'Lysobacter enzymogenes ssp. cookii ' Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov.

' Lysobacter enzymogenes ssp. cookii ' was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of ' L. enzymogenes ssp. cookii ' PAGU 1119 (GenBank accession number ATCC29488 ) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsici YC5194 ^T (99.4%) rather than L. enzymogenes DSM2043 ^T (97.2%). The value for whole genome DNA–DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043^T or L. capsici YC5194 ^T was 20.7–26.1% or 60.9–62.0%, respectively. Although PAGU 1119 and L. capsici YC5194 ^T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119^T ( ATCC29488 ).     

Comparison of the phosphatases of Lysobacter enzymogenes with those of related bacteria

Lysobacter enzymogenes ATCC 29487 (UASM 495) produces an outer-membrane-associated phosphatase and an excreted phosphatase. The cell-associated enzyme was compared to phosphatases of nine other Gram-negative gliding bacteria and to that of Escherichia coli. The other three species of the genus Lysobacter also produce a particulate, cell-associated phosphatase. Antiserum prepared against the phosphatase from the outer membrane of L. enzymogenes effectively precipitated the phosphatases of two other L. enzymogenes strains and the enzymes of L. antibioticus, L. brunescens and L. gummosus. Some inhibition of the enzyme by the antiserum also was observed. No significant reaction could be detected between the antiserum and the cell-associated phosphatases of species of Cytophaga johnsonae, 'C. compacta', Myxococcus xanthus, E. coli and the excreted phosphatase of L. enzymogenes. The results indicate that the four species of the genus Lysobacter are closely related despite their physiological differences and that the outer-membrane-associated phosphatases of these organisms have different structural characteristics than the phosphatases of the other Gram-negative bacteria that were used. Furthermore, differences in the amino acid compositions of the cell-associated and the excreted phosphatase of L. enzymogenes confirm the immunological results and are in agreement with the physical and chemical differences noted between the two enzymes.     

Structural Investigations and Identification of the Extracellular Bacteriolytic Endopeptidase L1 from Lysobacter sp. XL1

The N-terminal amino acid sequence (23 amino acid residues) and the amino acid composition of the extracellular bacteriolytic enzyme L1 of 21 kD from the bacterium Lysobacter sp. XL1 have been determined. The enzyme was hydrolyzed by trypsin, the resulting peptides were isolated, and their primary structures were determined. A high extent of homology (92%) of the N-terminal amino acid sequence and the primary structure of isolated peptides of the enzyme L1 (62 amino acid residues or 31% of protein sequence) to the corresponding sites of alpha-lytic proteinases (EC 3.4.21.12) of Lysobacter enzymogenes and Achromobacter lyticus was found. These data allowed identification of the endopeptidase L1 of Lysobacter sp. XL1 as alpha-lytic proteinase EC 3.4.21.12.     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

Functional evenness of N-to-P ratios of evergreen-deciduous mixtures predicts positive non-additive effect on leaf litter decomposition

Plant and Soil - To evaluate the functions of a new biocontrol bacterium, Lysobacter enzymogenes LE16, in the mineralization of soil organic phosphorus (P) and in the stimulation of plant P uptake...     

The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.     

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis

Endoproteinase Lys-C from Lysobacter enzymogenes, which is commercially available, proved to be useful in the determination of primary structures of proteins. The enzyme preferentially cleaves at the carboxyl side of lysine residues.     

The role of clp-regulated factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter enzymogenes C3

A global regulator was previously identified in Lysobacter enzymogenes C3, which when mutated, resulted in strains that were greatly reduced in the expression of traits associated with fungal antagonism and devoid of biocontrol activity towards bipolaris leaf-spot of tall fescue and pythium damping-off of sugarbeet. A clp gene homologue belonging to the crp gene family was found to globally regulate enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes C3 (Kobayashi et al. 2005). Here, we report on the expansion of the biocontrol range of L. enzymogenes C3 to summer patch disease caused by Magnaporthe poae. The clp- mutant strain 5E4 was reduced in its ability to suppress summer patch disease compared with the wild-type strain C3 and was completely devoid of antifungal activity towards M. poae. Furthermore, cell suspensions of 5E4 were incapable of colonizing M. poae mycelium in a manner that was distinct for C3. Strain C3 demonstrated biosurfactant activity in cell suspensions and culture filtrates that was associated with absorption into the mycelium during the colonization process, whereas 5E4 did not. These results describe a novel interaction between bacteria and fungi that intimates a pathogenic relationship.     

