bluuebunny/crossref_metadata_2025 huggingface dataset:
Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobiumlegume symbiosis
Authors: Mateos Pedro F , Baker David L , Petersen Maureen , Velázquez Encarna , Jiménez-Zurdo José I , Martínez-Molina Eustoquio , Squartini Andrea , Orgambide Guy , Hubbell David H , Dazzo Frank B
Published: April 2011

A central event of the infection process in the Rhizobiumlegume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobiumlegume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.Key words: Rhizobium leguminosarum, cellulase, cell wall, chitolipooligosaccharide, clover, root hair.
Selection of several classes of mimosine-degradation-defective Tn3Hogus-insertion mutants of Rhizobium sp. strain TAL1145 on the basis of mimosine-inducible GUS activity
Authors: Fox P M , Borthakur D
Published: April 2011

Rhizobium sp. strain TAL1145 that nodulates Leucaena leucocephala degrades mimosine, a toxin produced by this tree legume. A cosmid clone, pUHR263, containing ~25 kb cloned DNA was isolated by plating Escherichia coli cells containing the cosmid clone library of TAL1145 on a minimal medium in which 3-hydroxy-4-pyridone (HP), a degradation product of mimosine, was used as the source of nitrogen. Cosmid pUHR263 was mutagenized by random insertions of Tn3Hogus, a transposon that makes transcriptional gus fusions when it is inserted in a gene in the correct orientation. Various pUHR263::Tn3Hogus derivatives that showed mimosine-inducible or mimosine-repressible GUS activities when transferred to the Rhizobium sp. strain TAL1145 were selected. Mutants of TAL1145 were constructed by transferring these Tn3Hogus insertions into the TAL1145 chromosome through double-homologous recombination. These mutants were classified into five classes on the basis of defects in mimosine degradation. The growth of these mutants was inhibited to different extents by mimosine applied to the growth medium. Mimosine forms a red-colored Fe-mimosine complex when FeCl3 is added to the medium. The inhibitory effect of Fe-mimosine on growth of the mutants was much less than that of mimosine.Key words: mimosine, mid and pyd genes, Leucaena leucocephala, tree legume, Tn3Hogus.
Modification of the protein expression pattern induced in the nitrogen-fixing actinomyceteFrankiasp. strain ACN14a-tsr by root exudates of its symbiotic hostAlnus glutinosaand cloning of thesodFgene
Authors: Hammad Y , Maréchal J , Cournoyer B , Normand P , Domenach A -M
Published: April 2011

Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to detect proteins induced in Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host, Alnus glutinosa. The 5 most prominent proteins were purified from 2-D gels and characterized by N-terminal sequencing. All of these proteins had a high percentage of similarity with known stress proteins. One protein match was the Fe superoxide dismutase (Fe-SOD), another was a tellurite resistance protein (Ter), the third was a bacterioferritin comigratory protein (Bcp); and two matches, differing only by their isoelectric point, were the same small heat shock protein (Hsp), a major immune reactive protein found in mycobacteria. This suggests that the symbiotic microorganism Frankia, first responds with a normal stress response to toxic root products of its symbiotic host plant. To confirm its identity, the gene corresponding to the Fe-SOD protein, sodF was isolated from a genomic library by a PCR-approach and sequenced. It is the first stress response gene characterized in Frankia.Key words: Frankia, Alnus glutinosa, root-exudates, superoxide dismutase, tellurite resistance, bacterioferritin comigratory protein, heat shock protein.
Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in different formulations
Authors: Hynes Russell K , Jans Desirée C , Bremer Eric , Lupwayi Newton Z , Rice Wendell A , Clayton George W , Collins Mandy M
Published: April 2011

