Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> J23 of grape must origin

Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1–100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.     

Fusarium fractiflexum sp. nov. and two other species within theGibberella fujikuroi species complex recently discovered in Japan that form aerial conidia in false heads

Morphological and molecular phylogenetic analyses were conducted on 12 strains ofFusarium, deposited in MAFF asF. subglutinans (≡F. moniliforme var.subglutinans≡F. sacchari var.subglutinans) orFusarium sp. because they formed aerial conidia in false heads in the dark. These strains were resolved as three distinct species within theGibberella fujikuroi species complex. A new species,F. fractiflexum, and two species new to Japan,F. circinatum andF. concentricum, are described and illustrated and their morphological features are discussed.Fusarium fractiflexum, isolated from diseased yellow leaf spots ofCymbidium spp., is differentiated from other fusaria based on its yellowish colonies and aerial conidia formed in false heads in the dark and in zigzag-like conidial chains under black light. Japanese strains ofF. circinatum also formed elongate, coiled sterile hyphae. Phialidic aerial conidia with a pointed apex and a wedgeshaped base were found inF. concentricum cultured under black light and represent a new diagnostic character of the species, in addition to colonies with alternating concentric rings when cultured on PDA. Based on DNA sequences of the β-tubulin gene and two other loci, strains ofF. fractiflexum were resolved phylogenetically as members of the Asian clade of theG. fujikuroi species complex. In addition, Japanese strains ofF. circinatum andF. concentricum were phylogenetically identical to the ex-type strains.     

Sexual forms of yeasts in clinical samples

The sexual or teleomorphic state of yeasts has only been described in a few clinically involved species, mainly those of the Saccharomycetaceae family. With the aim of gathering information on their incidence in human pathology, a study has been made of a total of 2,135 strains isolated from clinical samples and cultivated in McClary agar. From these, 8 strains in teleomorphic state were identified: Kluyveromyces marxianus [1], Pichia anomala [2], Pichia farinosa [1], Pichia membranaefaciens [1] and Saccharomyces cerevisiae [3]. The two strains of P. anomala were responsible for fungemia; K. marxianus and the two strains of S. cerevisiae produced vaginitis; the other strains were oral cavity colonizers.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Dioxygenase activity and relative behaviour of Pseudomonas strains from soil in the presence of different aromatic compounds

Ten different Pseudomonas strains isolated from contaminated soils were tested for expression of active dioxygenases. Of these, two different clusters, related to strain origin were observed. The first included two P. fluorescens strains and two P. aeruginosa strains isolated from soils polluted with polyaromatic hydrocarbons and the second two P. cepacia strains and four P. chlororaphis strains from soils with polyphenols. All the isolates showed catechol 1,2-dioxygenase basal activity, while other dioxygenases (catechol 2,3-dioxygenase, protocatechuate 2,3-, 3,4- and 4,5-dioxygenases) were detected only after growth in the presence of suitable inducers (benzoate, catechol, salicylate, phenol). Significant induction of catechol 1,2-dioxygenase, the major activity of the tested strains, was also observed when combining starvation with the presence of high molecular weight aromatic hydrocarbons with recalcitrant structures (fluoranthene, chrysene, benzanthracene, pyrene).     

Identification of Bacillus azotofixans using API tests

The identification of Bacillus azotofixans strains using API tests is described. Twenty-two strains were studied according to their fermentation pattern on 49 different carbohydrates. A profile of the B. azotofixans type strain is presented, together with an average profile of all strains tested. The fermentation pattern for B. azotofixans is also compared to those of the closely similar species B. polymyxa and B. macerans. These profiles may be useful for the identification of new strains.     