Isolation of zoosporogenous actinomycetes from desert soils

We have undertaken a study to estimate the species diversity of zoosporogenous actinomycetes that can be isolated from an arid environment. The study site encompassed an area of approximately 22 000 square kilometers of the Mojave Desert along the California-Nevada border. A series of 29 soil samples was collected along two intersecting transects of approximately 190 and 240 km which traversed a number of distinct ecosystems. A₀ horizon soils were collected from the rhizosphere of the predominant vegetation at each sampling site and screened for the target genera using selective isolation techniques: chemoattraction (xylose and -collidine) and baiting with hair. Following incubation of primary isolation plates for 28 days at 28°C, all colonies that exhibited filamentous growth, presence of sporangia and/or motile spores upon direct microscopic observation (450 and 1000×) were further characterized by fatty acid analysis (FAME). Most of the isolates fell into three broad clusters that roughly correlated with presumptive genus assignments. Individual isolates could be assigned to 226 FAME biotypes based on chromatographic similarity (85%). The dominant species (514/826 isolates) belong to a previously undescribed taxon that morphologically resemblesGeodermatophilus but possesses unique FAME profiles that include at least three novel lipids. The remainder of the isolates were species ofActinoplanes, indeterminate species or vagrant isolates ofStreptomyces.     

<Emphasis Type="Italic">Lysobacter daecheongensis</Emphasis> sp. nov., isolated from sediment of stream near the Daechung dam in South Korea

A Gram-negative, aerobic, rod shaped, non-spore-forming bacterial strain, designated Dae08^T, was isolated from sediment of the stream near Daechung dam in South Korea, and was characterized in order to determine its taxonomic position, using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain Dae08^T belongs to the family Xanthomonadaceae of the Gammaproteobacteria, and is related to Lysobacter brunescens ATCC 29482^T (97.3%). The phylogenetic distances from any other species with validly published names within the genus Lysobacter were greater than 3.7%. The G+C contents of the genomic DNA of strain Dae08^T was 69.3 mol%. The detection of a quinone system with Q-8 as the predominant compound and a fatty acid profile with iso-C_15:0, iso-C_17:1, ω9c, iso-C_17:0, iso-C_16:0, and iso-C_11:0 3-OH as the major acids supported the affiliation of strain Dae08^T to the genus Lysobacter. DNA-DNA relatedness between strain Dae08^T and its phylogenetically closest neighbour was 28%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Dae08^T (= KCTC 12600^T) should be classified in the genus Lysobacter as the novel species, for which the name Lysobacter daecheongensis sp. nov. is proposed.     

Characterization of Armillaria spp. from peach orchards in the southeastern United States using fatty acid methyl ester profiling

Limited information is available regarding the composition of cellular fatty acids in Armillaria and the extent to which fatty acid profiles can be used to characterize species in this genus. Fatty acid methyl ester (FAME) profiles generated from cultures of A. tabescens, A. mellea, and A. gallica consisted of 16–18 fatty acids ranging from 12–24 carbons in length, although some of these were present only in trace amounts. Across the three species, 9-cis,12-cis-octadecadienoic acid (9,12-C18:2), hexadecanoic acid (16:0), heneicosanoic acid (21:0), 9-cis-octadecenoic acid (9-C18:1), and 2-hydroxy-docosanoic acid (OH-22:0) were the most abundant fatty acids. FAME profiles from different thallus morphologies (mycelium, sclerotial crust, or rhizomorphs) displayed by cultures of A. gallica showed that thallus type had no significant effect on cellular fatty acid composition (P > 0.05), suggesting that FAME profiling is sufficiently robust for species differentiation despite potential differences in thallus morphology within and among species. The three Armillaria species included in this study could be distinguished from other lignicolous basidiomycete species commonly occurring on peach (Schizophyllum commune, Ganoderma lucidum, Stereum hirsutum, and Trametes versicolor) on the basis of FAME profiles using stepwise discriminant analysis (average squared canonical correlation = 0.953), whereby 9-C18:1, 9,12-C18:2, and 10-cis-hexadecenoic acid (10-C16:1) were the three strongest contributors. In a separate stepwise discriminant analysis, A. tabescens, A. mellea, and A. gallica were separated from one another based on their fatty acid profiles (average squared canonical correlation = 0.924), with 11-cis-octadecenoic acid (11-C18:1), 9-C18:1, and 2-hydroxy-hexadecanoic acid (OH-16:0) being most important for species separation. When fatty acids were extracted directly from mycelium dissected from naturally infected host tissue, the FAME-based discriminant functions developed in the preceding experiments classified all samples (n = 16) as A. tabescens; when applied to cultures derived from the same naturally infected samples, all unknowns were similarly classified as A. tabescens. Thus, FAME species classification of Armillaria unknowns directly from infected tissues may be feasible. Species designation of unknown Armillaria cultures by FAME analysis was identical to that indicated by IGS-RFLP classification with AluI.     