The effect of inoculant formulation on the population dynamics of rhizobia in the pea rhizosphere was investigated using a streptomycin-resistant mutant of Rhizobium leguminosarum bv. viceae NITRAGIN128C56G (128C56G strR). The isolate was formulated into liquid, peat powder, and granular peat carriers, and was tested on pea at field sites near Saskatoon, Saskatchewan, and Beaverlodge, Alberta, in 1996 and 1997. The liquid and peat powder formulations were applied to seed while the granular inoculant was applied to soil. In three out of four site years, population dynamics were similar among formulations: an initial decline or lag period lasting 25 days followed by an increase to approximately 105 colony-forming units (CFU)/seedling by 1428 days after planting (DAP) and, where sampled, a continuing increase from 107 to 108 CFU/plant at 63 DAP. In these same site years, nodule number (not determined at Beaverlodge in 1997) and nodule occupancy at 60 days were not significantly different among formulations. In contrast, soil populations of 128C56G strR from the liquid formulation declined to near zero by 28 DAP at Beaverlodge in 1996, when soil moisture was excessive in spring because of high rainfall. Populations increased in this treatment after this time, but remained significantly lower than the populations of the other two formulations throughout the sampling period. Pea seed yields were not significantly different among treatments in either year at Beaverlodge, but were significantly higher with granular inoculant than the noninoculated control in Saskatoon. Within inoculated treatments at Saskatoon, there were no significant differences in grain yield.Key words: Rhizobium leguminosarum, rhizosphere, population dynamics.
Molecular strain typing ofCampylobacter jejuniby pulsed-field gel electrophoresis in a single day
Authors: Michaud Sophie , Arbeit Robert D , Gaudreau Christiane
Published: April 2011

Rapid molecular strain typing is critical for effective outbreak investigation and implementation of infection control measures. Pulsed-field gel electrophoresis is a highly discriminatory technique for Campylobacter jejuni, but generally requires 35 days. We describe a simplified protocol for pulsed-field gel electrophoresis that provides high quality typing of C. jejuni isolates in a single day.Key words: pulsed-field gel electrophoresis, Campylobacter jejuni, molecular epidemiology.
Debaryomyces hanseniigrowth in nonsterile seawater ClO2- peptone-containing medium
Authors: Ramírez-Orozco Martín , Hernández-Saavedra Norma Y , Ochoa José-Luis
Published: April 2011

We found that the marine yeast Debaryomyces hansenii strain C-11 (CIBNOR yeast collection, La Paz, México) is highly tolerant to chlorine dioxide (ClO2), a powerful biocide agent. A direct application of this observation is the fermentation of the yeast in a nonsterile medium with an initial concentration of 0.3 mg/L of ClO2. The disinfectant helps to avoid the growth of unwanted microorganisms while allowing the development of the yeast. Because the concentration of ClO2decreases during the fermentation, we ascribe to D. hansenii cells a "biocontrol" action that contributes to the collection of a contaminant-free yeast cell biomass.Key words: marine yeasts, Debaryomyces hansenii, biocide, biocontrol, Cl02disinfection, Cl02resistance, nonsterile medium.
Characterization of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots
Authors: Yu T , Nassuth A , Peterson R L
Published: April 2011

Phialocephala fortinii Wang & Wilcox is a member of root-inhabiting fungi known collectively as dark septate endophytes (DSE). Although very common and distributed worldwide, few studies have documented their interaction with roots on a structural basis. The objective of this study was to determine the early colonization events and formation of microsclerotia of P. fortinii in roots of Asparagus officinalis L., a species known to have DSE. A loose network of hyphae accumulated at the root surface, and coils formed around root hairs and external to epidermal cells overlying short cells of the dimorphic, suberized exodermis. Root penetration occurred via swollen, appressorium-like structures into epidermal cells where coiling of hyphae occurred along the periphery of the cells. Hyphae penetrated from the epidermis into short exodermal cells and from these into cortical cells. Hyphae colonized the cortex up to the endodermis and sometimes entered the vascular cylinder. Some root tips were colonized as well. Microsclerotia in epidermal and exodermal short cells accumulated glycogen, protein, and polyphosphate. Energy-dispersive X-ray spectroscopy on distinct bodies visible in microsclerotial hyphae revealed high levels of phosphorus.Key words: Mycelium radicis atrovirens, Phialocephala fortinii, microsclerotia, DSE.
Detection ofAeromonas hydrophilain a drinking- water distribution system: a field and pilot study
Authors: Chauret Christian , Volk Christian , Creason Robin , Jarosh John , Robinson Jeff , Warnes Carl
Published: April 2011

A 16-month study was conducted on the presence of Aeromonas hydrophila in drinking water in Indiana, U.S.A. Enumeration was conducted in source water, in various sites within a water treatment plant, and in the distribution system in both bulk water and biofilm, as well as in a simulated (annular reactors) drinking-water distribution system. Presumptive Aeromonas spp. counts on source waters regularly approached 103104CFU/100 mL, during summer months and granular activated carbon - filtered water counts ranged from <1 to 490 CFU/100 mL. In source water, presumptive Aeromonas levels were related to water temperature. Aeromonas hydrophila was never detected in the treatment plant effluent or distributed bulk water, showing disinfectant efficiency on suspended bacteria; however, isolates of A. hydrophila were identified in 7.7% of the biofilm samples, indicating a potential for regrowth and contamination of drinking-water distribution systems.Key words: Aeromonas hydrophila, distribution system, biofilm.
Effects of antecedent fermentative and respiratory growth on the detection of chloramine-stressed Escherichia coli and Salmonella typhimurium
Authors: Thunberg Richard L , Sexstone Alan J , Calabrese Joseph P , Bissonnette Gary K
Published: April 2011

