Extraction of the characteristics of Si nanocrystals by the charge pumping technique.

In this paper, the characteristics of silicon nanocrystals used as charge trapping centers in memory devices are examined using the two-level charge pumping (CP) technique performed as a function of frequency and energy filtered transmission electron microscopy (EFTEM). The parameters extracted from the two methods such as the depth location, density and effective diameter of the nanocrystals are in good quantitative agreement. These results validate the charge pumping approach as a non-destructive powerful technique to access most of the properties of nanocrystals embedded in dielectrics and located at injection distances from the substrate surface not limited to the direct tunneling regime.

Temperature-dependent low electric field charging of Si nanocrystals embedded within oxide-nitride-oxide dielectric stacks.

In this work we examine the current peaks and the negative differential resistance that appear in the low electric field regime of oxide-nitride-oxide structures with a two-dimensional band of silicon nanocrystals embedded in a nitride layer. The silicon nanocrystals were synthesized by low energy ion implantation (1 keV, 1.5 x 10(16) Si(+) cm(-2)) and subsequent thermal annealing (950 degrees C, 30 min). Electrical examination was performed at temperatures from 20 to 100 degrees C using constant voltage ramp-rate current measurements. This approach enables us to determine the origin of the observed current peaks as well as to extract the trapping location of the injected carriers within the dielectric stack. The results confirm that the carriers are trapped within the Si nanocrystal band, verifying that this region corresponds to energy minima of the dielectric stack.

Streptomyces turgidiscabies and Streptomyces reticuliscabiei: one genomic species, two pathogenic groups.

Three strains of Streptomyces reticuliscabiei and two strains of Streptomyces turgidiscabies were analysed, together with reference and type strains of other Streptomyces species, for phenotypic traits, DNA-DNA relatedness, comparison of 16S rRNA gene sequences and presence of necrotic protein gene (nec1) homologues in order to clarify their phylogenetic relationships. A numerical analysis of phenotypic characteristics showed that S. reticuliscabiei and S. turgidiscabies belong to the same cluster and share almost all morphological and biochemical traits that are important in the identification of Streptomyces species. DNA-DNA hybridization and phylogenetic comparisons of 16S rRNA gene sequences confirmed that the two species are genomically closely related. In contrast, pathological data showed that S. turgidiscabies and S. reticuliscabiei cause two distinct diseases. Gene homologues of nec1 were detected in S. turgidiscabies and other common scab species (Streptomyces scabiei, Streptomyces europaeiscabiei and Streptomyces stelliscabiei), but not in S. reticuliscabiei. To avoid confusion between agents causing separate diseases, it is proposed that the existing distinct species names are retained: S. turgidiscabies involved in common scab and S. reticuliscabiei involved in netted scab.

Relationship between spatial and genetic distance in Agrobacterium spp. in 1 cubic centimeter of soil.

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place. Agrobacterium spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 microm in diameter were sampled with spatial coordinates in 1 cm(3) of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10(3) agrobacteria g(-1)), a high genetic diversity was maintained. In two independent microsamples (500 microm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.

Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purées.

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25 degrees C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis. A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans. Storage conditions at 4 degrees C favored the development of "B. macroides/B. maroccanus" and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25 degrees C favored the development of B. subtilis group (B. licheniformis and B. subtilis) and B. cereus group strains. At 10 degrees C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus, and B. pumilus varied with the experiment.

Combined use of a specific probe and PCAT medium to study Burkholderia in soil.

Due to its pathogenic traits and agricultural benefits, there is some challenge in detecting Burkholderia in the soil environment. In this perspective, an existing semi-selective medium, (PCAT), was combined with a Burkholderia specific molecular probe. Using the complete 16S rRNA sequences of all available Burkholderia species type strains, we selected the following sequence: 5'-ACCCTCTGTTCCGACCATTGTATGA-3'. The probe was validated against GenBank sequences, with dot blots and colony hybridization tests. A diversity study of all strains growing on a PCAT plate after plating a soil dilution (75 strains) was carried out with ARDRA analysis and colony hybridization tests. All the hybridizing strains belonged to genus Burkholderia. The major type of non-hybridizing isolates belonged to Pseudomonas (16S rRNA sequencing). Both tools were combined to compare the Burkholderia populations in a rhizosphere (maize) and a non-rhizosphere soil. Based on hybridizing PCAT isolates, we were able to show an increase in Burkholderia populations in the maize rhizosphere. This genus represented 2% and 16% of the total cultivable microflora in the non-rhizosphere and rhizosphere soils, respectively. Although PCAT was shown not to be appropriate to routinely enumerate Burkholderia populations in soil, it allowed environmental investigations at the genus level, when combined with a molecular specific probe.