Partial characterization and dynamics of synthesis of high molecular mass exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

Exopolysaccharide (EPS) preparations from Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) strains LBB.B26 and LBB.B332 and Streptococcus thermophilus strains LBB.T54 and LBB.T6V were characterized using ion-exchange chromatography and gel filtration. All four preparations contained a neutral EPS with molecular mass in the range of 1.3−1.6 × 10⁶ Da (HMM-EPS). The EPS preparations from the two L. bulgaricus strains also contained an acidic low molecular mass EPS fraction (LMM-EPS) comprising from 10% to 34% of the total EPS yield. HMM-EPS preparations were subjected to High Pressure Liquid Chromatography (HPLC) analysis of monomer sugars after complete hydrolysis. Glucose, galactose and/or rhamnose in different ratios proved to be the principal sugars building the HMM-EPS from all four strains. The chemical composition of HMM-EPS was strictly strain-specific. The LMM-EPS contained galactose. The viscosifying properties of the four different HMM-EPS varied greatly with intrinsic viscosity in the range from 0.26 (strain B26) to 2.38 (strain T6V). For 24 h the two L. bulgaricus strains accumulated more HMM-EPS in milk (>70 mg l⁻¹) than S. thermophilus strains T54 and T6V (<30 mg l⁻¹), but maximal yields were reached earlier with cocci (8 h) than with rods (16–24 h). The contribution of HMM-EPS production to increased viscosity of fermented milk was demonstrated for all of the tested strains grown as monocultures or as mixed yogurt starters compared to non-EPS producing S. thermophilus LBB.A and poor EPS-producer L. bulgaricus LBB.B5. The extent of increased viscosity was strongly dependent on the nature of the produced HMM-EPS, rather than simply on polymer yield.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol

In this study, the M13 primer was used to distinguish Geotrichum candidum from the anamorphic and teleomorphic forms of other arthrospore-forming species (discriminatory power = 0.99). For intraspecific characterization, the GATA4 primer showed the highest level of discrimination for G. candidum among the 20 microsatellite primers tested. A molecular typing protocol (DNA concentration, hybridization temperature and type of PCR machine) was optimized through a series of intra- and interlaboratory trials. This protocol was validated using 75 strains of G. candidum, one strain of G. capitatum and one strain of G. fragrans, and exhibited a discrimination score of 0.87. This method could therefore be used in the agro-food industries to identify and to evaluate biodiversity and trace strains of G. candidum. The results show that the GATA4 primer might be used to differentiate strains according to their ecological niche.     

我国沿海拟菱形藻属的2新记录种及其产毒特征分析

为澄清我国沿海拟菱形藻属(Pseudo-nitzschia)的物种多样性,并确认中国海域拟菱形藻属是否具有产生多莫酸(Domoicacid)的能力,采用毛细管显微操作技术从我国沿海水体中分离、纯化拟菱形藻细胞,建立了单克隆培养株系,并基于核糖体转录间隔区ITS1-5.8S-ITS2 序列构建了分子系统树。结果表明,结合在光学显微镜和透射电镜下观察的形态学特征和分子系统发育分析数据,鉴定到我国拟菱形藻属的2新记录种:银河拟菱形藻(P. galaxiae Lundholm & Moestrup)和微孔拟菱形藻(P. micropora Priisholm, Moestrup & Lundholm),对其形态学特征进行了详细描述,并与相似种类进行了比较研究。利用高效液相色谱(HPLC)技术对多莫酸特征进行了检测,结果表明培养株系并不产生多莫酸。这些为我国拟菱形藻属物种多样性和产毒特征研究提供了基础数据。     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.     

Simple sequence repeat (SSR) markers in the ectomycorrhizal fungus Laccaria bicolor for environmental monitoring of introduced strains and molecular ecology applications

Simple sequence repeat (SSR) markers were developed from SSR-enriched genome libraries for the ectomycorrhizal basidiomycete Laccaria bicolor. Seven markers were single-locus and amplified unambiguously in L. bicolor. The seven SSR markers were further characterized using an array of 15 L. bicolor strains representative of diverse origins worldwide. The observed number of alleles per locus varied from 5–9 and the values of observed heterozygosity from 0.167 to 0.667. The seven SSR loci could be amplified from DNA extracted from root tips of L. bicolor inoculated pine seedlings. All the L. bicolor ectomycorrhizas analysed exhibited the same SSR multi-locus profile as that detected for the UAMH8232 inoculant strain. The set of markers described represents a potent tool for the monitoring of introduced strains of L. bicolor and for molecular ecology applications.     

