Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S

<p>Abstract</p> <p>Background</p> <p>Information regarding the Alkali-Tolerance Response (AlTR) in <it>Listeria monocytogenes </it>is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively.</p> <p>Results</p> <p>In this study, <it>L. monocytogenes </it>was shown to exhibit a significant AlTR following a 1-h exposure to mild alkali (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The observed variability between results of cDNA arrays and 2D gel electrophoresis may be accounted for by posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses.</p> <p>Conclusion</p> <p>Alkali pH provides therefore <it>L. monocytogenes </it>with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where alkali conditions prevail. This study showed strong evidence that the AlTR in <it>L. monocytogenes </it>functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources.</p

Hepatoprotective activity of Cayratia trifolia (L.) domin against nitrobenzene induced hepatotoxicity

The hepatoprotective activity of ethanolic extract of the whole plant of Cayratia trifolia (L.) Domin was evaluated against nitrobenzene (NB) (50 mg/Kg bw, orally) induced hepatic damage in rats. The extract at the dose of 200 mg/kg body weight were administered orally once every day for 7 successive days. The treatment restores the elevated levels of the liver marker enzymes such as alanine amino transferase, aspartate amino transferase, alkaline phosphatase in serum and lipid peroxidation in tissue homogenate. Whereas the antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase remains decreased in carcinogen induced group and after treatment the levels of antioxidant enzymes increased. Histopathological analysis of the NB induced animals showed severe necrosis and fatty infiltration in liver. After treatment with C. trifolia the rats showed regeneration of hepatocytes. The results thus support the use of C. trifolia as a hepatoprotective agent.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Collection and Conservation of Leguminous Crops and Their Wild Relatives in Tamil Nadu, India, 2008

Based on the Memorandum of Understanding between the National Institute of Agrobiological Sciences, Japan and the Tamil Nadu Agricultural University, India, a field survey was conducted in Tamil Nadu State, India from March 1 to 16, 2008. As a result, 32 accessions of leguminous plants consist of the genus Vigna, Lablab, Phaseolus and Macrotyloma were recorded and seed samples of 29 accessions consisting of 17 cultivated and 12 wild accessions were collected. All the seed materials collected were deposited at Tamil Nadu Agricultural University, India.本報告は，独立行政法人農業生物資源研究所ジーンバンクとインド，タミルナドゥ農業大学の間で2007年4月に締結した協同研究協定（MOU）に基づいて行われたインド，タミルナドゥ州における植物遺伝資源の調査報告である．調査は，2008年3月1日～16日にかけて行った．調査の結果，在来作物の栽培は著しく減少しつつあることが明らかになったが，乾燥が厳しい地域で現地農民がモスビーンの野生型（匍匐性で茎が長く伸び，葉の切れ込みが大きく耐乾性が強いという）といっている系統の栽培や，やや栽培化が進んだ状態（種子がやや大型化し，裂莢性が低下しているという）と思われるVigna stipulacea の多目的栽培（食用種子生産，飼料生産，被覆作物）など，興味深い特性を持つと考えられる遺伝資源も残存していた．Vigna（ササゲ）属野生植物遺伝資源に関しても，高い多様性の収集が期待できる地域である．また，Lablab（フジマメ）属の野生種も豊富であることが今回の調査で明らかになった．菜食主義者が多いインドにおいては，マメ科作物はタンパク供給源として重要な位置を占めており，なかでもVigna属作物（ケツルアズキとリョクトウ）の安定的な生産と品種改良等による収量増に関する研究には高い優先順位をつけて研究を推進している．協同研究の相手であるタミルナドゥ農業大学においても，ケツルアズキとリョクトウの育種，栽培，分子遺伝学的解析などの課題に多くの研究者が取り組んでいた．今後の共同研究による大きな成果が期待できる

Collection and Conservation of Leguminous Crops and Their Wild Relatives in Tamil Nadu, India, 2009