Modification of the protein expression pattern induced in the nitrogen-fixing actinomycete Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host Alnus glutinosa and cloning of the sodF gene.

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.

Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina).

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism.

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.

Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode.

Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 10(5)-10(6) counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using 15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.

High-resolution phylogenetic analysis of NO2--oxidizing Nitrobacter species using the rrs-rrl IGS sequence and rrl genes.

A high-resolution phylogenetic analysis of Nitrobacter strains and their neighbours was made using the rrs-rrl intergenic spacer sequence and the hypervariable part of the rrl gene. The phylogenetic tree obtained was consistent with that which was obtained previously but was much more discriminating, permitting the design of genus-specific primers.

Microscale diversity of the genus Nitrobacter in soil on the basis of analysis of genes encoding rRNA.

We looked at the diversity of [NO(2)](-) oxidizers at field scale by examining isolates at clump scale and in microsamples of soil (diameter, 50 microm). The genetic distances (as determined by amplified ribosomal DNA restriction analysis performed with Nitrobacter-specific primers) in a small clump of soil were as large as those between reference strains from large geographical areas. Diversity in individual microsamples was shown by serotyping.

DNA relatedness among strains of Streptomyces pathogenic to potato in France: description of three new species, S. europaeiscabiei sp. nov. and S. stelliscabiei sp. nov. associated with common scab, and S. reticuliscabiei sp. nov. associated with netted scab.

The genomic relatedness was evaluated by DNA-DNA hybridization for 23 strains (21 were pathogenic and two were saprophytic strains) isolated from lesions of common and netted scab in France and 19 strains from other countries, including type strains of Streptomyces species. Three genomospecies were defined within the conventional species of Streptomyces scabies, and these genomospecies were different from other pathogenic described species (Streptomyces acidiscabies, Streptomyces caviscabies) based on previously published phenotypic data. Two of these genomospecies (1 and 3) correspond to new species, for which the names Streptomyces europaeiscabiei sp. nov. (with type strain CFBP 4497T) and Streptomyces stelliscabiei sp. nov. (with type strain CFBP 4521T) are proposed. Genomospecies 2 corresponds to S. scabies (with type strain CFBP 4517T = ATCC 49173T), and includes only one French strain. The pathogenic strains associated with netted scab lesions constituted a new species that was named Streptomyces reticuliscabiei sp. nov. (with type strain CFBP 4531T). The G+C content of DNA from the three strains CFBP 4497T (S. europaeiscabiei), CFBP 4521T (S. stelliscabiei), CFBP 4531T (S. reticuliscabiei) was 71.3, 71.0 and 69.8 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that the type strain CFBP 4497T was very similar to the type strain of S. scabies, whereas, the type strain of S. stelliscabiei, CFBP 4521T, was very similar to the type strain of Streptomyces bottropensis. On the basis of 16S rRNA gene sequences, the type strain of S. reticuliscabiei, CFBP 4531T, differed extensively from the other strains of Streptomyces tested.

Stimulation of the ionic transport system in Brassica napus by a plant growth-promoting rhizobacterium (Achromobacter sp.).

A plant growth-promoting rhizobacterium belonging to the genus Achromobacter was isolated from the oil-seed-rape (Brassica napus) root. Growth promotion bioassays were performed with oilseed rape seedlings in a growth chamber in test tubes containing attapulgite and mineral nutrient solution, containing NO3- as N source. The presence of this Achromobacter strain increased shoot and root dry weight by 22-33% and 6-21%, respectively. Inoculation of young seedlings with the Achromobacter bacteria induced a 100% improvement in NO3- uptake by the whole root system. Observations on the seminal root of seedlings 20 h after inoculation showed that there was an enhancement of both the number and the length of root hairs, compared to non-inoculated seedlings. Electrophysiological measurements of NO3- net flux with ion-selective microelectrodes showed that inoculation resulted in a specific increase of net nitrate flux in a root zone morphologically similar in inoculated and non-inoculated plants. The root area increased due to root hair stimulation by the Achromobacter bacteria, which might have contributed to the improvement of NO3- uptake by the whole root system, together with the enhancement of specific NO3- uptake rate. Moreover, inoculated plants showed increased potassium net influx and proton net efflux. Overall, the data presented suggest that the inoculation of oilseed-rape with the bacteria Achromobacter affects the mineral uptake.

A recA gene phylogenetic analysis confirms the close proximity of Frankia to Acidothermus.

The closer proximity of Frankia and Acidothermus cellulolyticus relative to the morphologically close Geodermatophilus found previously was confirmed by resequencing the rrs gene of Acidothermus cellulolyticus and the housekeeping gene, recA. The diagnostic sugar 2-O-methyl-D-mannose was detected only in Frankia, while hopanoid lipids were present at high levels in both Acidothermus and Frankia.

Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov.

The taxonomic position of Photorhabdus strains was examined through the results of DNA relatedness (S1 nuclease method) studies associated with the determination of delta Tm, 16S rRNA phylogenetic inferences and phenotypic characterization, including morphological, auxanographic, biochemical and physiological properties. Three genomic species were delineated on a consensus assessment. One of these species corresponded to Photorhabdus luminescens, since strains were at least 50% related to the type strain of this species with delta Tm less than 7 degrees C. The two other species were novel genomic species II and III, which were less than 40% related to each other with delta Tm higher than 9 degrees C. A comparison of the complete 16S rDNA sequences of several representatives of genomic species II and genomic species III revealed that each of them formed a stable lineage independent of the cluster generated by P. luminescens strains. The genomic species differed in their maximum temperatures for growth. A correlation with the ecological origin of the bacterial samples was noticed. The heat-tolerant group I (maximum growth temperature 35-39 degrees C) corresponded to the symbionts of Heterorhabditis bacteriophora groups Brecon and HP88 and Heterorhabditis indica, nematodes living in warm and tropical countries, respectively. Group II (maximum growth temperature 33-35 degrees C) encompassed symbionts from Heterorhabditis megidis, Heterorhabditis zealandica and group NC1 of H. bacteriophora, nematodes isolated in temperate climates. Group III were bacteria isolated from human specimens. Two new species, Photorhabdus temperata sp. nov. (type strain CIP 105563T) and Photorhabdus asymbiotica sp. nov. (type strain ATCC 43950T), are proposed for genomic species II and III, respectively. Species I and II can be separated into sub-groups on the basis of high DNA-DNA relatedness (more than 80% DNA binding with delta Tm < 1.5 degrees C), 16S rDNA branching and phenotypic characters. Therefore, we propose that the two species P. luminescens and P. temperata should be subdivided into subspecies as follows: P. luminescens subsp. luminescens subsp. nov. (type strain ATCC 29999T), P. luminescens subsp. akhurstii subsp. nov. (type strain CIP 105564T), P. luminescens subsp. laumondii subsp. nov. (type strain CIP 105565T) and P. temperata subsp. temperata subsp. nov.

Disruption of narG, the gene encoding the catalytic subunit of respiratory nitrate reductase, also affects nitrite respiration in Pseudomonas fluorescens YT101.

The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

Comparative phylogeny of rrs and nifH genes in the Bacillaceae.

The rrs (16S rDNA) gene sequences of nitrogen-fixing endospore-forming bacilli isolated from the rhizosphere of wheat and maize were determined in order to infer their phylogenetic position in the Bacillaceae. These rhizosphere strains form a monophyletic cluster with Paenibacillus azotofixans, Paenibacillus polymyxa and Paenibacillus macerans. Two of them (RSA19 and TOD45) had previously been identified as Bacillus circulans (group 2) by phenotypic characterization (API 50CH). Evidence for nitrogen fixation by P. azotofixans, P. polymyxa, P. macerans and putative B. circulans strains RSA19 and TOD45 was provided by acetylene-reduction activity, and confirmed by amplifying and sequencing a nifH fragment (370 nt). The phylogenetic tree of nifH-derived amino acid sequences was compared to the phylogenetic tree of rrs sequences. All Paenibacillus nifH sequences formed a coherent cluster distinct from that of related nitrogen-fixing anaerobic clostridia and Gram-positive high-G+C-content frankiae. The nifH gene was neither detected in the B. circulans type strain (ATCC 4513T) nor in the type strains of Bacillus subtilis, Bacillus cereus, Bacillus alcalophilus, Bacillus simplex, Brevibacillus brevis and Paenibacillus validus. Accordingly, nitrogen fixation among aerobic endospore-forming Firmicutes seems to be restricted to a subset of species in the genus Paenibacillus.

Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium.

Lindane-degrading activity under aerobic conditions has been observed in two bacterial strains: UT26, phenotypically identified as Sphingomonas paucimobilis, and a new single unidentified isolate named RP5557T. The rrs (16S rDNA) sequences for both strains and the phenotypic characteristics for the unidentified isolate RP5557T were determined. RP5557T does not have high identity (less than 90% in all cases) with any sequence in the GenBank or RDP databases. A phylogenetic analysis based on rrs sequences indicated that RP5557T belongs to the gamma-Proteobacteria in a coherent phylum that includes the genera Xanthomonas and Xylella (100% bootstrap), whereas UT26 is clearly separate from the Xanthomonas cluster. Based on the phylogenetic analyses and on the phenotypic characteristics, a new genus, Rhodanobacter, containing a single species, Rhodanobacter lindaniclasticus, is proposed for strain RP5557T (= LMG 18385T), which becomes the type strain.