)-phosphorylated signal transducer and activator of transcription 3 [PTyr(705)STAT3] could be inversely correlated to that of PTEN as well.In vitrophosphatase assays suggested the presence of an increased level of a PTyr(705)STAT3 phosphatase in papilloma extract. Immunodepletion of PTEN from papilloma extracts resulted in a reduction of the PTyr(705)STAT3 phosphatase activity. Transfection of PTEN cDNA into HeLa cells attenuated STAT3 phosphorylation at Tyr(705) in a dose-dependent manner. This attenuation of STAT3 phosphorylation was independent of the STAT3 kinase. Interestingly, introduction of a lipid phosphatase mutant of PTEN (G129E) resulted in heightened PTyr(705)STAT3 phosphatase activity, relative to that obtained from wild-type PTEN transfection. These data indicate that PTEN negatively regulates STAT3 activation in HPV-infected papilloma cells. Induction of PTEN and reduction of activated STAT3 might be a result of a host defence mechanism or a virus-directed strategy to alter normal epithelial differentiation programming.
Analysis of the desialidation process of the haemagglutinin protein of influenza B virus: the host-dependent desialidation step
Authors: Luo C. , Nobusawa E. , Nakajima K.
Published: August 2015

It was reported previously that haemadsorption by the haemagglutinin (HA) protein of influenza B virus required that the protein must undergo desialidation. When MDCK and COS cells were infected with influenza B/Kanagawa/73 virus in the presence of a neuraminidase (NA) inhibitor, Zanamivir, haemadsorption on MDCK cells was inhibited but that on COS cells was not. The activity of the NA protein of the two types of infected cells was similar and both were inhibited by Zanamivir in a dose-dependent manner. A comparison of the desialidation of the HA protein was made on MDCK and COS cells in the presence of bacterial NA and both cells were found to have similar sensitivity. On the accumulation of the HA and NA proteins in the trans-Golgi network of MDCK cells by means of low-temperature treatment, desialidation of the HA protein in the presence of Zanamivir was detected by two-dimensional gel electrophoresis. Because this agent was reported to be unable to penetrate cells, these data suggest that, in MDCK cells, desialidation of the HA protein occurs on the cell surface but, in COS cells, the HA and NA proteins might accumulate in the trans-Golgi network, thus allowing NA desialidation before their migration to the cell surface.
Uncoupling of virus-induced inflammation and anti-viral immunity in the brain parenchyma
Authors: Stevenson P. G. , Austyn J. M. , Hawke S.
Published: August 2015

Non-neuroadapted influenza virus confined to the brain parenchyma does not induce antigen-specific immunity. Nevertheless, infection in this site upregulated major histocompatibility complex (MHC) class I and MHC class II expression and recruited lymphocytes to a perivascular compartment. T cells recovered from the brain had an activated/memory phenotype but did not respond to viral antigens. In contrast, T cells recovered from the brain after infection in a lateral cerebral ventricle, which is immunogenic, showed virus-specific responses. As with infectious virus, influenza virus-infected dendritic cells elicited virus-specific immunity when inoculated into the cerebrospinal fluid but not when inoculated into the brain parenchyma. Thus, inflammation and dendritic cell function were both uncoupled from immune priming in the microenvironment of the brain parenchyma and neither was sufficient to overcome immunological privilege.
Genetic vaccines protect against Sin Nombre hantavirus challenge in the deer mouse (Peromyscus maniculatus)
Authors: Bharadwaj Mausumi , Mirowsky Katy , Ye Chunyan , Botten Jason , Masten Barbara , Yee Joyce , Lyons C. Richard , Hjelle Brian
Published: August 2015

We used a deer mouse (Peromyscus maniculatus) infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice (Bharadwaj et al., Vaccine 17, 2836–2843, 1999). Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene also was protective. The protective efficacy of glycoprotein gene fragments correlated with splenocyte proliferation in the presence of cognate antigen, but none induced neutralizing antibodies. Genetic vaccines against SN virus can protect outbred deer mice from infection even in the absence of a neutralizing antibody response.
Suppression of Japanese encephalitis virus infection by non-steroidal anti-inflammatory drugs
Authors: Chen Chun-Jung , Raung Shue-Ling , Kuo Ming-Der , Wang Yu-Ming
Published: August 2015