In vitro laboratory studies were performed to assess the effects of antecedent growth conditions on the recovery of Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 following chloramine disinfection. Six- and 18-h cultures of each organism were grown under aerobic, fermentative, and nitrate-reducing conditions prior to disinfection. At predetermined time intervals during a 10-min exposure to chloramine, survivors were surface plated on nonselective recovery media to determine Cnt values. It was observed that nitrate-reducing growth predisposed the test organisms towards an increased sensitivity to chloramine stress over cells grown under fermentation or aerobic conditions (p < 0.01).Key words: Escherichia coli, Salmonella typhimurium, chloramine, survival, antecedent growth conditions.
Rhizobitoxine production and symbiotic compatibility of Bradyrhizobium from Asian and North American lineages of Amphicarpaea
Authors: Parker Matthew A , Peters N Kent
Published: April 2011

Reciprocal inoculations with Bradyrhizobium sp. isolates from the North American legume Amphicarpaea bracteata (L.) Fern. (Phaseoleae-Glycininae) and from a Japanese population of its close relative Amphicarpaea edgeworthii (Benth.) var. japonica were performed to analyze relative symbiotic compatibility. Amphicarpaea edgeworthii plants formed few or no nodules with any North American bradyrhizobial strains isolated from A. bracteata, but all A. bracteata lineages formed effective nitrogen-fixing nodules with Japanese Bradyrhizobium isolates from A. edgeworthii. However, one group of A. bracteata plants (lineage Ia) when inoculated with Japanese bradyrhizobia developed a striking leaf chlorosis similar to that known to be caused by rhizobitoxine. The β-cystathionase inhibition assay demonstrated that significant amounts of rhizobitoxine were present in nodules formed by these Japanese bradyrhizobia. No North American bradyrhizobial isolate from A. bracteata induced chlorosis on any plants, and the β-cystathionase assay failed to detect rhizobitoxine in nodules formed by these isolates. The role of rhizobitoxine in A. edgeworthii nodulation development was tested by inoculating plants with a Bradyrhizobium elkanii rhizobitoxine-producing strain, USDA 61, and two mutant derivatives, RX17E and RX18E, which are unable to synthesize rhizobitoxine. Amphicarpaea edgeworthii inoculated with wild-type USDA 61 developed >150 nodules per plant, while plants inoculated with RX17E and RX18E developed fewer than 10 nodules per plant. Thus, efficient nodule development in A. edgeworthii appears to be highly dependent on rhizobitoxine production by Bradyrhizobium strains.Key words: Bradyrhizobium elkanii, geographic variation, Leguminosae, mutualism.
RAPD analysis, serotyping, and esterase typing indicate that the population ofListeria monocytogenesstrains recovered from cheese and from patients with listeriosis in Belgium are different
Authors: Malak M , Vivier A , André P , Decallonne J , Gilot P
Published: April 2011

Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period.Key words: Listeria, typing, RAPD, esterase, serovar.
Endophytic fungi from Amomum siamense
Authors: Bussaban Boonsom , Lumyong Saisamorn , Lumyong Pipob , McKenzie Eric HC , Hyde Kevin D
Published: April 2011

Endophytic fungi were isolated from apparently healthy organs of the wild ginger Amomum siamense Criab., including leaves, pseudostems, and rhizomes, collected from two sites in Doi Suthep-Pui National Park, Thailand. Endophytes were relatively common with an isolate prevalence of 70%83% at the two sites sampled in the wet and dry seasons. The endophyte assemblages from the two sites were diverse and comprised 7 Ascomycetes and 26 mitosporic fungi. Colletotrichum "gloeosporioides" (Penz.) Penz. & Sacc. in Penz., Glomerella spp., xylariaceous fungi, and Phomopsis spp. were consistently dominant as endophytes on Amomum siamense, but many rare species and mycelia sterilia were also recorded. There was no significant difference between the number of isolates recovered from leaves containing vein tissues and those containing intervein tissues, independent of leaf age. Most taxa showed a preference for either leaf tissue or pseudostems. Two new Ascomycetes species, Gaeumannomyces amomi and Leiosphaerella amomi, were discovered from leaves and rhizomes, respectively, and four species of Pyricularia, including three new species, were isolated from leaves.Key words: Zingiberaceae endophytes, isolate prevalence, tissue specificity, seasonal effects.
Mycelial differentiation and spore formation by Streptomyces brasiliensis in submerged culture
Authors: Rueda Begoña , Miguélez Elisa M , Hardisson Carlos , Manzanal Manuel B
Published: April 2011

