bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries
Authors: Gaetti-Jardim Elerson , Marcelino Silvia L. , Feitosa Alfredo C. R. , Romito Giuseppe A. , Avila-Campos Mario J.
Published: November 2009

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum–periodonticum–simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60–100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0 %, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum–periodonticum–simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.
Alterations to the structure of Leishmania major induced by N-arylisoquinolines correlate with compound accumulation and disposition
Authors: Ponte-Sucre Alicia , Gulder Tanja , Gulder Tobias A. M. , Vollmers Gerina , Bringmann Gerhard , Moll Heidrun
Published: November 2009

Naphthylisoquinoline alkaloids equipped with aN,C-hetero-‘biaryl’ axis, and, in particular, simplified synthetic analogues thereof, kill intracellularLeishmania majorat concentrations in the low submicromolar range, while being significantly less toxic to their major host cell, the macrophage, at the same concentrations. To further investigate their mechanism of action we evaluated the morphological and ultrastructural changes induced by specificN-arylisoquinolines inL. major, and the correlation of these changes with compound accumulation and disposition by the parasite. After 24 h of treatment with the synthetic arylisoquinolinium salts 3 or 4, dramatic structural changes and cell death were observed. Furthermore, the auto-fluorescent derivative salt 3 accumulates continually in intracellular compartments. Our results thus suggest that the leishmanicidal effect of arylisoquinolinium salts may involve their ability to accumulate and precipitate in intracellular organelles, form a huge vacuole and eventually promote cell lysis.
DNA sequence analysis of cagA 3′ motifs of Helicobacter pylori strains from patients with peptic ulcer diseases
Authors: Salih Barik A. , Bolek Bora Kazim , Arikan Soykan
Published: November 2009

The Helicobacter pylori cagA gene is a major virulence factor that plays an important role in gastric pathologies. DNA sequence data for the cagA 3′ region of Western isolates differ markedly in their EPIYA motifs from those of East Asian isolates. An increase in the number of these motifs is known to be associated with gastric cancer. Whether such an association is also the case for peptic ulceration was investigated in this study. Gastric biopsies were collected from 96 patients with duodenal ulcer (DU), gastric ulcer (GU) and gastritis. The types of EPIYA motif detected by PCR among 28 DU strains were 13 ABC, eight ABCC, six ABCCC, and in one patient both ABC and ABCCCCC; among nine GU strains were two ABC, five ABCC and two ABCCC; and among 40 gastritis strains were 35 ABC and five ABCC. DNA sequencing was carried out to confirm the detection of the EPIYA motif types and to analyse their peptide sequences. A significant association was found between the number of the EPIYA-C motifs (≥2) and peptic ulceration (P=0.00001) compared with gastritis. In conclusion, this study shows that our patients harboured cagA-positive H. pylori strains with EPIYA motifs of the Western type and that the increase in the number of EPIYA-C motifs was significantly associated with DU and GU but not with gastritis, indicating predictive association with the severity of the disease.
The changing aetiology of paediatric bacteraemia in England and Wales, 1998–2007
Authors: Henderson Katherine L. , Johnson Alan P. , Muller-Pebody Berit , Charlett André , Gilbert Ruth , Sharland Mike
Published: November 2009