Japanese encephalitis virus (JEV) infection generates a rapid inflammatory response including peripheral neutrophil leucocytosis and infiltration of neutrophils into extraneural tissue. The level of inflammation correlates well with the clinical outcome in Japanese encephalitis patients. Non-steroidal anti-inflammatory drugs (NSAIDs), used medicinally for their analgesic and anti-inflammatory properties, are being considered for prevention of cardiovascular disease and cancer, as well as for treatment of human immunodeficiency virus infection. Apart from their ability to inhibit prostaglandin synthesis, the mechanisms underlying the beneficial therapeutic effects are largely unknown. We used aspirin, indomethacin and sodium salicylate to study the role of NSAIDs in JEV propagationin vitro. We found that NSAIDs suppressed JEV propagation in neuronal and non-neuronal cells. Blockade of cyclooxygenase activity by NSAIDs caused decreased production of free radicals and prostaglandins. However, these pharmacological alterations did not seem to correlate well with the antiviral effects. When cells were treated with the mitogen-activated protein kinase (MAPK) inhibitors PD 98059 and SB 203580, salicylate lost its antiviral effect. The activation of MAPK by anisomycin mimicked the action of salicylate in suppressing JEV-induced cytotoxicity. The decreased phosphorylation of extracellular signal-regulated kinase (ERK) was induced by JEV infection and the decrease in ERK was reversed by salicylate. Our data suggest that the signalling pathways of MAPK play a role in the antiviral action of salicylate.
Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad
Authors: Takahashi Masaharu , Nishizawa Tsutomu , Yoshikawa Akira , Sato Shin , Isoda Norio , Ido Kenichi , Sugano Kentaro , Okamoto Hiroaki
Published: August 2015

Two distinct hepatitis E virus (HEV) isolates, designated HE-JI3 and HE-JI4, were identified in a single patient with acute hepatitis in Japan, who had not travelled abroad. The HEV load of HE-JI3 at admission was 102 copies/ml, but that of HE-JI4 was tenfold higher at 103 copies/ml. The viraemia of HE-JI4 persisted for up to 16 days from admission, whereas HE-JI3 disappeared at 9 days after admission. The entire nucleotide sequence of the HE-JI4 isolate and partial nucleotide sequences of open reading frames (ORFs) 1 and 2 of the HE-JI3 isolate were determined. The full-length nucleotide sequence of HE-JI4 consisted of 7171 nucleotides excluding the poly(A) tail and contained ORF1 encoding 1684 amino acids, ORF2 encoding 671 amino acids and ORF3 encoding 114 amino acids. Sequence and phylogenetic analyses of the HEV genomes indicated that HE-JI4 was most closely related to an HEV isolate (T1) of genotype IV with the same strategy for translation of ORF2 and ORF3, but which differed from it by 16·5% over the entire genome. The HE-JI3 isolate showed the highest nucleotide identity (88·6–95·1%) to the genotype III HEVs, having higher identity to human and swine HEV isolates from the United States (US1, US2 and swUS1) than to those reported thus far from Japan (JRA1 and swJ570). The two co-infecting strains of HE-JI3 and HE-JI4 identified from the single patient shared only 80·1% nucleotide identity. These results indicate that multiple genotypes of HEV co-circulate in Japan, and that genotype IV comprises a remarkably heterogeneous group of HEVs.
Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes
Authors: Tscharke David C. , Reading Patrick C. , Smith Geoffrey L.
Published: August 2015