Comparison of batch culture growth and fermentation of a poultry Veillonella isolate and selected Veillonella species grown in a defined medium

The objective of this study was to develop a defined medium for quantitating nutritional requirements and fermentation products of a poultry cecal isolate of Veillonella and to compare these parameters with representative Veillonella species. The poultry isolate is one of 29 organisms from a continuous-flow culture that has been shown to be effective against Salmonella colonization in broilers. When the Veillonella species were grown in anaerobic batch culture, propionate and acetate were the only volatile fatty acids detected. Lactate was needed to provide energy for the growth of the Veillonella in the defined medium. The poultry isolate had significantly (p< 0.05) higher Y(lactate)(g of dry cell weight per mole of lactate utilized) and dry cell weight than the other Veillonella species when grown on amino acid supplemented defined media. Cultures of the Veillonella species in the defined medium grown with supplemented amino acids aspartate, threonine, arginine, and serine indicated that these amino acids were metabolized to acetate and propionate. Amino acid analysis on media inoculated with either V. atypica or the poultry isolate also indicated that these organisms may have different amino acid preferences. For nearly all of the amino acid supplemented media combinations the poultry isolate utilized significantly (p< 0.05) more threonine and serine whereas V. atypica utilized significantly (p< 0.05) more aspartate. The defined medium supported growth of all of the Veillonella species tested and should enable further in-depth physiological studies to be conducted on the poultry Veillonella studies.     

An Endoreticulatus species from Ocinara lida (Lepidoptera: Bombycidae) in Taiwan

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zw?lfer.     

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

Cultivation-dependent characterization of rhizobacterial communities from field grown Chinese cabbage Brassica campestris ssp pekinensis and screening of traits for potential plant growth promotion

The composition of the bacterial community associated with plant roots is influenced by a variety of plant, environmental factors and also management practices. Our study aimed at detecting the root associated bacterial communities of Chinese cabbage under different fertilization regimes using cultivation dependent methods. The cultivable population was studied using plate count assay, fatty acid methyl ester (FAME) analysis and carbon substrate utilization␣(SU)using BIOLOG™ plates. Taxonomical identification of the isolates by FAME resulted in about 83% identification and they represented 9 and 14 different known bacterial genera from the rhizosphere and root interior respectively from Proteobacteria (α, β, and γ), firmicutes (actinobacteria and the Bacillus groups) and Bacteroidetes. Pseudomonas and Bacillus were associated with the plants grown under all the fertilized conditions and actinobacteria could be observed only in rhizosphere of plants grown on unfertilized plots. FAME and BIOLOG profiles of the rhizosphere and endophytic isolates could separate them with reference to fertilization. Principal component analysis (PCA) on the BIOLOG SU revealed that the isolates were metabolically dissimilar. The diversity, as revealed by the diversity indices was greater among the isolates obtained from unfertilized samples than that of fertilized ones. The isolates analyzed for different traits related to plant growth promotion revealed differences between rhizosphere and endophytic isolates and also with reference to the treatments. The highest percentage of phosphate solubilizing bacteria (PSB) and 1-aminocyclopropane-1-carboxylic acid (ACC) utilizers was recorded in chemical fertilizer treated samples, followed by the organic fertilizer treated. The results from this study indicate that fertilizers have an effect on the root associated bacterial communities of Chinese cabbage and also on their physiological characteristics related to plant growth promotion.