Bacteraemia in children is a potentially life-threatening condition. The objective of this study was to determine trends in the aetiology of bacteraemia in children aged 1 month–15 years in England and Wales by collecting data voluntarily reported by National Health Service hospital microbiology laboratories. Over the 10-year period 1998–2007, a total of 51 788 bacteraemia cases involving 105 genera/species of bacteria were reported. Total annual reports of bacteraemia increased from 4125 to 6916, with a mean increase of 6.5 % per year (95 % CI: 1.3–12.1 %). In 2007, just over half the cases were accounted for by four groups of organisms: coagulase-negative staphylococci (28 %), Staphylococcus aureus (10 %), non-pyogenic streptococci (9 %) and Streptococcus pneumoniae (7 %). These organisms along with a further 13 species/genera accounted for 90 % of the cases. The commonest Gram-negative organisms were Neisseria meningitidis and Escherichia coli, which each accounted for 5 % of total bacteraemia reports in 2007. There was a significant decrease in reports of bacteraemia due to the three vaccine-preventable pathogens Haemophilus influenzae, N. meningitidis and Strep. pneumoniae, following the introduction of each vaccine programme or catch-up campaign. This study identified the commonest causes of bacteraemia in children in England and Wales, and highlighted the shifts in trends observed over time.
Role and clinical course of verotoxigenic Escherichia coli infections in childhood acute diarrhoea in Argentina
Authors: Rivero Mariana Alejandra , Passucci Juan Antonio , Rodriguez Edgardo Mario , Parma Alberto Ernesto
Published: November 2009

The aim of this study was to investigate the role and clinical course of verotoxigenic Escherichia coli (VTEC) infections in children with acute diarrhoea from Argentina, the country with the highest worldwide incidence of haemolytic uraemic syndrome (HUS). To accomplish this objective, 437 samples from children up to 6 years old with acute diarrhoea were collected and processed. More than 60 % of the children studied presented watery or mucous diarrhoea without blood, and in 25.2 % of the cases the samples contained blood. In a first screening, a multiplex PCR was performed to detect the presence of the vt
1, vt
2, eae, ehxA and saa virulence genes. The strains were then isolated and analysed to characterize their serotypes, virulence genes, antibiotic susceptibility profiles and verotoxin (VT) production. Forty-four of the 437 samples (10.1 %) were positive for VTEC virulence genes. VTEC-infected patients presented different types of diarrhoea (27.3 % belonged to the non-bloody type). Several serotypes and virulence genotypes were found. Isolates belonged to the serotypes O157 : H7, O145 : H−, O26 : H11, O121 : H19, O111 : H2 and O118 : H2. HUS developed in 16 (36.4 %) patients positive for VTEC virulence genes. All of the VTEC isolates produced a cytopathic effect on Vero cell monolayers, confirming the ability to express VT. Despite most strains being sensitive to all of the antimicrobials studied, a positive association between clinical progression to HUS and antibiotic therapy was observed for the total number of patients studied, as well as for the VTEC+ group. In conclusion, the data obtained in this study increase our knowledge of the role and clinical course of VTEC infection in childhood acute diarrhoea beyond bloody diarrhoea, and might be considered for the prevention, diagnosis and management of this disease. It is possible that the optimal approach for VTEC diagnosis could be using multiplex PCR to search for the presence of the vt
1, vt
2, eae and ehxA genes.
The pneumococcal response to oxidative stress includes a role for Rgg
Authors: Bortoni Magda E. , Terra Vanessa S. , Hinds Jason , Andrew Peter W. , Yesilkaya Hasan
Published: November 2009

Streptococcus pneumoniaeresides in the oxygen-rich environment of the upper respiratory tract, and therefore the ability to survive in the presence of oxygen is an important aspect of itsin vivosurvival. To investigate howS. pneumoniaeadapts to oxygen, we determined the global gene expression profile of the micro-organism in aerobiosis and anaerobiosis. It was found that exposure to aerobiosis elevated the expression of 54 genes, while the expression of 15 genes was downregulated. Notably there were significant changes in putative genome plasticity and hypothetical genes. In addition, increased expression ofrgg, a putative transcriptional regulator, was detected. To test the role of Rgg in the pneumococcal oxidative stress response, an isogenic mutant was constructed. It was found that the mutant was sensitive to oxygen and paraquat, but not to H2O2. In addition, the absence of Rgg strongly reduced the biofilm-forming ability of an unencapsulated pneumococcus. Virulence studies showed that the median survival time of mice infected intranasally with therggmutant was significantly longer than that of the wild-type-infected group, and the animals infected with the mutant developed septicaemia later than those infected intranasally with the wild-type.
Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates
Authors: Shu Hung-Yu , Fung Chang-Phone , Liu Yen-Ming , Wu Keh-Ming , Chen Ying-Tsong , Li Ling-Hui , Liu Tze-Tze , Kirby Ralph , Tsai Shih-Feng
Published: November 2009

Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5′ end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3′ end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.
Major impacts on the primary metabolism of the plant pathogen Cryphonectria parasitica by the virulence-attenuating virus CHV1-EP713
Authors: Dawe Angus L. , Van Voorhies Wayne A. , Lau Tannia A. , Ulanov Alexander V. , Li Zhong
Published: November 2009

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the familyHypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration ofC. parasiticacolonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both CO2production and O2consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profiﬁling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi.
RelA regulates virulence and intracellular survival of Francisella novicida
Authors: Dean R. E. , Ireland P. M. , Jordan J. E. , Titball R. W. , Oyston P. C. F.
Published: November 2009

Analysis of the genome ofFrancisella tularensishas revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of therelAgene inFrancisellaby generating a mutant inFrancisella novicida. Under amino acid starvation conditions, therelAmutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. TherelAmutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance toin vitrostress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis ofF. novicida.
Deletion and overexpression of the Aspergillus nidulans GATA factor AreB reveals unexpected pleiotropy
Authors: Wong Koon Ho , Hynes Michael J. , Todd Richard B. , Davis Meryl A.
Published: November 2009

TheAspergillus nidulanstranscription factor AreA is a key regulator of nitrogen metabolic gene expression. AreA contains a C-terminal GATA zinc finger DNA-binding domain and activates expression of genes necessary for nitrogen acquisition. Previous studies identified AreB as a potential negative regulator of nitrogen catabolism showing similarity withPenicillium chrysogenumNreB andNeurospora crassaASD4. TheareBgene encodes multiple products containing an N-terminal GATA zinc finger and a leucine zipper motif. We deleted theareBgene and now show that AreB negatively regulates AreA-dependent nitrogen catabolic gene expression under nitrogen-limiting or nitrogen-starvation conditions. AreB also acts pleiotropically, with functions in growth, conidial germination and asexual development, though not in sexual development. AreB overexpression results in severe growth inhibition, aberrant cell morphology and reduced AreA-dependent gene expression. Deletion of either the DNA-binding domain or the leucine zipper domain results in loss of both nitrogen and developmental phenotypes.
A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response
Authors: Weber Mary M. , French Christa L. , Barnes Mary B. , Siegele Deborah A. , McLean Robert J. C.
Published: November 2009

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm−2) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).
Expression of phosphofructokinase in Neisseria meningitidis
Authors: Baart Gino J. E. , Langenhof Marc , van de Waterbeemd Bas , Hamstra Hendrik-Jan , Zomer Bert , van der Pol Leo A. , Beuvery E. C. , Tramper Johannes , Martens Dirk E.
Published: November 2009

Neisseria meningitidisserogroup B is a pathogen that can infect diverse sites within the human host. According to theN. meningitidisgenomic information and experimental observations, glucose can be completely catabolized through the Entner–Doudoroff pathway and the pentose phosphate pathway. The Embden–Meyerhof–Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this,in silicoflux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineeredN. meningitidisstrain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with apfkA-deficient control strain, which was associated mainly with an increase in CO2production, whereas production of by-products was similar in the two strains. This might explain why thepfkAgene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression ofpfkAwere observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated.
The hrcA and hspR regulons of Campylobacter jejuni
Authors: Holmes Christopher W. , Penn Charles W. , Lund Peter A.
Published: November 2009