Previously, we developed a model for testing the virulence and immunogenicity of vaccinia virus (VV) mutants based on the intradermal injection of BALB/c mouse ear pinnae. The model is characterized by a local infection in the inoculated skin without signs of systemic illness, mimicking dermal vaccination with VV. Here a further characterization of this model is presented, including the responses of mice to infectious virus doses as low as 10 p.f.u., a quantification of the infiltrate at the site of infection and use of different virus and mouse strains. The model was then used to compare the pathogenesis of six mutants of VV strain Western Reserve (WR) lacking genes A36R, A40R, A44L, B12R, B13R or B15R with that of appropriate control viruses. All of these genes except B12R and B15R influence the outcome of dermal infection with WR and for A40R and B13R this is the first role that has been reported after infection of mammals. A comparison of new and published results from intradermal and intranasal models is presented, showing that out of 16 gene deletion or insertion mutants of VV, half have phenotypes distinct from controls in only one of these models. Thus, the intranasal and intradermal models are complementary tools for dissecting the genetic basis of VV virulence.
The expansion of a hypervariable, non-hr ori-like region in the genome of Cryptophlebia leucotreta granulovirus provides in vivo evidence for the utilization of baculovirus non-hr oris during replication
Authors: Jehle Johannes A.
Published: August 2015

In this report a naturally occurring hypervariable region within the genome of different Cryptophlebia leucotreta granulovirus genotypes is characterized. The region consists of a stretch of direct repeats, short palindromes and an unusual AT-rich region. Although the organization of these repeat sequences is unique to baculoviruses, it has the structural features of a ‘non-hr’ origin of DNA replication (ori). Restriction analysis and Southern hybridization revealed that this region is expanded during virus replication. Sequence comparison of different isolated genotypes indicated that the expansion is caused by concatenation of short repeats within the region or by concatenation of the complete region. These findings indicate that the expansion of non-hr origin-like regions is not restricted to defective-interfering particles, as was found previously for baculoviruses propagated in cell culture. Moreover, it appears that non-hr complexity contributes to the natural heterogeneity and genetic plasticity of baculovirus genomes. Also, circumstantial evidence is discussed that hr oris might have developed from internal rearrangement and multiplication of a non-hr ori during baculovirus evolution.
Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America
Authors: Arauz-Ruiz Patricia , Norder Helene , Robertson Betty H. , Magnius Lars O.
Published: August 2015

The complete genomes were sequenced for ten hepatitis B virus (HBV) strains. Two of them, from Spain and Sweden, were most similar to genotype D, although encoding d specificity. Five of them were from Central America and belonged to genotype F. Two strains from Nicaragua and one from Los Angeles, USA, showed divergences of 3·1–4·1% within the small S gene from genotype F strains and were recognized previously as a divergent clade within genotype F. The complete genomes of the two genotype D strains were found to differ from published genotype D strains by 2·8–4·6%. Their S genes encoded Lys122, Thr127 and Lys160, corresponding to the putative new subtype adw3 within this genotype, previously known to specify ayw2, ayw3 or, rarely, ayw4. The complete genomes of the three divergent strains diverged by 0·8–2·5% from each other, 7·2–10·2% from genotype F strains and 13·2–15·7% from other HBV strains. Since pairwise comparisons of 82 complete HBV genomes of intratypic and intertypic divergences ranged from 0·1 to 7·4% and 6·8 to 17·1%, respectively, the three sequenced strains should represent a new HBV genotype, for which the designation H is proposed. In the polymerase region, the three strains had 16 unique conserved amino acid residues not present in genotype F strains. So far, genotype H has been encountered in Nicaragua, Mexico and California. Phylogenetic analysis of the complete genomes and subgenomes of the three strains showed them clustering with genotype F but forming a separate branch supported by 100% bootstrap. Being most similar to genotype F, known to be an Amerindian genotype, genotype H has most likely split off from genotype F within the New World.
Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis
Authors: Iqbal Munir , McCauley John W.
Published: August 2015

