Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Ume?), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Ume? (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.     

Analysis of cellular fatty acids in Orientia tsutsugamushi as taxonomic markers

Six Orientia strains including 3 prototype strains such as Gilliam, Karp, and Kato, and 3 strains (Boryong, Pajoo, and Yongworl) isolated in Korea, were studied for the profiles of their cellular fatty acids. All tested strains contained octadecenoic acid C (18: 1) omega 9 c(57.3 +/- 3.5%), octadecanoic acid C (18: 0) (15.3 +/- 1.5%), and hexadecanoic acid C (16: 0) (12.7 +/- 1.7%) as major components; however, interestingly, eicosenoic acid C (20: 1) omega 9 c(2.6 +/- 0.6%) was found in all strains except the Yongworl strain. Furthermore none of the strains contained 3-hydroxy fatty acids. The ratios of total saturated fatty acid (SFA) to total unsaturated fatty acid (UFA) were within the range of 0.34 to 0.54. These results showed that the cellular fatty acid profile should provide more reliable information for the identification of these bacteria.     

Polar lipid and fatty acid profiles – Re-vitalizing old approaches as a modern tool for the classification of mycoplasmas?

A set of 20 Mollicutes strains representing different lines of descent, including the type species of the genus Mycoplasma, Mycoplasma mycoides, Acholeplasma laidlawii and a strain of Mesoplasma, were subjected to polar lipid and fatty acid analyses in order to evaluate their suitability for classification purposes within members of this group. Complex polar lipid and fatty acid profiles were detected for each examined strain. All strains contained the polar lipids phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (MfGL-I), 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine (MfEL), sphingomyelin (SphM), 1-O-alkyl/alkenyl-glycero-3-phosphocholine (lysoMfEL), the unknown aminophospholipid APL1 and the cholesterol Chol2. A total of 19 strains revealed the presence of phosphatidylethanolamine (PE) and/or phosphatidylglycerol (PG), and the presence of diphosphatidylglycerol (DPG) was detected in 13 strains. The unknown aminolipid AL1 was found in the extracts of 17 strains. Unbranched saturated and unsaturated compounds predominated in the fatty acid profiles. Major fatty acids were usually C16:0, C18:0, C18:1 omega9c and 'Summed feature 5' (C18:2 omega6, 9c/C18:0 anteiso). Our results demonstrated that members of the M. mycoides cluster showed rather homogenous polar lipid and fatty acid profiles. In contrast, each of the other strains was characterized by a unique polar lipid profile and significant quantitative differences in the presence of certain fatty acids. These results indicate that analyses of both polar lipid and fatty acid profiles could be a useful tool for classification of mycoplasmas.     

Whole cell fatty acid patterns of Xenorhabdus species

Thirty-three strains of the nematode-associated bacterium Xenorhabdus were characterized by traditional biochemical tests and whole cell fatty acid analysis. In traditional tests 26 strains were found to belong to X. luminescens and 7 to X. nematophilus (sensu latu). No further subdivision could be made. In fatty acid analysis, however, X. luminescens strains could be divided into three subgroups. The amount of distinction in fatty acids is similar to that at subspecies or species level found in other bacteria. Xenorhabdus nematophilus could be clearly differentiated from X. luminescens , key acids are 12: 0, 15: 0 iso, 16: 0, 17: 0 iso, 17: 0 cyclo, 18: 1 cis 11 and 19: 0 cyclo. Separation is almost at genus level. The presence of branched and hydroxy acids in Xenorhabdus and its aberrant morphology make the placement of this genus in the Enterobacteriaceae questionable. This is the first report on fatty acid profiles of Xenorhabdus species.     

Use of Cellulose Acetate Electrophoresis in the Taxonomy of Steinernematids (Rhabditida,Nematoda)