The human pathogenCampylobacter jejunihas a classic heat shock response, showing induction of chaperones and proteases plus several unidentified proteins in response to a small increase in growth temperature. The genome contains two homologues to known heat shock response regulators, HrcA and HspR. Previous work has shown that HspR controls several heat-shock genes, but thehrcAregulon has not been defined. We have constructed single and double deletions ofC. jejuni hrcAandhspRand analysed gene expression using microarrays. Only a small number of genes are controlled by these two regulators, and the two regulons overlap. Strains mutated inhspR, but not those mutated inhrcA, showed enhanced thermotolerance. Some genes previously identified as being downregulated in a strain lackinghspRshowed no change in expression in our experiments.
VEJφ, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes
Authors: Campos Javier , Martínez Eriel , Izquierdo Yovanny , Fando Rafael
Published: November 2009

A novel filamentous bacteriophage, designated VEJφ, was isolated from strain MO45 of Vibrio cholerae of the O139 serogroup. A molecular characterization of the phage was carried out, which included sequencing of its whole genome, study of the genomic structure, identification of the phage receptor, and determination of the function of some of the genes, such as those encoding the major capsid protein and the single-stranded DNA-binding protein. The genome nucleotide sequence of VEJφ, which consists of 6842 bp, revealed that it is organized in modules of functionally related genes in an array that is characteristic of the genus Inovirus (filamentous phages). VEJφ is closely related to other previously described filamentous phages of V. cholerae, including VGJφ, VSK and fs1. Like these phages, VEJφ uses as a cellular receptor the type IV fimbria called the mannose-sensitive haemagglutinin (MSHA). It was also demonstrated that VEJφ, like phage VGJφ, is able to transmit the genome of phage CTXφ, and therefore the genes encoding the cholera toxin (CT), horizontally among populations of V. cholerae expressing the MSHA receptor fimbria. This suggests that the variety of phages implicated in the horizontal transmission of the CT genes could be more diverse than formerly thought.
Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice
Authors: Gong Hao , Vu Gia-Phong , Bai Yong , Yang Edward , Liu Fenyong , Lu Sangwei
Published: November 2009

The type III secretion system (T3SS) encoded bySalmonellapathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development ofSalmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studiedin vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinantSalmonellastrains that contain a FLAG epitope inserted in-frame to genesinvJ,prgJ,sipC,sipD,sopAandsopB, and investigated the expression of the tagged proteins bothin vitroandin vivoduring murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the taggedSalmonellastrains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed inSalmonellacolonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially inSalmonellacolonizing the ileum but not the liver, while SipC is expressed preferentially inSalmonellacolonizing the liver but not the ileum. Thus,Salmonellaappears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.
AI-2 quorum-sensing inhibitors affect the starvation response and reduce virulence in several Vibrio species, most likely by interfering with LuxPQ
Authors: Brackman Gilles , Celen Shari , Baruah Kartik , Bossier Peter , Van Calenbergh Serge , Nelis Hans J. , Coenye Tom
Published: November 2009

The increase of disease outbreaks caused by Vibrio species in aquatic organisms as well as in humans, together with the emergence of antibiotic resistance in Vibrio species, has led to a growing interest in alternative disease control measures. Quorum sensing (QS) is a mechanism for regulating microbial gene expression in a cell density-dependent way. While there is good evidence for the involvement of auto-inducer 2 (AI-2)-based interspecies QS in the control of virulence in multiple Vibrio species, only few inhibitors of this system are known. From the screening of a small panel of nucleoside analogues for their ability to disturb AI-2-based QS, an adenosine derivative with a p-methoxyphenylpropionamide moiety at C-3′ emerged as a promising hit. Its mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of Vibrio harveyi AI-2 QS mutants. Our results indicate that this compound, as well as a truncated analogue lacking the adenine base, block AI-2-based QS without interfering with bacterial growth. The active compounds affected neither the bioluminescence system as such nor the production of AI-2, but most likely interfered with the signal transduction pathway at the level of LuxPQ in V. harveyi. The most active nucleoside analogue (designated LMC-21) was found to reduce the Vibrio species starvation response, to affect biofilm formation in Vibrio anguillarum, Vibrio vulnificus and Vibrio cholerae, to reduce pigment and protease production in V. anguillarum, and to protect gnotobiotic Artemia from V. harveyi-induced mortality.
SigC sigma factor is involved in acclimation to low inorganic carbon at high temperature in Synechocystis sp. PCC 6803
Authors: Gunnelius Liisa , Tuominen Ilona , Rantamäki Susanne , Pollari Maija , Ruotsalainen Virpi , Tyystjärvi Esa , Tyystjärvi Taina
Published: November 2009