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein Ernsinteracts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant Ernswas undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of Ernswas essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in Ernsreduced the binding of Ernsto immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal Erns, Ernsthat was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with Erns. It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of Ernsconstitutes a GAG-binding site.
Quasispecies in the 5′ untranslated genomic region of bovine viral diarrhoea virus from a single individual
Authors: Jones Leandro Roberto , Zandomeni Rubén , Weber E. Laura
Published: August 2015

The variability of the 5′ untranslated genomic region (5′UTR) of bovine viral diarrhoea virus (BVDV) RNA obtained from a single individual was analysed. Lung, kidney and spleen tissues from a naturally infected foetus were used as the source of viral RNA. A fragment of 288 bases of the internal ribosome entry site from the BVDV 5′UTR was amplified by RT–PCR using a proofreading DNA polymerase. PCR products were cloned into pGem and, subsequently, transformed intoEscherichia coli. The single-strand conformational polymorphisms of 158 lung-derived clones were analysed; a total of 11 banding patterns was observed. DNAs corresponding to all patterns were sequenced. Of the randomly selected clones, 11 and 10 clones derived from the kidney and spleen, respectively, were also sequenced. All sequences presented differences ranging from 1 to 6 nt substitutions. Analysis of the secondary structure of the variant sequences and comparisons to variant nucleotide sites from the 5′UTR of several BVDV isolates showed that the observed changes were almost free of randomness. Clustering and phylogenetic analyses suggested the existence of low-kinetic variants. BVDV quasispecies may be involved in establishing persistent infections by means of eluding maternal antibodies. The methods described here may be adapted easily both to analyse large numbers of samples from other genomic regions and for the study of BVDV quasispecies evolution in other systems.
Poliovirus transcytosis through M-like cells
Authors: Ouzilou Laurent , Caliot Elise , Pelletier Isabelle , Prévost Marie-Christine , Pringault Eric , Colbère-Garapin Florence
Published: August 2015

During the digestive-tract phase of infection, poliovirus (PV) is found in the oropharynx and the intestine. It has been proposed that PV enters the organism by crossing M cells, which are scattered in the epithelial sheet covering lymphoid follicles of Peyer’s patches. However, PV translocation through M cells has never been demonstrated. A model of M-like cells has been previously developed using monolayers of polarized Caco-2 enterocytes cocultured with lymphocytes isolated from Peyer’s patches. In this model, lymphoepithelial interactions trigger the appearance of epithelial cells having morphological and functional characteristics of M cells. We have demonstrated efficient, temperature-dependent PV transcytosis in Caco-2 cell monolayers containing M-like cells. This experimental evidence is consistent with M cells serving as gateways allowing PV access to the basal face of enterocytes, the underlying immune follicle cells, and PV transport toward mesenteric lymph nodes.
Intramolecular disulfide bonding is essential for betanodavirus coat protein conformation
Authors: Krondiris John V. , Sideris Diamantis C.
Published: August 2015

Here we report on the conformational changes that are responsible for the appearance of theDicentrarchus labraxencephalitis virus (DlEV) coat protein as a doublet in SDS–PAGE. Wild-type and mutated forms of the coat protein cDNA were expressed inE. coli. The study of the resulting recombinant molecules excluded the possibility of the involvement of a precursor autocatalysis mechanism or a ribosomal frameshifting event in the doublet formation. The appearance of the coat protein doublet was found to be β-mercaptoethanol sensitive. Based on this observation, we carried out substitution of all cysteine residues. The obtained results demonstrated the importance of intramolecular disulfide bonding between cysteines 187 and 201 on coat protein conformational changes.
Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes
Authors: Potgieter A. C. , Steele A. D. , van Dijk A. A.
Published: August 2015

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.
LPS-binding protein and CD14-dependent attachment of hepatitis B surface antigen to monocytes is determined by the phospholipid moiety of the particles
Authors: Vanlandschoot Peter , Van Houtte Freya , Roobrouck Annelies , Farhoudi Ali , Stelter Felix , Peterson Darell L. , Gomez-Gutierrez Julian , Gavilanes Francisco , Leroux-Roels Geert
Published: August 2015