A steinernematid nematode was isolated from soil samples collected near St. John''s, Newfoundland, Canada. On the basis of its morphometry and RFLPs in ribosomal DNA spacer, it was designated as a new strain, NF, of Steinernema feltiae. Cellulose acetate electrophoresis was used to separate isozymes of eight enzymes in infective juveniles of S. feltiae NF as well as four other isolates: S. feltiae Umeå strain, S. feltiae L1C strain, Steinernema carpocapsae All strain, and Steinernema riobravis TX strain. Based on comparisons of the relative electrophoretic mobilities (μ) of the isozymes, one of the eight enzymes (arginine kinase) yielded zymograms that were distinctive for each of the isolates, except for the Umeå and NF strains of S. feltiae, which had identical banding patterns. Four enzymes (fumarate hydratase, phosphoglucoisomerase, phosphoglucomutase, and 6-phosphogluconate dehydrogenase) yielded isozyme banding patterns that were characteristic for all isolates, except for the L1C and NF strains of S. feltiae, which were identical. Two enzymes (aspartate amino transferase and glycerol-3-phosphate dehydrogenase) yielded zymograms that permitted S. carpocapsae All strain to be discriminated from the other four isolates, while the remaining enzyme (mannose-6-phosphate isomerase) was discriminatory for S. riobravis TX strain. Except for one enzyme, the isozyme banding pattern of the NF isolate of S. feltiae was the same as in the L1C strain, isolated 13 years previously from Newfoundland. Cellulose acetate electrophoresis could prove invaluable for taxonomic identification of isolates of steinernematids, provided that a combination of enzymes is used.     

Intraspecies variability of cellular fatty acids among soil and intestinal strains of Desulfovibrio desulfuricans.

A comparison of cellular fatty acid profiles of Desulfovibrio desulfuricans DSM 642 and 14 wild strains of this species, isolated from two completely different environments, soil and the human intestine, was carried out. All the D. desulfuricans strains grown on lactate and sulfate indicated the presence of considerable amounts of i-C15:0, i-C17:1 and C16:0. Although differences in the quantities of individual fatty acids present in each strain were clear in the group of soil strains (similarity, 67.6%), in contrast to almost identical fatty acid patterns (similarity, near 100%) in the intestinal strains, the results were variable within the limits acceptable for species demonstration. The higher similarity of the fatty acid profiles of intestinal strains may be a result of the similarity of biocenoses in the human digestive tract. The coefficients of variability of i-C17:1 and i-C15:0 (the major branched-chain fatty acids), as well as clustering of the investigated strains compared with strains described in the literature after plotting percentages of i-C17:1 fatty acid against i-C15:0 fatty acid, confirmed a certain heterogeneity of cellular fatty acid profiles within the group of soil strains, in contrast to almost ideal homogeneity within the group of intestinal isolates. Intestinal strains contained a higher ratio of saturated to unsaturated fatty acids (2.2 +/- 0.14) than did soil strains (1.6 +/- 0.2; in one case, 2.7). We propose that intestinal D. desulfovibrio bacteria should be assumed to be a highly homogeneous group and should be represented by the strain D. desulfuricans subsp. intestinus in collections of microbial cultures.     

Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring

During screening for amylase-producing bacteria, a strain designated OnlT was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growth were 55 degrees C and pH 7. Strain On1(T) grew well in minimal medium containing starch as the sole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1(T) is a member of beta-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1(T) were 16:0 (about 30%), 18:1 omega 7c (about 20%) and summed feature 3 (16:1 omega 7c or 15:0 iso 2OH or both [about 31%]). Its DNA base ratio was 65.9 mol% G + C. We propose to classify strain On1(T) (= BCRC 17405T = LMG 22827T) as Caldimonas taiwanensis sp. nov.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20: 4 omega 6) can stimulate platelet aggregation after conversion to prostaglandin G2 and H2 and thence to thromboxane A2. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca2+-free buffer. Their stimulation by C 20: 4 6 was followed by 14C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca2+ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [14C]-5HT release. Other C 20 polyunsaturated fatty acids could not substitute for C 20: 4 omega 6 in inducing aggregation, and Ca2+ was found to be a prerequisite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use of the lipoxygenase inhibitor BW 755 C did not prevent C 20: 4 omega 6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these conditions, C 20: 4 omega 6-induced platelet aggregation might be due to an increased membrane permeability to Ca2+ induced by this fatty acid in the absence of oxidation.     

Diaphorobacter nitroreducens gen nov,sp nov,a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge

Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized. All of the isolates had almost identical phenotypic characteristics. They were motile gram-negative rods with single polar flagella and grew well with simple organic compounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions. However, none of the sugars tested supported their growth. The cellular fatty acid profiles showed the presence of C16:1omega7cis and C16:0 as the major components and of 3-OH-C10:0 as the sole component of hydroxy fatty acids. Ubiquinone-8 was detected as the major respiratory quinone. A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of beta-Proteobacteria, but formed no monophyletic cluster with any previously known species of this family. The closest relative to our strains was an unidentified bacterium strain LW1 (=DSM 13225) (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium. DNA-DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50%. The G+C content of genomic DNA of the new strains was 64 to 65 mol%. Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we propose the name Diaphorobacter nitroreducens. The type strain is strain NA10B (=JCM 11421=CIP 107294). We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter.     