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Sporogenic hyphae formed under these conditions were morphologically similar and developmentally equivalent to aerial hyphae and metamorphosed into chains of spores by following a sequence of ultrastructural changes similar to that observed during growth on solid media. In addition, our electron microscopy study revealed two previously unrecognized aspects of hyphal development in streptomycetes: the formation of sporogenic hyphae was always preceded by changes in the structure of the nucleoid, and the sheath that characteristically covered these hyphae was not deposited coincidently with wall formation in the apical growing portion of the hypha.Key words: Streptomyces brasiliensis, mycelial differentiation, sporulation.
Mapping of genes encoding glycoside hydrolases on the chromosome of Cellulomonas fimi
Authors: Stoll Dominik
Published: April 2011

Cellulomonas fimi genomic DNA was digested with HpaI, MunI, HindIII, and NsiI, producing fragments ranging in size from 20 to 1400 kbp that were resolved by pulsed field gel electrophoresis. Genetic and physical linkages were determined by Southern blotting and were used to construct a genome map. Cellulomonas fimi has a single circular chromosome of approx. 4000 kbp. Except for two closely linked genes, cbh6A and cel5A, the genes known to encode glycoside hydrolases are scattered widely on the chromosome.Key words: Cellulomonas fimi, genome map, pulsed field gel electrophoresis, glycoside hydrolases.
Osmoregulation inBacillus subtilisunder potassium limitation: a new inducible K+-stimulated, VO43-inhibited ATPase
Authors: ebestian Jirí , Petrmichlová Zdenka , ebestianová tepánka , Náprstek Josef , Svobodová Jaroslava
Published: April 2011

Bacillus subtilis exhibited an inducible K+-transporting ATPase activity with apparent Kmand maximum velocity Vmaxof 12.9 µM and 25.1 µmol·min1·(g cell protein)1, respectively, when cultivated on a synthetic medium containing less than 400 µM K+. Due to this enzyme, the growth rate of the bacterium in synthetic medium was not changed down to 115 µM K+, and the bacterium was able to grow down to 20 µM K+. The limiting K+concentration was higher in media with osmolarity increased by NaCl or sucrose. The ATPase was inhibited by micromolar concentrations of vanadate (Ki= 1.6 µM). The ATPase activity was not stimulated by any other monovalent cation. The subunit of this ATPase, with an Mrof 52 000, covalently bound the gamma phosphate group of ATP. This phosphorylated intermediate was unstable in neutral and basic pH as well as in the presence of potassium and was stable in acid pH. The enzyme did not show immunological cross-reactivity with antibody against Kdp ATPase of Escherichia coli.Key words: Kdp-like, potassium transport, Bacillus subtilis, transport ATPase, P-type ATPase.
DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil
Authors: Thomassin-Lacroix Eric JM , Yu Zhongtang , Eriksson Mikael , Reimer Kenneth J , Mohn William W
Published: April 2011

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82°30'N, 62°19'W). The enrichment culture was grown on Jet A-1 fuel at 7°C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.Key words: ARDRA, biodegradation, bioremediation, fuel, MPN-PCR, Pseudomonas, psychrotolerant, Rhodococcus, Sphingomonas, 16S rRNA.
Biphenyl-associatedmeta-cleavage dioxygenases fromComamonas testosteroniB-356
Authors: Hein Paul , Powlowski Justin , Barriault Diane , Hurtubise Yves , Ahmad Darakshsan , Sylvestre Michel
Published: April 2011