β-Lactamase activity and resistance to penicillins in Myxococcus xanthus

Several mutants and other variants of Myxococcus xanthus HP100 were obtained with differences in their sensitivity to carbenicillin and other penicillin derivatives. The specific activities of -lactamase in different resistant organisms varied from strain to strain but were consistently higher than in HP100. The relative molecular mass (M _r ) of the enzyme in M. xanthus HP100 was found to be 22,300. In certain carbenicillin resistant strains a second fraction of -lactamase activity of molecular weight 186,000 presumed to be an octamer of the other form was present. The enzyme was found in cell free extracts and also in culture supernatants of all carbenicillin resistant mutants but not in culture supernatants of strain HP100. In all the carbenicillin resistant mutants a part of the intracellular enzyme activity was released by osmotic shock and this activity may be periplasmic. The forms of the enzyme present in the culture supernatants and released by osmotic shock were monomeric. Carbenicillin resistance was not transferable between strains by conjugation. One resistance allele inhibited the transfer of the R factor Sa between myxococci.Non-standard abbreviations C^S C^R sensitivity and resistance to carbenicillin - C _u ^R C _S ^R unstable and stable resistance to carbenicillin     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

块菌(松露)宿主可培养内生菌群落结构与多样性研究

探究四川凉山彝族自治州块菌主产区华山松内生菌群的结构及多样性。在会东县选取4个点(新田乡、新云乡、淌塘镇、雪山乡)的块菌宿主华山松的根、茎、叶为实验材料,通过不同培养基分离样品根、茎、叶的内生细菌、真菌、放线菌,DNA分子鉴定分离菌株的种属,最终分离得到细菌46株,其中欧文氏菌属(Erwinia)5株,沙门氏菌属(Salmonell)1株,杆菌属(Bacillus)22株,葡萄球菌属(Staphylococcus)2株,假单胞菌属(Pseudomonas)2株,类芽胞杆菌属(Paenibacillus)2株,布丘氏菌属(Buttiauxella)2株,肠杆菌属(Enterobacte)3株,爱文氏菌属(Ewingella)1株,泛菌属(Rahnella)1株,拉恩氏菌属(Rahnella Izard)2株,其他属3株;真菌19株,均为子囊菌门(Ascomycota),其中青霉属(Penicillium)4株,疱霉属(Phoma)1株,子囊菌门未知菌1株,篮状菌属(Cladosporium)1株,分枝孢子菌属(Sydowia)1株,曲霉属(Aspergillus)1株,其他属10株;放线菌33株,为链霉菌属(Streptomyces)22株,短小杆菌属(Curtobacterium)2株,短杆菌属(Brevibacterium)1株,其他属8株。研究结果表明,来自不同地点、不同植株部位、不同培养基分离得到的块菌宿主华山松内生菌有差异,证实微生物在土壤、块菌、宿主植物之间有着复杂的相互作用,为块菌的人工栽培提供参考。     

Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

Thirty-five strains of the entomopathogenic bacterium Bacillus thuringiensisactive on Spodoptera exigua, were characterized by means of serological identification and determination of crygene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exiguaand tested against two other species of the same genus: S. littoralisand S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstakiwere the most frequent within S. exigua-active strains and that serovar aizawaihad the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in crygenes as determined by PCR suggests a non random distribution of some crygenes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodopteraspecies, were very common within S. exigua-active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralisor S. frugiperda; and some strains lacking all of the Spodoptera-active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodopteraspecies. Since strains sharing the same crygenes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensisstrains.     

Biological species and morphological characteristics ofArmillaria mellea complex in Hokkaido:A. sinapina and two new species,A. jezoensis andA. singula

Three intersterility groups ofArmillaria mellea sensu lato were discovered by examining all pairwise combinations of monosporous isolates of basidiomes collected in Hokkaido. One of them, group IV, was identified asA. sinapina by mating it with tester strains. Two new species, groups III and V, were namedA. jezoensis andA. singula, respectively. Their morphological forms and the ecology of their basidiomes are described.