Erythrobacter vulgaris sp. nov., a novel organism isolated from the marine invertebrates

Four yellow-pigmented, gram-negative, chemoorganotrophic aerobic bacteria were isolated from starfish Stellaster equestris (strains 022-2-10T, 022-2-9, and 022-2-12) and soft coral (unidentified species) (strain 022-4-7) collected in the South China Sea. 16S rRNA gene sequence-based analyses of the new organisms revealed that Erythrobacter spp. were the closest relatives and shared the highest similarity of 98.7% to E. citreus, 98.5% to E. flavus, 97.9% to E. litoralis and 97.6% to E. longus. The novel organisms were tolerant to 3-6% NaCl, grew between 10 degrees C and 40 degrees C, and were not able to degrade gelatin, casein, and agar, while degraded Tween 80. Two strains (022-2-9 and 022-2-12) could weakly degrade starch. All strains produced a large pool of carotenoids and did not have Bacteriochlorophyll a. Phosphatidylethanolamine (30-36%), phosphatidylglycerol (39-46%), and phosphatidylcholine (21-27%) were the predominant phospholipids. Sphingoglycolipid was not detected. The major fatty acids were 16:0 (6-11%), 16:1omega7 (12-15%), and 18:1omega7 (46-49%). The two-hydroxy fatty acids, 13:0-2OH, 14:0-2OH, 15:0-2OH, 16:0-2OH were also present. The G + C content of the DNAs ranged from 61 to 62 mol%. The level of DNA similarity among four strains was conspecific and ranged from 94% to 98%. Even though new strains and other species of the genus had rather high level of 16S rRNA gene sequence similarities, DNA-DNA hybridization experiments showed only 33-39% of binding with the DNA of the type strains. On the basis of these results and the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the new organisms be classified as a novel species; the name Erythrobacter vulgaris sp. nov. is proposed. The type strain is 022-2-10T (= KMM 3465T = CIP 107841T).     

Protein synthesis and degradation in isolated muscle. Effect of omega 3 and omega 6 fatty acids.

The ability of derivatives of the essential fatty acids linoleic acid (C18:2, omega 6) and alpha-linolenic acid (C18:3, omega 3) to stimulate rates of protein synthesis and degradation was investigated in isolated intact muscles from fasted rabbits. Both omega 6 derivatives examined, arachidonic acid (C20:4, omega 6) and dihomo-gamma-linolenic acid (C20:3, omega 6), when added at concentrations up to 1 microM, stimulated the rate of protein synthesis and the release of prostaglandin F2 alpha (PGF2 alpha). Metabolites of the omega 6 series, namely eicosapentaenoic acid (C20:5, omega 3) and docosahexaenoic acid (C22:6, omega 3), were without effect on the rate of protein synthesis and resulted in a decrease in the release of PGF2 alpha. None of the fatty acids had a significant effect on the rate of protein degradation. Although insulin (100 mu units/ml) also stimulated rates of protein synthesis when added alone, none of the omega 3 or omega 6 fatty acids, when added with insulin at concentrations of 0.2 microM, potentiated the effect of the hormone.     

A novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus indica sp. nov., symbiotically associated with entomopathogenic nematode Steinernema thermophilum Ganguly and Singh, 2000

In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28(T) = DSM 17382(T), strain 42 = DSM 17383 and strain 43 = DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28(T) and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28(T) were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28(T), DSM 17382(T), CIP 108830(T).     

Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions

Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 degrees C) with or without NaCl (7%), and their fatty acid compositions were analysed. The results showed that low thermal adaptation response of L. monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids. However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0. In hyperosmotic medium and over the entire temperature range (4 degrees C, 20 degrees C and 37 degrees C) the two L. monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0. In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain. These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.     