In addition to 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O), biphenyl-grown cells of Comamonas testosteroni B-356 were shown to produce a catechol 2,3-dioxygenase (C2,3O). B1,2O showed strong sequence homology with B1,2Os found in other biphenyl catabolic pathways, while partial sequence analysis of the C2,3O of B-356 suggested a relationship with xylEII-encoded C2,3O. The coexistence of two meta-cleavage dioxygenases in this strain prompted a comparison between the catalytic properties of the two enzymes. C2,3O has a much broader substrate specificity than native or His-tagged B1,2O: both enzymes were inhibited by chlorocatechols, but B1,2O was more sensitive than C2,3O. The results are discussed in terms of the physiological implications of interaction between metabolites from the lower biphenyl-chlorobiphenyl pathway and enzymes of the upper pathway.Key words: chlorobiphenyl, catabolism, dioxygenase, nucleotide sequence, enzyme kinetics.
Sequencing ofporAfrom clinical isolates ofNeisseria meningitidisdefines a subtyping scheme and its genetic regulation
Authors: Arhin Francis F , Moreau France , Coulton James W , Mills Elaine L
Published: April 2011

Subtyping Neisseria meningitidis by methods that rely on monoclonal antibody (mAb) reactivity results in an unusually high number of strains that are not subtypeable. To subtype 48 strains isolated (1993-1994) in the province of Quebec that were not subtypeable by mAb-based techniques, we used DNA sequencing of the variable regions of porA, a gene that encodes the class 1 outer membrane protein. We assigned subtypes to all the previously nonserosubtypeable isolates and identified some novel subtypes. Because our sequencing strategy included the promoter region of porA, different isolates were compared in their sequences of the porA promoter region. A poly(G) stretch lies between the -10 and -35 regions of the promoter; replacement of a G residue by an A residue in this region resulted in loss of expression of porA. No correlation was found between the number of G residues in the poly(G) stretch and the level of expression; a minimum of 10 G residues is required in this stretch for expression of porA. One isolate expressed no class 1 outer membrane protein because of the insertion sequence IS1301 in the coding region of porA. Another isolate did not express the protein owing to a frame-shift mutation within the coding region of porA. Sequencing of porA allowed assignments of subtypes to previously uncharacterized isolates and provided insights about the regulation of expression of this gene in N. meningitidis.Key words: Neisseria meningitidis, outer membrane proteins, subtyping, PorA, DNA sequencing.
Halotolerant Bacillus diversity in hypersaline environments
Authors: Garabito M J , Márquez M C , Ventosa A
Published: April 2011

A total of 99 Gram-positive aerobic endospore-forming halotolerant rods were isolated from different hypersaline sources in Spain (salterns as well as saline soils). They were characterized taxonomically for a total of 178 features, including the utilization of 95 different substrates determined by the Biolog microbial identification system. These results were analysed by numerical techniques using the Jaccard (SJ) coefficient and clustering was achieved using the unweighted pair group method with averages algorithm. At 60% similarity level, the majority of the isolates were clustered in seven phenons. The guanine plus cytosine (G + C) content of some representative strains selected from each phenon ranged from 36.0 to 50.4 mol%. The isolates were identified as members of the genus Bacillus, and were assigned to the species B. pantothenticus, B. firmus, B. alcalophilus, B. megaterium, and B. laterosporus. They were extremely halotolerant, most were able to grow in media with up to 20 or 25% salt. The diversity of groups and characteristics of the halotolerant Bacillus isolated in this study indicate that they may play an important ecological role and contribute to the microbial diversity of hypersaline environments.Key words: halotolerant bacteria, Bacillus, taxonomy, hypersaline environments.
Root nodules ofCeanothus caeruleuscontain both the N2-fixingFrankiaendophyte and a phylogetically related Nod-/Fix-actinomycete
Authors: Ramírez-Saad Hugo , Janse Jaap D , Akkermans Antoon DL
Published: April 2011

Attempts to isolate the N2-fixing endophyte of Ceanothus caeruleus (Rhamnaceae) root nodules, led to the isolation of nine actinomycetous strains. Owing to their inability to fix nitrogen (Fix-) and nodulate (Nod-), they could not be regarded as the effective endophyte. Characterization was done based on morphological and physiological features and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and sequencing of nearly full length 16S rDNA and partial nifH genes. Phylogenetic analysis based on 16S rDNA revealed that both the effective endophyte and the isolated actinomycetes belong to two different but well-defined lineages within the family Frankiaceae. One lineage is formed mainly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix-/Nod-isolates. Application of temperature gradient gel electrophoresis techniques to actinorhizal nodules allowed us to detect and identify 16S rDNA sequences from both the Fix+and the Fix-nodule inhabitants. Interestingly, these same two sequences were detected on Hippophae rhamnoides nodules obtained after inoculation with Ceanothus caeruleus nodule suspensions. The isolates were located in the outer layers of the nodule.Key words: Frankia, Ceanothus, 16S rDNA, nifH, temperature gradient gel electrophoresis (TGGE), Fix-/Nod-strains.
The effect of suspending solution supplemented with marine cations on the oxidation of Biolog GN MicroPlateTMsubstrates byVibrionaceaebacteria
Authors: Noble Lisa D , Gow John A
Published: April 2011