Based on the Memorandum of Understanding between the National Institute of Agrobiological Sciences, Japan and Tamil Nadu Agricultural University, India, a field survey was conducted in Tamil Nadu State, India from 29^ January 10^ February, 2009. As a result, 134 accessions of leguminous plants consist of the genus Cajanus , Lablab , Macrotyloma and Vigna , were recorded and seed samples consisting of 99 cultivated and 35 wild accessions were collected. All the seed materials collected were deposited at Tamil Nadu Agricultural University, India. Genetic erosion of traditional pulse landraces is rapidly proceeding in Tamil Nadu mainly due to an increase in the area of cash crops.本報告は, 独立行政法人農業生物資源研究所ジーンバンクとインド・タミルナドゥ農業大学の間で2007年4月に締結した協同研究協定（MOU）に基づいて行われたインド・タミルナドゥ州における二回目のマメ科植物遺伝資源の調査報告である．調査は，2009年1月29日～2月10日にかけて行った．調査の結果，灌漑水が利用可能になった地域において在来作物の栽培は著しく減少していることが明らかになった．タミルナドゥ州では, 政府の援助により，井戸による灌漑施設が急速に普及してきており，トウモロコシやキャッサバなどの商品作物栽培も急速に広がってきていることから, 在来作物の消失が懸念される．乾燥が厳しい天水農業地域では，ソルガムとマメ科在来作物（リョクトウ，ササゲ，モスビーン，フジマメ，キマメ等）の混作が残っていた．ホースグラムも同様の環境で栽培されていたが，単作されている例が多かった．このような環境においては，耐乾性に優れた野生種V. trilobataとV.radiata var. sublobataの自生も見られた．V. trilobataに関しては，今回の調査で，その栽培と人による食用としての利用が確認された他，ある程度栽培化が進んだと思われるような系統も収集された．また，これまでその存在が不明であったモスビーン（V. aconitifolia）の野生種と思われる3系統も，同様の乾燥環境下で発見された．これら3系統の分類学的取り扱いについては，今後詳細に検討する必要がある．これら乾燥地のマメ科植物とは対照的に，これまでV. trilobat と混同されてきたV.stipulaceaは，水田等の湿った粘土質土壌の生息地で生育していた．作物の中では，ケツルアズキ（V. mungo）が水田の畦で栽培される例が多かったことから，加湿な環境に適応している栽培種であると考えられた．菜食主義者が多いインドにおいては，マメ科作物はタンパク供給源として重要な位置を占めており，なかでもVigna属作物（ケツルアズキとリョクトウ）の安定的な生産と品種改良等による収量増は緊急の課題となっている．近年，インドにいおいては，リョクトウの生産量が不足しており，中国等からの輸入に依存していることから，その取引価格は高騰しているため，農民の生産意欲は高い．今後の共同研究による大きな成果が期待できる

Rapid Microbiological Testing: Monitoring the Development of Bacterial Stress

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H2O2. The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery

Collection and Conservation of Wild Leguminous Crop Relatives in Hokkaido, Japan, 2008

Based on the Memorandum of Understanding between the National Institute of Agrobiological Sciences, Japan and the Tamil Nadu Agricultural University, India, a field survey was conducted in southwestern part of Hokkaido island, Japan from 29^ September to 3^ October, 2008. As a result, 31 accessions of leguminous plants consist of the genus Amphicarpaea , Glycine and Lotus were recorded and seed samples were collected. All the seed materials collected were deposited at NIAS genebank, Japan.本報告は，独立行政法人農業生物資源研究所ジーンバンクとインド，タミルナドゥ農業大学の間で2007年4月に締結した協同研究協定（MOU）に基づいて招聘したM. Pandiyan博士をメンバーに加えて行った北海道南西部におけるマメ科植物遺伝資源の調査報告である．調査は，2008年9月29日～10月3日にかけて行った．調査の結果， 野生ダイズであるツルマメ（Glycinesoja）12点， かつてアイヌ民族による利用がみられたヤブマメ（Amphicarpaea bracteata）15点，ミヤコグサ属植物4点，合計31点の遺伝資源を収集保存した．これらの遺伝資源は，2009年度につくば市の農業生物資源研究所において栽培し，特性評価，種子増殖を行い配布可能なアクティブコレクションとして生物研ジーンバンクにおいて保存する予定である

Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus

Background: Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus. Methods: Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates. Results: On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol. Conclusion: Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus

Staphylococcal phenotypes induced by naturally occurring and synthetic membrane-interactive polyphenolic β-lactam resistance modifiers.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability