Williopsis saturnusand Williopsis beijerinckiiAre Recognized as Distinct Taxa by Means of the Polymerase Chain Reaction with Nonspecific Primers

Fifteen strains of the yeast Williopsissensu stricto were analyzed by means of UP-PCR. With the N21 universal primer, this approach showed that the strains could be clearly divided into two groups corresponding to the species W. saturnus(Klöcker) Zender and W. beijerinckii(van der Walt) Naumov et Vustin. The results obtained are in good agreement with data of genetic and isoenzyme analyses and provide no support for the conspecificity of W. saturnusand W. beijerinckiicommonly accepted in modern yeast taxonomic manuals.     

Identification of Zygowilliopsis californicaStrains of Different Origin by Means of Polymerase Chain Reaction with Universal Primers

After reevaluation of the taxonomic position of 27 yeast collection strains of different origin by UP-PCR followed by dot-hybridization, only 22 strains were assigned to the biological species Zygowilliopsis californica(Lodder) Kudriavzev. Four strains were identified as Williopsis suaveolens(Klöcker) Naumov et al. Universal primers L45 and N21 are recommended for identification of the Z. californicayeasts.     

Microbial production of (S)-1-phenyl-1,3-propanediol by stereospecific reduction of 3-hydroxy-1-phenylpropane-1-one

A novel microbial method of synthesizing (S)-1-phenyl-1,3-propanediol [(S)-PPD] was developed in this study. Our laboratory stock cultures were screened for microorganisms that stereospecifically produced (S)-PPD from 3-hydroxy-1-phenylpropane-1-one (HPPO) using an intact cell system. Of the 828 strains examined (321 bacteria, 233 yeasts and 274 molds), certain strains of Williopsis saturnus var. mrakii and Cryptococcus albidus were found to produce (S)-PPD with over 99% enantiomeric excess (e.e.). Screening identified W. saturnus var. mrakii AJ-5620 as the most productive strain, and this strain was used for further experiments. The (S)-PPD-producing reaction using intact W. saturnus var. mrakii AJ-5620 cells was carried out by successive feeding of HPPO. A total (S)-PPD yield of 9.9 g/l was produced in 20 h. The molar yield was 81% and the optical purity of the (S)-PPD produced was over 99% e.e.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Structure and allele-specific expression variation of novel α/β hydrolase fold proteins in gentian plants

Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to α/β hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB281493 and AB281494.     

Growth increment ofAlnus glutinosa upon dual inoculation with effective and ineffectiveFrankia strains

Investigations on the ecological function of ineffectiveFrankia strains and their behaviour in competition with effectiveFrankia strains indicated an enhanced plant growth upon dual inoculation with increasing amounts of effective (i.e. N₂-fixing)Frankia strains and simultaneous inoculation with a constant amount of an ineffectiveFrankia strain. Enhanced plant growth was measured as increase in plant height and total dry weight at constant shoot/root ratio. The stimulating effect of the ineffective strain was independent of the plant clone and was obtained with bothAlnus glutinosa clones W I and B II, which were resistant and susceptable, respectively, to the ineffective strain. Stimulation was also independent of the nodulation conditions. Short-term studies (7 weeks) under axenic conditions and greenhouse experiments during 3 months showed comparable results, not only in plant growth but also in nodule formation. Increment in plant growth was not necessarily correlated to higher nodule formation with the effectiveFrankia strains.     

Sexual fertility of fortyFusarium strains from the EuropeanFusarium sambucinum project

Fungus strains designated asFusarium sambucinum, F. torulosum, orFusarium sp. nov. were crossed withMAT1-1 andMAT1–2 tester strains ofGibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated asF. torulosum was fertile with a tester strain ofG. pulicaris, suggesting that this is an intraspecies cross and that the strain isG. pulicaris, and, consequently,F. sambucinum rather thanF. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are notG. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.     