Bacteria belonging to the family Vibrionaceae were suspended using saline and a solution prepared from a marine-cations supplement. The effect of this on the profile of oxidized substrates obtained when using Biolog GN MicroPlatesTMwas investigated. Thirty-nine species belonging to the genera Aeromonas, Listonella, Photobacterium, and Vibrio were studied. Of the strains studied, species of Listonella, Photobacterium, and Vibrio could be expected to benefit from a marine-cations supplement that contained Na+, K+, and Mg2+. Bacteria that are not of marine origin are usually suspended in normal saline. Of the 39 species examined, 9 were not included in the Biolog data base and were not identified. Of the 30 remaining species, 50% were identified correctly using either of the suspending solutions. A further 20% were correctly identified only when suspended in saline. Three species, or 10%, were correctly identified only after suspension in the marine-cations supplemented solution. The remaining 20% of species were not correctly identified by either method. Generally, more substrates were oxidized when the bacteria had been suspended in the more complex salts solution. Usually, when identifications were incorrect, the use of the marine-cations supplemented suspending solution had resulted in many more substrates being oxidized. Based on these results, it would be preferable to use saline to suspend the cells when using Biolog for identification of species of Vibrionaceae. A salts solution containing a marine-cations supplement would be preferable for environmental studies where the objective is to determine profiles of substrates that the bacteria have the potential to oxidize. If identifications are done using marine-cations supplemented suspending solution, it would be advisable to include reference cultures to determine the effect of the supplement. Of the Vibrio andListonella species associated with human clinical specimens, 8 out of the 11 studied were identified correctly when either of the suspending solutions was used.Key words: Biolog, cations, salts, marine bacteria, Vibrionaceae.
Degradation of hydrocarbons in crude oil by the ascomycetePseudallescheria boydii(Microascaceae)
Authors: April T M , Abbott S P , Foght J M , Currah R S
Published: April 2011

Four unique strains of Pseudallescheria boydii were isolated from oil-soaked soils in British Columbia and Alberta and compared to strains from cattle dung and raw sewage. Considerable variability in morphology, colony appearance, colony diameter, and temperature tolerance occurred among the strains. They also varied in the sporogenous states produced in culture; all strains had a Scedosporium anamorph and either the Graphium anamorph or cleistothecial teleomorph. Conspecificity of the six isolates was inferred from their morphology and supported by restriction fragment length polymorphism profiles of the internally transcribed spacer region of rDNA and comparing these to Petriella sordida, a similar taxon in the Microascaceae. Three of the strains isolated from oil-contaminated soil and the strain from sewage were tested for their ability to utilize hydrocarbons by incubation with Prudhoe Bay Crude oil as the sole carbon source. Gas chromatographic analysis of the residual oil revealed that the strains isolated from oil-contaminated soil degraded the linear aliphatics. The strain from sewage, previously shown by others to utilize the volatile n-alkanes (i.e., ethane, propane, and butane), did not utilize the liquid saturate compounds. None of the strains was observed to degrade compounds in the aromatic fraction. Pseudallescheria boydii may be an important agent for in situ bioremediation of saturates in oil-contaminated sites.Key words: bioremediation, filamentous fungi, Graphium, hydrocarbon degradation, Scedosporium.
Amplification of putative chlorocatechol dioxygenase gene fragments from α- and β-Proteobacteria
Authors: Leander Michelle , Vallaeys Tatiana , Fulthorpe Roberta
Published: April 2011

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and chlorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.Key words: 2,4-dichlorophenoxyacetate, chlorobenzoate, biodegradation, ortho cleavage, detection.
Transformation of Penicillium griseoroseum nitrate reductase mutant with the nia gene from Fusarium oxysporum
Authors: de Queiroz Marisa V , Barros Andréa O , de Barros Everaldo G , Guimarães Walter V , de Araújo Elza F
Published: April 2011