Desulfobulbus mediterraneus sp. nov., a sulfate-reducing bacterium growing on mono- and disaccharides

A new sulfate-reducing bacterium, strain 86FS1, was isolated from a deep-sea sediment in the western Mediterranean Sea with sodium lactate as electron and carbon source. Cells were ovoid, gram-negative and motile. Strain 86FS1 contained b- and c-type cytochromes. The organism was able to utilize propionate, pyruvate, lactate, succinate, fumarate, malate, alanine, primary alcohols (C(2)-C(5)), and mono- and disaccharides (glucose, fructose, galactose, ribose, sucrose, cellobiose, lactose) as electron donors for the reduction of sulfate, sulfite or thiosulfate. The major products of carbon metabolism were acetate and CO(2), with exception of n-butanol and n-pentanol, which were oxidized only to the corresponding fatty acids. The growth yield with sulfate and glucose or lactate was 8.3 and 15 g dry mass, respectively, per mol sulfate. The temperature limits for growth were 10 degrees C and 30 degrees C with an optimum at 25 degrees C. Growth was observed at salinities ranging from 10 to 70 g NaCl l(-1). Sulfide concentrations above 4 mmol l(-1) inhibited growth. The fatty acid pattern of strain 86FS1 resembled that of Desulfobulbus propionicus with n-14:0, n-16:1omega7, n-16:1 omega5, n-17:1 omega6 and n-18:1 omega7 as dominant fatty acids. On the basis of its phylogenetic position and its phenotypic properties, strain 86FS1 affiliates with the genus Desulfobulbus and is described as a new species, Desulfobulbus mediterraneus sp. nov.     

Production of 5,8,11-Eicosatrienoic Acid (Mead Acid) by a (Delta)6 Desaturation Activity-Enhanced Mutant Derived from a (Delta)12 Desaturase-Defective Mutant of an Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3(omega)9) was attained by a mutant fungus, Mortierella alpina M209-7, derived from (Delta)12 desaturase-defective M. alpina Mut48. The 20:3(omega)9 production by M209-7 was 1.3 times greater than that by its parent strain, Mut48. This is thought to be due to its enhanced (Delta)6 desaturation activity, which was 1.4 times higher than that of Mut48. In both strains, 87 to 88% of the total lipids comprised triacylglycerol (TG) and 85% of 20:3(omega)9 was contained in TG. On optimization of the culture conditions for M209-7, earlier glucose feeding and shifting of the growth temperature from 28 to 19(deg)C on the second day were shown to be effective. Under the optimal conditions with a 10-liter jar fermentor, 20:3(omega)9 production reached 1.65 g/liter of culture medium (corresponding to 118 mg/g of dry mycelia and 28.9% of total fatty acids), which is about twice that reported previously (0.8 g/liter).     

Production of 5,8,11-eicosatrienoic acid by a Δ5 and Δ6 desaturation activity-enhanced mutant derived from a Δ12 desaturation activity-defective mutant of Mortierella alpina 1S-4

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from delta12 desaturation activity-defective and delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28 degrees C to 20 degrees C on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-l jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).     

Paracoccus bengalensis sp. nov., a novel sulfur-oxidizing chemolithoautotroph from the rhizospheric soil of an Indian tropical leguminous plant

Paracoccus versutus-like isolates from the rhizosphere of Clitoria ternatea, a slender leguminous herb (family--Papilionaceae), found ubiquitously in waste places and village forests of the Lower Gangetic plains of India, presented a case of graduated infraspecific variation that was capped by the identification of a new species Paracoccus bengalensis (type strain JJJ(T) = LMG 22700(T) = MTCC 7003(T)). The diverged phenetic and genetic structure of these sulfur-oxidizing chemolithoautotrophs presented a case of apparent nonconformity of 16S rRNA gene sequence similarities with results of DNA-DNA hybridization. Despite high 16S rRNA gene sequence similarity with P. versutus one of the newly isolated strains, viz., JJJ(T) was identified as a new species of Paracoccus by virtue of its explicitly low DNA-DNA hybridization (42-45%) with the type strain of the closest species P. versutus (), distinct G + C content (65.3 mol%), physiological and biochemical differences amounting to <60% phenetic similarity with strains of P. versutus as well as new isolates akin to the species. The newly described species also had a unique fatty acid profile that was distinguished by the absence of 18:1 omega9c, unique possession of Summed feature 3 (16:1omega7c & 15:0 iso 2-OH), 19:0 10 methyl, and a much higher concentration of 19:0 cycloomega8c.     