It was observed recently that recombinant yeast-derived hepatitis B surface antigen (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. It is shown here that binding requires the presence of the LPS receptor CD14. Furthermore, evidence is presented that a domain on CD14 that is identical to or largely overlaps with the LPS-binding pocket is instrumental for the attachment of rHBsAg. Additionally, it is shown that the heat-labile LPS-binding protein (LBP) catalyses the binding of rHBsAg to the cells. Remarkably, natural plasma-derived HBsAg (pHBsAg) does not have this property. pHBsAg devoid of its lipids and reconstituted with phosphatidylserine or phosphatidylglycerol acquires the characteristic of yeast-derived HBsAg. Clearly, the interaction of rHBsAg with the cell membrane is determined by the presence of charged phospholipids that are absent in pHBsAg. Although a lipid–receptor interaction is suggested, antibody-inhibition experiments suggest a possible involvement of the C-terminal region of the S protein in the interaction with monocytes. The possible implications of these observations for hepatitis B virus (HBV) infection and HBV vaccine efficiency are discussed.
Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1·3 kb non-coding region between the E2 and L2 open reading frames
Authors: Terai Masanori , Burk Robert D.
Published: August 2015

We have characterized the complete genome (8300 bp) of an isolate of Felis domesticus papillomavirus (FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the nucleotide sequence of the L1 open reading frame demonstrated that the FdPV genome was most closely related to canine oral papillomavirus (COPV). A 384 bp non-coding region (NCR) was found between the end of L1 and the beginning of E6, and a 1·3 kbp NCR was located between the end of E2 and the beginning of L2. Phylogenetic analysis placed FdPV in the E3 clade with COPV. Both viruses contain the atypical second NCR, which has no homology with sequences in existing databases.
Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore The GenBank accession number for the sequence reported in this paper is AF361942.
Authors: González Carreró Manuel I , Sangari Félix J , Agüero Jesús , Garcı́a Lobo Juan M
Published: August 2015

Brucella abortusis known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions. In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5in the virulentB. abortusstrain 2308. This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA. Production of catecholic compounds by both theBrucellamutant and parental strains under iron-deprivation conditions was assayed by TLC. Two catecholic substances were identified in the supernatant of the parental strain 2308. The faster migrating spot showed the same retention factor (Rf) as that of purified 2,3-DHBA. The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol. This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308. The genomic region containing Tn5in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing. The sequence revealed that the insertion had occurred at a gene with homology toEscherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin. Intracellular survival and growth rates of theB. abortuswild-type andentFmutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection. It is concluded thatB. abortussynthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.
Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451
Authors: Fernández Marisol , Sánchez Jesús
Published: August 2015

The presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20–22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20–22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20–22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.
Physical and genetic map of the Lactobacillus sakei 23K chromosome
The GenBank accession numbers for the sequences reported in this paper are given in Table 2 and the legend to Fig. 3.
Authors: Dudez Anne-Marie , Chaillou Stéphane , Hissler Lionel , Stentz Régis , Champomier-Vergès Marie-Christine , Alpert Carl-Alfred , Zagorec Monique
Published: August 2015

The Lactobacillus
sakei 23K chromosome was analysed by pulsed-field gel electrophoresis after digestion with the restriction enzymes AscI, NotI and SfiI. The chromosome size was estimated to be 1845±80 kb. The use of I-CeuI, specific for rrn genes encoding 23S rRNAs, showed that seven rrn loci were present, on 40% of the chromosome. The seven rrn clusters were mapped and their orientation was determined, allowing the position of the replication origin to be estimated. Partial I-CeuI digestions were used to construct a backbone and the different restriction fragments obtained with AscI, NotI and SfiI were assembled to a physical map by Southern hybridization. Eleven L. sakei gene clusters previously identified were mapped, as well as 25 new loci located randomly on the chromosome and 11 regions flanking the rrn gene clusters. A total of 47 clusters were thus mapped on L. sakei chromosome. The new loci were sequenced, allowing the identification of 73 complete or incomplete coding sequences. Among these 73 new genes of L. sakei, the function of 36 could be deduced from their similarity to known genes described in databases. However, 10 genes had no homologues, 10 encoded proteins similar to proteins of unknown function and 17 were similar to hypothetical proteins.
A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components
The GenBank accession number for the sequence reported in this paper is AF120104.
Authors: Roos Stefan , Jonsson Hans
Published: August 2015