A heterologous transformation system for Penicillium griseoroseum has been developed. This system is based on nia, the structural gene from Fusarium oxysporum encoding nitrate reductase. Penicillium griseoroseum niaD mutants have been selected from chlorate-resistant colonies. Among 24 chlorate-resistant colonies analyzed, 2 were confirmed to be niaD mutants. Transformation frequencies of 8 transformants/µg of DNA were obtained. DNA hybridization analyses of five transformants showed distinct integration patterns of the plasmid and in all of them the integration occurred at tandem arrays. The transformation system established in this work will be useful for genetic studies of the pectinolytic complex genes from P. griseoroseum.Key words: Penicillium griseoroseum, heterologous transformation, nitrate reductase.
Inactivation of enzymes within spores ofBacillus megateriumATCC 19213 by hydroperoxides
Authors: Palop Alfredo , Rutherford Glenn C , Marquis Robert E
Published: April 2011

The organic hydroperoxides t-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores of Bacillus megaterium ATCC 19213 concomitant with mortality. Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays. The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of differences in the types of radicals generated. However, each agent inactivated each of the enzymes, albeit at different rates. Comparative assessments of enzyme inactivation by lethal levels of H2O2or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant. The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat. Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.Key words: bacterial spores, hydroperoxides, Bacillus megaterium, enzyme inactivation.
Bacterial community structure at defined locations ofPinus sylvestris-Suillus bovinusandPinus sylvestris-Paxillus involutusmycorrhizospheres in dry pine forest humus and nursery peat
Authors: Timonen Sari , Jørgensen Kirsten S , Haahtela Kielo , Sen Robin
Published: April 2011

Bacteria were isolated and characterized from uncolonized soil, nonmycorrhizal and mycorrhizal short roots, and soil-colonizing external mycelium from intact Pinus sylvestris - Suillus bovinus and Pinus sylvestris - Paxillus involutus mycorrhizospheres developed in microcosms containing dry pine forest humus or nursery peat. Total numbers of colony-forming units (CFU/mg dry weight) in the different locations from all ectomycorrhizospheres indicated an overall bacterial-enrichment gradient towards the roots, whereas sporeformers were more evenly distributed. Fluorescent pseudomonads were commonly isolated from all mycorrhizosphere locations in nursery peat, but they were nearly absent from the forest humus community. In contrast, sporeformers were more abundant at all locations in the latter growth substrate. The bacterial species composition of forest and nursery mycorrhizospheres was clearly divergent when characterized according to their carbon source utilization patterns in Biolog®GN or GP microplates. Factorial-designed ANOVA of a principal component analysis of the carbon source utilization data showed significant differences between isolates from the two soil types and, to a lesser extent, between S. bovinus and Paxillus involutus mycorrhizospheres. Bacterial communities from mycorrhizospheres and uncolonized soil were distinguished by their preferential utilization of carbohydrates and organic and amino acids, respectively. Suillus bovinus associated bacteria appeared to favour mannitol and Paxillus involutus associated bacteria appeared to favour fructose as carbon sources. This study demonstrates the combined effect of soil type, fungal symbiont, and precise location on bacterial communities associated with Pinus sylvestris ectomycorrhizospheres.Key words: Biolog, carbon source utilization, ectomycorrhiza, Scots pine, soil bacteria.
Killing two birds with one stone: simultaneous extraction of DNA and RNA from activated sludge biomass
Authors: Yu Zhongtang , Mohn William W
Published: April 2011

DNA and RNA are usually extracted from activated sludge samples using two separate methods developed for soil and sediment samples. However, activated sludge differs from soil and sediment in at least three aspects: high biomass density, low humic acid content, and the presence of bacterial aggregate flocs. Taking these characteristics into consideration, we developed a simple and rapid method allowing simultaneous DNA and RNA extraction from activated sludge samples. This method combines (i) mini-bead beating, which is most efficient in breaking bacterial aggregate flocs and cells, (ii) protection of RNA with diethyl pyrocarbonate, and (iii) precipitation of impurities with ammonium acetate. Phenol/chloroform extraction and column purification are not necessary. The resulting DNA and RNA are suitable for PCR and reverse transcriptase - PCR, respectively. The efficiencies of cell lysis and nucleic acid recovery were high enough to permit detection by PCR of 102cells/mL of mixed liquor. By simultaneously extracting both DNA and RNA from a single sample, this method eliminates variability in cell lysis between extraction of DNA and RNA using two different methods. This extraction method is rapid, and within 1 h, one person can process four or more samples. This simple method makes it easier to analyze a large number of activated sludge samples.Key words: activated sludge, DNA, extraction, PCR, RNA.
Biodegradation of cyanide compounds by aPseudomonasspecies (S1)
Authors: Dhillon J K , Shivaraman N
Published: April 2011