Inactivation of thesigCgene (sll0184), encoding the group 2 sigma factor SigC, leads to a heat-sensitive phenotype ofSynechocystissp. PCC 6803. Cells of the ΔsigC strain grew poorly at 43 °C at pH 7.5 under ambient CO2conditions. Addition of inorganic carbon in the form of 3 % CO2or use of an alkaline growth medium (pH 8.3) restored the growth of the ΔsigC strain at 43 °C. These treatments compensate for the low concentration of inorganic carbon at high temperature. However, addition of organic carbon as glucose, pyruvate, succinate or 2-oxoglutarate did not restore growth of the ΔsigC strain at 43 °C. In the control strain, the amount of the SigC factor diminished after prolonged incubation at 43 °C if the pH of the growth medium was 7.5 or 6.7. Under alkaline conditions, the amount of the SigC factor remained constant at 43 °C and cells of the control strain grew better than at pH 7.5 or pH 6.7. The pH dependence of high-temperature growth was associated with changes in photosynthetic activity, indicating that the SigC factor is involved in adjustment of photosynthesis according to the amount of available inorganic carbon. Our results indicate that acclimation to low inorganic carbon is a part of acclimation to prolonged high temperature and that the SigC factor has a central role in this acclimation.
Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum
Authors: Janus Danielle , Hoff Birgit , Kück Ulrich
Published: November 2009

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrialβ-lactam antibiotic producerPenicillium chrysogenumusing theDsRedreporter gene under the control of the constitutivetrpCpromoter or the induciblexylPpromoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenousPcbrlAmorphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi inP. chrysogenumis dependent on a Dicer peptide was provided with a strain lackingPcdcl2. In the ΔPcdcl2 background, silencing of thePcbrlAgene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system inP. chrysogenumwas finally demonstrated by silencing thePcku70gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.
DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium
Authors: Lin Dongxia , Kim Byoungkwan , Slauch James M.
Published: November 2009

Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria.Salmonella entericaserovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of theSalmonellapathogenicity island 1 (SPI1) type three secretion system genes and activation of the RcsCDB system, as well as ASST activity. In contrast to DsbA/DsbB, however, the DsbL/DsbI system cannot catalyse the disulfide bond formation required for flagellar assembly. Phylogenic analysis suggests that theassT dsbL dsbIgenes are ancestral in theEnterobacteriaceae, but have been lost in many lineages. Deletion ofassTconfers no virulence defect during acuteSalmonellainfection of mice.
Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria
Authors: Barth Kenneth R. , Isabella Vincent M. , Clark Virginia L.
Published: November 2009

SinceNeisseria gonorrhoeaeandNeisseria meningitidisare obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensalNeisseriagenome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. OnlyNeisseria mucosacontained a nitrate reductase (Nar), and onlyNeisseria lactamica,Neisseria cinerea,Neisseria subflava,Neisseria flavescensandNeisseria siccacontained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested exceptN. mucosahad a two- to fourfold lower nitrite reductase activity thanN. gonorrhoeae, whileN. meningitidisand most of the commensalNeisseriaspecies had a two- to fourfold higher nitric oxide (NO) reductase activity. ForN. meningitidisand most of the commensalNeisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared withN. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that ofN. gonorrhoeae. The greatest difference between theNeisseriaspecies was the lack of a functional Nos system in the pathogenic speciesN. gonorrhoeaeandN. meningitidis.
Genetic determinants of Pseudomonas aeruginosa biofilm establishment
Authors: Müsken Mathias , Di Fiore Stefano , Dötsch Andreas , Fischer Rainer , Häussler Susanne
Published: November 2009