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0·1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3–7·4, with maximum binding at pH 4–5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.
Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-‘Synechococcus’ clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon
The GenBank accession numbers for the ITS amplicon sequences reported in this paper are AF387607 (PCC 9511T), AF387610 (PCC 6307), AF387608 (PCC 7001) and AF387609 (PCC 7941).
Authors: Laloui Wassila , Palinska Katarzyna A , Rippka Rosmarie , Partensky Frédéric , Tandeau de Marsac Nicole , Herdman Michael , Iteman Isabelle
Published: August 2015

PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of ‘Synechococcus’ (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511T, although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511T, PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-‘Synechococcus’ strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.
Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets
Authors: Skuce Robin A , McCorry Thomas P , McCarroll Julie F , Roring Solvig M. M , Scott Alistair N , Brittain David , Hughes Stephen L , Hewinson R. Glyn , Neill Sydney D
Published: August 2015

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of theMycobacterium tuberculosiscomplex, particularly the monitoring of strains ofMycobacterium bovis. With the recent availability of genome sequences for strains of theM. tuberculosiscomplex, novel PCR-basedM. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci inM. tuberculosisH37Rv, the copy number of which varies in other strains of theM. tuberculosiscomplex. Six of these VNTRs were applied to a panel of 100 differentM. bovisisolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well betweenM. bovisstrains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of theM. tuberculosiscomplex. The novel VNTR targets identified in this study should additionally increase the power of this approach.
Role of spoT-dependent ppGpp accumulation in the survival of light-exposed starved bacteria
Authors: Gong Lan , Takayama Kathy , Kjelleberg Staffan
Published: August 2015

In bacteria, cytoplasmic levels of the effector nucleotide ppGpp are regulated in response to changes in growth conditions. This study describes the involvement of SpoT-mediated ppGpp accumulation in the survival of light-exposed bacteria during fatty acid starvation. In contrast to isogenic wild-type strains andrelAmutants, the ‘Vibrio angustum’ S14spoTandEscherichia colirelAspoTmutants displayed significant losses in viability in response to cerulenin-induced fatty acid starvation under cool-white fluorescent light. However, when starvation experiments were performed in complete darkness, or under light filtered through a UV-resistant perspex sheet, only a minor decline in viability was observed for the wild-type and mutant strains. This finding indicated that the lethal effect was mediated by weak UV emission. In contrast to theE. coli relA spoTmutant, which lacks ppGpp, the ‘V. angustum’ S14spoTmutant exhibited higher ppGpp levels and lower RNA synthesis rates during fatty acid starvation, features that might be correlated with its lethality. In agreement with this finding, fatty acid starvation lethality also occurred upon induction of ppGpp overaccumulation inE. coli. These data suggest that the precise regulation of ppGpp levels in the stressed cell is crucial, and that both the absence and the overaccumulation of ppGpp impair fatty acid starvation survival of light-exposed cells. Moreover, the UV-induced lethal effect during fatty acid starvation was also observed forE. colistrains mutated inrpoSanddps, which, in the wild-type, are regulated directly or indirectly by ppGpp, respectively. The restoration of viability of fatty-acid-starvedspoTmutant cells through the addition of exogenous catalase suggested that the observed light-dependent lethal effect was, at least in part, caused by UV-imposed oxidative stress. Based on these results, it is proposed that fatty acid starvation adaptation of light-exposed bacterial cells depends on the development of resistance to UV-induced oxidative stress. This stress resistance was found to require appropriate ppGpp levels, ppGpp-induced RpoS expression and, hence, upregulation of RpoS-regulated stress-defending genes, such asdps.
Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli
Authors: Amer Amal O , Valvano Miguel A
Published: August 2015