A Pseudomonas sp. (S1), isolated from soil by an enrichment technique was tested for its potential to degrade different cyanide compounds. Further, biodegradation/biotransformation of binary mixtures of the cyanide compounds by the culture was also studied. The results indicated that the culture could grow on the following nitriles by using them as carbon and nitrogen sources: acetonitrile, butyronitrile, acrylonitrile, adiponitrile, benzonitrile, glutaronitrile, phenylacetonitrile, and succinonitrile. Studies on the biodegradation of these cyanide compounds in binary mixtures showed that the presence of acrylonitrile or KCN delayed the degradation of acetonitrile in a mixture, while none of the other cyanide compounds affected the degradation of one another. The transformation products of the nitriles were their corresponding acids, and similarly, KCN was also directly transformed to formic acid. Studies on the transformation of these cyanide compounds showed that the rate of transformation of nitriles to their corresponding carboxylic acids was acrylonitrile > acetonitrile > adiponitrile > benzonitrile > KCN. This culture has the unique characteristic of transforming representatives of saturated aliphatic, aliphatic olefinic, aromatic, and aralkyl nitriles, as well as alkali cyanide, to their corresponding carboxylic acids.Key words: Pseudomonas sp.(S1), biodegradation, biotransformation, nitriles, cyanide.
Purification and characterization of the heat-labile α-amylase secreted by the psychrophilic bacterium TAC 240B
Authors: Chessa Jean-Pierre , Feller Georges , Gerday Charles
Published: April 2011

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce α-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified α-amylase shows a molecular mass of about 50 000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other α-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic α-amylase requires both Cl-and Ca2+for its amylolytic activity. Br-is also quite effecient as an allosteric effector. The comparison of the amino acid composition with those of other α-amylases from various organisms shows that the cold α-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. Key words: α-amylase, psychrophilic microorganisms, Antarctic.
Role of adhesion in the colonization of barley leaves by the yeastRhodosporidium toruloides
Authors: Buck James W , Andrews John H
Published: April 2011

To investigate whether leaf-surface yeasts adhere to the phylloplane in a two-phase manner, with weak, nonspecific attachment followed by stronger, time-dependent adhesion, we observed adhesion kinetics of the basidiomycete yeast Rhodosporidium toruloides to barley. While 50-60% of the cells adhered in short-term assays (up to 3 h), fewer than 10% were adherent after 10 days. Ten attachment-minus (Att-) mutants, deficient in phase I attachment, did not adhere after 5-day incubations, further suggesting a lack of stronger, independent phase II adhesion. Long-term (5 day) adhesion was similar for two isolates of R. toruloides and the ubiquitous leaf-surface fungus Aureobasidium pullulans. Long-term adhesion of R. toruloides to leaves of a waxless barley mutant was significantly greater than to leaves of the wild-type cv. Bonus. Application of exogenous nutrients (dilute yeast carbon base) to resident, wild-type R. toruloides populations resulted in both a rapid recolonization to the apparent carrying capacity of the leaves and an increase in the total adherent populations. Att-mutants recolonized barley segments, when supplied with nutrients, after more than 99% of the cells had been removed by agitation. Therefore, adhesion of R. toruloides to leaves was not required for subsequent colonization of the phylloplane. Overall, these data suggest that the frequency of yeast emigration from leaf surfaces, microbial growth rates, and leaf surface characteristics are major factors influencing colonization of leaf surfaces.Key words: epiphyte, phylloplane, Rhodotorula glutinis.
The use of babassu oil as substrate to produce bioemulsifiers byCandida lipolytica
Authors: Sarubbo L A , Porto ALF , Campos-Takaki G M
Published: April 2011

Candida lipolytica IA 1055 produced an extracellular emulsifier when using babassu oil as its sole carbon source during batch and fed batch fermentations at 27°C. Emulsification activity was detected after 60 h of growth in all conditions studied. The bioemulsifier was isolated after 144 h of fermentation from the best condition studied. The biopolymer seems to be a polysaccharide-protein-lipid complex.Key words: bioemulsifiers, biopolymer, Candida lipolytica, babassu oil, fermentation.
Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid
Authors: Yu Zhongtang , Mohn William W
Published: April 2011

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55°C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30°C to 60°C with maximum growth at 50°C; while, DhA-73 grew on DhA from 37°C to 60°C with maximum growth at 55°C. At 55°C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in theRubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.Key words: biodegradation, resin acid, semi-continuous enrichment, thermophiles.