The establishment of bacterial biofilms on surfaces is a complex process that requires various factors for each consecutive developmental step. Here we report the screening of the comprehensive Harvard Pseudomonas aeruginosa PA14 mutant library for mutants exhibiting an altered biofilm phenotype. We analysed the capability of all mutants to form biofilms at the bottom of a 96-well plate by the use of an automated confocal laser-scanning microscope and found 394 and 285 genetic determinants of reduced and enhanced biofilm production, respectively. Overall, 67 % of the identified mutants were affected within genes encoding hypothetical proteins, indicating that novel developmental pathways are likely to be dissected in the future. Nevertheless, a common theme that emerged from the analysis of the genes with a predicted function is that the establishment of a biofilm requires regulatory components that are involved in survival under microaerophilic growth conditions, arginine metabolism, alkyl-quinolone signalling, pH homeostasis and the DNA repair system.
SMc01553 is the sixth acyl carrier protein in Sinorhizobium meliloti 1021
Authors: Dávila-Martínez Yadira , Ramos-Vega Ana Laura , Contreras-Martínez Sandra , Encarnación Sergio , Geiger Otto , López-Lara Isabel M.
Published: November 2009

Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. InSinorhizobium meliloti1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence ofS. meliloti1021 also reveals the ORF SMc01553, annotated as a putative ACP.smc01553is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed inEscherichia coliwas labelledin vivowith [3H]β-alanine, a biosynthetic building block of the 4′-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4′-phosphopantetheine in the presence ofS. melilotiholo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase fromS. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures ofS. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle.
First report of a tetracycline-inducible gene expression system for mollicutes
Authors: Breton Marc , Sagné Evelyne , Duret Sybille , Béven Laure , Citti Christine , Renaudin Joël
Published: November 2009

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2
from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2
tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2
promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2
system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.
Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation
Authors: Mukaiyama Hiroyuki , Kajiwara Shiro , Hosomi Akira , Giga-Hama Yuko , Tanaka Naotaka , Nakamura Taro , Takegawa Kaoru
Published: November 2009

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome–vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeastSchizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in theatg1Δ andatg12Δ mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.
Fungal physiology – a future perspective
Authors: Wilson Richard A. , Talbot Nicholas J.
Published: November 2009

The study of fungal physiology is set to change dramatically in the next few years as highly scalable technologies are deployed allowing accurate measurement and identification of metabolites, proteins and transcripts within cells. The advent of next-generation DNA-sequencing technologies will also provide genome sequence information from large numbers of industrially relevant and pathogenic fungal species, and allow comparative genome analysis between strains and populations of fungi. When coupled with advances in gene functional analysis, protein–protein interaction studies, live cell imaging and mathematical modelling, this promises a step-change in our understanding of how fungal cells operate as integrated dynamic living systems.
Hibiscus chlorotic ringspot virus upregulates plant sulfite oxidase transcripts and increases sulfate levels in kenaf (Hibiscus cannabinus L.)
Authors: Zhang Xin , Wong Sek-Man
Published: November 2009

Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is required for encapsidation and virus systemic movement. To better understand the roles of HCRSV CP in virus infection and its interactions with host proteins, a cDNA library of kenaf (Hibiscus cannabinus L.) was constructed and screened by using a yeast two-hybrid system (YTHS) to identify CP-interacting proteins. One protein identified was sulfite oxidase (SO) and the interaction was confirmed in vitro and in vivo. The interaction was found to be associated with peroxisomes by immunofluorescent labelling of peroxisomes by an anti-SKL signal peptide antibody. Our YTHS results showed that only the P and S domains of CP interacted with SO from kenaf. This is probably due to the exposure of these two domains on the outer surface of the capsid. Peroxisomes were observed to aggregate in HCRSV-infected cells, and biochemical assays of total protein from kenaf leaf extracts showed that SO activity and SO-dependent H2O2-generating activity in the HCRSV-infected leaves increased compared with that in mock-inoculated kenaf plants.
Spontaneous tumour development in human papillomavirus type 8 E6 transgenic mice and rapid induction by UV-light exposure and wounding
Authors: Marcuzzi Gian Paolo , Hufbauer Martin , Kasper Hans Udo , Weißenborn Sönke Jan , Smola Sigrun , Pfister Herbert
Published: November 2009