Novosphingobium naphthalenivorans sp. nov., a naphthalene-degrading bacterium isolated from polychlorinated-dioxin-contaminated environments

Three strains of strictly aerobic, Gram-negative, naphthalene-degrading bacteria isolated from polychlorinated-dioxin-contaminated soil and sediment were characterized. These isolates grew well with naphthalene as the sole carbon and energy source, degrading it completely within 24 h of incubation. The isolates also degraded dibenzofuran co-metabolically in the presence of naphthalene with the concomitant production of yellow intermediate metabolite(s). A 16S rRNA gene sequence analysis revealed that the isolates affiliated to the genus Novosphingobium with Novosphingobium pentaromativorans and Novosphingobium subarcticum as their nearest phylogenetic neighbors (97.4-97.5% similarity). The isolates had a genomic DNA G+C ratio of 64.5-64.6 mol% and formed a genetically coherent group distinguishable from any established species of the genus Novosphingobium at a DNA-DNA hybridization level of less than 46%. The cellular fatty acids were characterized by the predominance of 18 : 1omega7c with significant proportions of 16 : 0, 16 : 1omega7c, 17 : 1omega6c and 2-OH 14 : 0. Sphingoglycolipids were present. The major respiratory quinone was ubiquinone-10. Spermidine was detected as the major polyamine. The distinct taxonomic position of the isolates within the Novosphingobium was also demonstrated by physiological and biochemical testing. Based on these phylogenetic and phenotypic data, we propose Novosphingobium naphthalenivorans sp. nov. to accommodate the novel isolates. The type strain is strain TUT562(T) (DSM 18518(T), JCM 13951(T), NBRC 102051(T)).     

Hansschlegelia plantiphila gen. nov. sp. nov., a new aerobic restricted facultative methylotrophic bacterium associated with plants

A new genus, Hansschlegelia, and a new species, Hansschlegelia plantiphila, are proposed for three strains of methanol-utilizing bacteria isolated from lilac buds (strain S(1)(T)), linden buds (strain S(2)) and blue spruce needles (strain S(4)), which were selected in winter at -17 degrees C. These bacteria are aerobic, Gram-negative, colorless, non-motile short rods that multiply by binary fission and employ the ribulose bisphosphate (RuBP) and the serine pathways for C(1) assimilation. The strains have a limited number of growth substrates and use methanol, methylamine, formate, CO(2)/H(2) and glycerol as carbon and energy sources. Only strain S(1)(T) grows with ethanol and inulin. The strains are neutrophilic and mesophilic, and synthesize phytohormones (auxins and cytokinins) and vitamin B(12). Their major cellular fatty acids are saturated C(16:0), straight-chain, unsaturated C(18:1)(omega)(7) and cyclopropane C(19 cyc) acids. The main ubiquinone is ubiquinone-10 (Q-10). The dominant phospholipids are phosphatidylethanolamine, phosphatidylcholine and diphosphatidylglycerol (cardiolipin). The DNA G+C content is 68.5+/-0.2 mol%. The strains share almost identical 16S rRNA gene sequences, a high DNA-DNA hybridization value (72-86%) and represent a novel lineage of autotrophic methanol-utilizing bacteria within the Alphaproteobacteria. Collectively, these strains comprise a new genus and species H. plantiphila gen. nov., sp. nov., with strain S(1)(T) (VKM B-2347(T), NCIMB 14035(T)) as the type strain.     

Distribution of Polyunsaturated Fatty Acids in Bacteria Present in Intestines of Deep-Sea Fish and Shallow-Sea Poikilothermic Animals

The lipid and fatty acid compositions in nine obligate and facultative barophilic bacteria isolated from the intestinal contents of seven deep-sea fish were determined. Phospholipid compositions were simple, with phosphatidylethanolamine and phosphatidylglycerol predominating in all strains. Docosahexaenoic acid (DHA; 22:6n-3), which has not been reported in procaryotes except for deep-sea bacteria, was found to be present in eight strains at a level of 8.1 to 21.5% of total fatty acids. In the other strain, eicosapentaenoic acid (EPA; 20:5n-3) was present at a level of 31.5% of total fatty acids. Other fatty acids observed in all strains were typical of marine gram-negative bacteria. Subcultures from pouches prepared from intestinal contents of five deep-sea fish by the most-probable-number (MPN) method were analyzed for fatty acids, and all subcultures contained DHA and/or EPA. Accordingly, viable cell counts of bacteria containing DHA and EPA were estimated at a maximum of 1.3 x 10(sup8) and 2.4 x 10(sup8) cells per ml, respectively, and accounted for 14 and 30%, respectively, of the total cell counts in the intestinal contents of the deep-sea fish. In the case of 10 shallow-sea poikilothermic animals having bacterial populations of 1.1 x 10(sup6) to 1.9 x 10(sup9) CFU per ml in intestinal contents, no DHA was found in the 112 isolates examined, while production of EPA was found in 40 isolates from cold- and temperate-sea samples. These results suggest that DHA and EPA are involved in some adaptations of bacteria to low temperature and high pressure.