The integral membrane protein WecA mediates the transfer ofN-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.
Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment
Authors: Waleron Małgorzata , Waleron Krzysztof , Podhajska Anna J , Łojkowska Ewa
Published: August 2015

Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi.
Umboniibacter roseus sp. nov., isolated from coastal seawater
Authors: Sung Hye-Ri , Kim Mibang , Shin Kee-Sun
Published: August 2015

A Gram-reaction-negative, non-motile, strictly aerobic, dark pink-pigmented and rod-shaped bacterial isolate, designated 14-121-B13T, was isolated from surface seawater off the coast of the South Sea at Namhae-gun, Republic of Korea. Cells were catalase- and oxidase-positive and required NaCl for growth. Strain 14-121-B13T grew optimally at 30 °C, in the presence of 2 % (w/v) NaCl and at pH 7.5–8.0.Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain 14-121-B13T clustered with the type strain of Umboniibacter marinipuniceus, with which it exhibited 96.7 % sequence similarity. The DNA G+C content of strain 14-121-B13T was 48.9 mol%. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The major respiratory quinone was ubiquinone Q-7 and the polar lipids detected in strain 14-121-B13T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, unidentified phospholipids, unidentified aminophospholipids and unidentified lipids. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain 14-121-B13T is considered to represent a novel species of the genus, Umboniibacter for which the name Umboniibacter roseus sp. nov. is proposed. The type strain is 14-121-B13T ( = DSM 29882T = KCTC 42467T).
Leaf traits drive differences in biomass partitioning among major plant functional types
Authors: Duursma Remko A. , Falster Daniel S.
Published: August 2015

The partitioning of biomass into leaves and stems is one of the most uncertain and influential components of global vegetation models (GVMs). Although GVMs typically assume that the major woody plant functional types (PFTs) differ in biomass partitioning, empirical studies have not been able to justify these differences. Here we test for differences between PFTs in partitioning of biomass between leaves and stems.

We use the recently published Biomass And Allometry Database (BAAD), a large database including observations for individual plants. The database covers the global climate space, allowing us to test for direct climate effects in addition to PFT.

The leaf mass fraction (LMF, leaf / total aboveground biomass) varied strongly between PFTs (as defined by deciduous vs. evergreen and gymnosperm vs. angiosperm). We found that LMF, once corrected for plant height, was proportional to leaf mass per area across PFTs. As a result, the PFTs did not differ in the amount of leaf area supported per unit above ground biomass. We found only weak and inconsistent effects of climate on biomass partitioning.

Combined, these results uncover fundamental rules in how plants are constructed and allow for systematic benchmarking of biomass partitioning routines in GVMs.
Systems Biology Approach to Model the Life Cycle ofTrypanosoma cruzi
Authors: Carrea Alejandra , Diambra Luis
Published: August 2015

Due to recent advances in reprogramming cell phenotypes, many efforts have been dedicated to developing reverse engineering procedures for the identification of gene regulatory networks that emulate dynamical properties associated with the cell fates of a given biological system. In this work, we propose a systems biology approach for the reconstruction of the gene regulatory network underlying the dynamics of theTrypanosoma cruzi’s life cycle. By means of an optimisation procedure, we embedded the steady state maintenance, and the known phenotypic transitions between these steady states in response to environmental cues, into the dynamics of a gene network model. In the resulting network architecture we identified a small subnetwork, formed by seven interconnected nodes, that controls the parasite’s life cycle. The present approach could be useful for better understanding other single cell organisms with multiple developmental stages.