Cutaneous human papillomavirus type 8 (HPV8) is carcinogenic in patients with epidermodysplasia verruciformis. Transgenic mice with the complete early region (CER) of HPV8 spontaneously developed papillomas, dysplasia and squamous cell carcinomas of the skin. To characterize the role of individual early genes in carcinogenesis, the E6 and E6/E7 genes were expressed separately in transgenic mice. Nearly all HPV8-E6-positive mice spontaneously developed multifocal tumours, characterized by papillomatosis, hyperkeratosis and varying degrees of epidermal dysplasia. In 6 % of the cases, the tumours became malignant, comparable with HPV8-CER mice. Thus, in the murine epidermis, E6 is the major oncogene necessary and sufficient to induce spontaneous tumour development up to the level of squamous cell carcinoma. To evaluate the synergistic effects of UV light and wound healing, the skin of HPV8 mice was irradiated with UVA/UVB light or wounded with punch biopsies. These treatments induced papillomatosis in HPV8-CER and -E6 mice within 3 weeks. Irradiation with UVA alone did not induce papillomatosis and UVB alone had a weaker effect than UVA/UVB, indicating a synergistic role of UVA in UVB-induced papillomatosis. An HPV8 infection persisting over decades in interaction with sun burns and wound healing processes may be a relevant cause of skin cancer in humans.
The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants
Authors: Sunpapao Anurag , Nakai Takashi , Dong Fang , Mochizuki Tomofumi , Ohki Satoshi T.
Published: November 2009

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Δ2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Δ2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Δ2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Δ2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Δ2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Δ2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Δ2b, whereas accumulation of Δ2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.
Major tegument protein VP8 of bovine herpesvirus 1 is phosphorylated by viral US3 and cellular CK2 protein kinases
Authors: Labiuk Shaunivan L. , Babiuk Lorne A. , van Drunen Littel-van den Hurk Sylvia
Published: November 2009

The UL47 gene product, VP8, is one of the major tegument proteins of bovine herpesvirus 1 (BoHV-1) and is subject to phosphorylation. Analysis of protein bands co-immunoprecipitated with VP8 from BoHV-1-infected cells by mass spectroscopy suggested that VP8 interacts with two protein kinases: cellular CK2 and viral US3. CK2 is a highly conserved cellular protein, expressed ubiquitously and known to phosphorylate numerous proteins. The US3 gene product is one of the viral kinases produced by BoHV-1 during infection. Interactions of CK2 and US3 with VP8 were confirmed outside the context of infection when FLAG–VP8 was expressed alone or co-expressed with US3–haemagglutinin tag in Cos-7 cells. Furthermore, VP8 and US3 were found to co-localize in the nucleus during viral infection. To explore the significance of these interactions, anin vitrokinase assay was performed, which demonstrated that VP8 is heavily phosphorylated by CK2. In the presence of the highly specific CK2 kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), phosphorylation of VP8 was significantly reduced. Phosphorylation of VP8 was also inhibited by the presence of kenpaullone, a less specific CK2 inhibitor, but not by protein kinase CK1 or protein kinase C inhibitors. When VP8 and US3 were both included in the kinase assay in the presence of DMAT, phosphorylation of VP8 was again observed. Autophosphorylation of US3 was also detected and was not inhibited by DMAT. Based on these results, it is proposed that VP8 interacts with cellular CK2 and viral US3 in BoHV-1-infected cells, and is in turn subject to kinase activities associated with both of these proteins.