Protein enrichment of lignocellulose resulting from the growth of two Streptomyces strains

Two Streptomyces strains were grown on sugarcane bagasse and groundnut hulls lignocelluloses in semi-solid state culture at 37°C for 12 weeks. Best results gave a 45% depletion of sugarcane bagasse lignocellulose with a 21% crude protein content of final material. The possibility of using S. viridosporus to improve the protein content of both lignocelluloses for use as an animal feedstock supplement is discussed.At the time of this research the authors were with the Department of Biological Sciences, Rivers State University of Science & Technology, PMB 5080, Port Harcourt, Nigeria and the Department of Biological Sciences, University of Calabar, PMB 1115, Calabar, Nigeria. Dr lyo is now with the Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.     

Completion of the IS map in E. coli: IS186 positions on the E. coli K12 chromosome

Summary Insertion sites of the transposable element IS186 were physically mapped in the genome of E. coli K12 strain BHB2600. This strain maintains four IS186 copies of which three, assigned to 0.3, 14.1 and 51.8 map min., share common map positions with the three IS186 copies in strains W3110 and HB101. The fourth, unique IS copy in BHB2600 maps at 49.3 min. The IS186 data complete the BHB2600 map for all chromosomal sites of known K12-associated IS types.     

3种无脊椎动物对近江牡蛎Crassostrea ariakensis和熊本牡蛎C. sikamea的捕食研究

捕食是影响牡蛎种群建立和牡蛎礁发育的重要生物因子之一。通过室内受控实验测定了日本蟳（Charybdis japonica）、脉红螺（Rapana venosa）和黄口荔枝螺（Thais luteostoma）对4组规格（W1：壳高10-20mm;W2：壳高20-30mm;W3：壳高30-40mm;W4：壳高>40mm）近江牡蛎（Crassostrea ariakensis）和熊本牡蛎（C.sikamea）的捕食偏好性和捕食效率。双因子方差分析结果表明,日本蟳对2种牡蛎的捕食效率没有显著性差异（P>0.05）,但牡蛎规格大小显著影响着日本蟳的捕食效率（P<0.05）,即日本蟳对W1组近江牡蛎的捕食效率显著高于W2和W4组（P<0.05）,W3组的被捕食效率介于中间（P>0.05）;日本蟳对W1组熊本牡蛎的捕食效率显著高于W2和W3组（P<0.05）,W4组的被捕食效率与其他处理组均没有显著性差异（P>0.05）。牡蛎种类（P=0.590）和规格大小（P=0.357）对脉红螺的捕食效率均无显著性影响,不同规格的两种牡蛎均呈现较低的被捕食效率。黄口荔枝螺对2种牡蛎的捕食效率无显著性差异（P=0.917）,但牡蛎规格大小显著影响其捕食效率（P=0.035）,即对W1组熊本牡蛎捕食效率显著高于其他3个规格组（P<0.05）,但其对不同规格近江牡蛎的捕食效率没有显著性差异（P>0.05）。2种牡蛎的壳厚与其壳高之间均存在极显著的正相关关系（P<0.001）。研究结果表明,3种无脊椎动物捕食者对近江牡蛎和熊本牡蛎并未表现出差异性的捕食偏好,但对不同规格牡蛎的捕食效率具有种间差异。     

丛枝菌根真菌促进南美蟛蜞菊生长及对难溶磷的吸收

为了解2种丛枝菌根真菌(AMF)摩西管柄囊霉(Funneliformis mosseae, FM)和地表球囊霉(Glomus versiforme, GV)对入侵植物南美蟛蜞菊(Wedelia trilobata)的生长和对难溶性磷酸盐利用的影响,采用沙培盆栽方式,研究了南美蟛蜞菊在接种AMF与添加难溶性磷酸盐的生长和磷含量的变化。结果表明,在磷限制环境下FM对南美蟛蜞菊的侵染率达55%～69%,GV的侵染率达到63%～80%。添加难溶性磷酸盐后,2种AMF均促进了南美蟛蜞菊茎的伸长(FM:+46%; GV:+65%)、总生物量的增加(FM:+27.2%; GV:+40%)和磷含量的增加(FM:+36.6%; GV:+40.7%)。对比FM,GV对植物利用难溶性磷有更显著的促进作用。因此,南美蟛蜞菊与2种AMF形成的共生体系可以促进植物生长和对营养资源的利用,提高对难溶性磷的吸收效率可能使得南美蟛蜞菊在营养贫乏的环境中更好地建立种群。     

Restriction site variation,length polymorphism and changes in gene order in the mitochondrial DNA of the yeast Kluyveromyces lactics

The purpose of this work was to compare mitochondrial DNA restriction endonuclease patterns in strains of the yeast Kluyveromyces lactis, from different sources, to see how conserved is the organization of this organellar genome. The mitochondrial DNA of five independently-isolated strains and one of unknown origin were compared. Strains NRRL Y-1205, NRRL Y-8279 and NRRL Y-1140 gave identical patterns. Strain NRRL Y-1564 showed an insertion, with respect to the other three, of approximately 1250 bp. Strain W600B had also an insertion with extra restriction sites for EcoRI, HpaI, HaeIII, HincII and XbaI. On the other hand, strain Y-123 showed a restriction pattern quite different from the others.Sequences putatively encoding apocytochrome b, ATPase subunit 9 and ribosomal RNA large subunit, were localized on the physical maps of three strains. Results demonstrated that the order of these three genes shows a common feature in strains W600B and WM37 (auxotroph of Y-1140) but a different distribution in WM27 (auxotroph derived from Y-123). All these facts explain the extensive intraspecific polymorphism observed in the mtDNA of this yeast.     

Microbial formation of caparrapidiol and derivatives from trans-nerolidol

Of 60 fungi and 40 bacteria, five strains, all belonging to the genus Fusarium, were able to hydrate (S)-trans-nerolidol to caparrapidiol. Fusarium moniliforme DSM 764 and F. tabacinum DSM 2125 oxidized trans-nerolidol to six new derivatives of caparrapidiol which were characterized. These products served to elucidate the absolute configuration of the naturally occurring caparrapidiol. Six further strains, again all Fusarium spp., hydrated geranylacetone at the inner double bond. The ecological implications of this rather rare biotransformation reaction are briefly discussed.W.-R. Abraham is with the Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, W-3300 Braunschweig, Germany     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

Formation of biogenic amines as criteria for the selection of wine yeasts

This work deals with biogenic amine production by yeast strains isolated from grapes and wines. A total of 50 strains were tested for their capacity to produce biogenic amines in wine. In general, all the species produced very low or non-detectable amounts of histamine, whereas methylamine and agmatine were formed by all the species considered. The highest concentration of total biogenic amines was formed by Brettanomyces bruxellensis, with an average value of 15 mg/l, followed by Saccharomyces cerevisiae with an average of 12.14 mg/l. The other species formed less than 10 mg of total biogenic amines per litre. Wines fermented with the most fermentative strains of S. cerevisiae species had the highest contents of ethanolamine, from 2.3 to 16 mg/l, and of agmatine, from 3.1 to 7.5 mg/l. The strains of the other species, which exhibited a low fermentative ability, Kloeckera apiculata, B. bruxellensis and Metschnikowia pulcherrima, varied in the production of agmatine and phenylethylamine. A significant variability in the production of cadaverine was characteristic of Candida stellata strains, which varied also in ethanolamine production. Our results emphasize the importance of using selected strains of S. cerevisiae, not only for the expression of desirable technological traits, but also to avoid potentially negative effects on human health. Therefore, the characterization of strains of S. cerevisiae for the 'production of biogenic amines' becomes of applicative interest.     

Numerical phenetic study of the genus Carnobacterium

Eighty-nine strains representing the genus Carnobacterium, Enterococcus durans, Vagacoccus salmoninarum and atypical Lactobacillus strains MT12 and MT13 were examined for 92 unit characters. Computer analysis of the data resulted in the recovery of four major, five minor and thirteen single membered clusters. Three cluster-groups contained seventy-four of the Carnobacterium strains, Enterococcus durans NCFB 596^T and Lactobacillus maltaromicus NCFB 2382^T. Cluster-group A was equated with Carnobacterium piscicola and cluster-group B with Carnobacterium divergens. Lactobacillus maltaromicus NCFB 2382^T shared many properties in common with the C. piscicola strains. The recovery of several Carnobacteriumstrains as single membered clusters suggests that the genus Carnobacterium is underspeciated. Further work is also required to determine the subspecific structure of Carnobacterium divergens and Carnobacterium piscicola.