Abbreviations

GRN

gene regulatory network

SVD

singular value decomposition

TS

trans-sialidase
Hirshfeld surface analysis of two new phosphorothioic triamide structures
Authors: Alamdar Amir Hossein , Pourayoubi Mehrdad , Saneei Anahid , Dušek Michal , Kučeráková Monika , Henriques Margarida S.
Published: August 2015

Hirshfeld surfaces and two-dimensional fingerprint plots are used to analyse the intermolecular interactions in two new phosphorothioic triamide structures, namelyN,N′,N′′-tris(3,4-dimethylphenyl)phosphorothioic triamide acetonitrile hemisolvate, P(S)[NHC6H3-3,4-(CH3)2]3·0.5CH3CN or C24H30N3PS·0.5CH3CN, (I), andN,N′,N′′-tris(4-methylphenyl)phosphorothioic triamide–3-methylpiperidinium chloride (1/1), P(S)[NHC6H4(4-CH3)]3·[3-CH3-C5H9NH2]+·Cl−or C21H24N3PS·C6H14N+·Cl−, (II). The asymmetric unit of (I) consists of two independent phosphorothioic triamide molecules and one acetonitrile solvent molecule, whereas for (II), the asymmetric unit is composed of three components (molecule, cation and anion). In the structure of (I), the different components are organized into a six-molecule aggregate through N—H...S and N—H...N hydrogen bonds. The components of (II) are aggregated into a two-dimensional array through N—H...S and N—H...Cl hydrogen bonds. Moreover, interesting features of packing arise in this structure due to the presence of a double hydrogen-bond acceptor (the S atom of the phosphorothioic triamide molecule) and of a double hydrogen-bond donor (the N—H unit of the cation). For both (I) and (II), the full fingerprint plot of each component is asymmetric as a consequence of the presence of three fragments. These analyses reveal that H...H interactions [67.7 and 64.3% for the two symmetry-independent phosphorothioic triamide molecules of (I), 30.7% for the acetonitrile solvent of (I), 63.8% in the phosphorothioic triamide molecule of (II) and 62.9% in the 3-methylpiperidinium cation of (II)] outnumber the other contacts for all the components in both structures, except for the chloride anion of (II), which only receives the Cl...H contact. The phosphorothioic triamide molecules of both structures include unsaturated C atoms, thus presenting C...H/H...C interactions: 17.6 and 21% for the two symmetry-independent phosphorothioic triamide molecules in (I), and 22.7% for the phosphorothioic triamide molecule of (II). Furthermore, the N—H...S hydrogen bonds in both (I) and (II), and the N—H...Cl hydrogen bonds in (II), are the most prominent interactions, appearing as large red spots on the Hirshfeld surface maps. The N...H/H...N contacts in structure (I) are considerable, whereas for (II), they give a negligible contribution to the total interactions in the system.
Positional isomeric effect on the crystallization of chlorine-substitutedN-phenyl-2-phthalimidoethanesulfonamide derivatives
Authors: Köktaş Koca Serap , Sevinçek Resul , Akgül Özlem , Aygün Muhittin
Published: August 2015

Theortho-,para- andmeta-chloro-substitutedN-chlorophenyl-2-phthalimidoethanesulfonamide derivatives, C16H13ClN2O4S, have been structurally characterized by single-crystal X-ray crystallography.N-(2-Chlorophenyl)-2-phthalimidoethanesulfonamide, (I), has orthorhombic (P212121) symmetry,N-(4-chlorophenyl)-2-phthalimidoethanesulfonamide, (II), has triclinic (P\overline{1}) symmetry andN-(3-chlorophenyl)-2-phthalimidoethanesulfonamide, (III), has monoclinic (P21/c) symmetry. The molecules of (I)–(III) are regioisomers which have crystallized in different space groups as a result of the differing intra- and intermolecular hydrogen-bond interactions which are present in each structure. Compounds (I) and (II) are stabilized by N—H...O and C—H...O hydrogen bonds, while (III) is stabilized by N—H...O, C—H...O and C—H...Cl hydrogen-bond interactions. The structure of (II) also displays π–π stacking interactions between the isoindole and benzene rings. All three structures are of interest with respect to their biological activities and have been studied as part of a programme to develop anticonvulsant drugs for the treatment of epilepsy.