A simple chromatographic route for the isolation of meso diaminopimelic acid.

Meso diaminopimelic acid is an important noncoded amino acid found in Gram-negative bacterial peptidoglycan. In spite of its importance, this stereoisomer is not available commercially. A simple, economical procedure was developed for the isolation of pure meso diaminopimelic acid via an high-performance liquid chromatography separation. In our new approach, the underivatized three isomers of diaminopimelic acid were separated on a crown ether-based chiral stationary phase. For the structure identification, (1)H NMR spectroscopy was applied.

Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method.

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a "meso" configuration such as diaminopimelic acid (A2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the DL-FDLA method, and the resulting elution behavior was summarized as follows: (1) the LL- and meso-isomers were eluted prior to the DD-isomer except for one case; (2) the LL- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the L- and D-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.

Desulfurispora thermophila gen. nov., sp. nov., a thermophilic, spore-forming sulfate-reducer isolated from a sulfidogenic fluidized-bed reactor.

A thermophilic, Gram-positive, endospore-forming, sulfate-reducing bacterium was isolated from a sulfidogenic fluidized-bed reactor treating acidic metal- and sulfate-containing water. The strain, designated RA50E1(T), was rod-shaped and motile. The strain grew at 40-67 degrees C (optimum growth at 59-61 degrees C) and pH 6.4-7.9 (optimum growth at pH 7.0-7.3). The strain tolerated up to 1 % NaCl. Sulfate, sulfite, thiosulfate and elemental sulfur were used as electron acceptors, but not nitrate, nitrite or iron(III). Electron donors utilized were H(2)/CO(2) (80 : 20, v/v), alcohols, various carboxylic acids and some sugars. Fermentative growth occurred on lactate and pyruvate. The cell wall contained meso-diaminopimelic acid and the major respiratory isoprenoid quinone was menaquinone MK-7. Major whole-cell fatty acids were iso-C(15 : 0) and iso-C(17 : 0). Strain RA50E1(T) was distantly related to representatives of the genera Desulfotomaculum, Pelotomaculum, Sporotomaculum and Cryptanaerobacter. On the basis of 16S rRNA gene sequence data, the strain cannot be assigned to any known genus. Based on the phenotypic and phylogenetic features of strain RA50E1(T), it is proposed that the strain represents a novel species in a new genus, for which the name Desulfurispora thermophila gen. nov., sp. nov. is proposed. The type strain of Desulfurispora thermophila is RA50E1(T) (=DSM 16022(T)=JCM 14018(T)).

Bacillus arsenicus sp. nov., an arsenic-resistant bacterium isolated from a siderite concretion in West Bengal, India.

Strain Con a/3(T) is a Gram-positive, motile, endospore-forming, rod-shaped and arsenic-resistant bacterium, which was isolated from a concretion of arsenic ore obtained from a bore-hole. The bacterium grew in the presence of 20 mM arsenate and 0.5 mM arsenite. Diaminopimelic acid was present in the cell wall peptidoglycan, MK-7 was the major menaquinone, and iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0) and C(16 : 1)(delta7cis) were the major fatty acids. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain Con a/3(T) was identified as a member of the genus Bacillus. It exhibited maximum similarity (97 %) at the 16S rRNA gene level with Bacillus barbaricus (DSM 14730(T)); however, the DNA-DNA relatedness value with B. barbaricus was 60 %. Strain Con a/3(T) also exhibited a number of phenotypic differences from B. barbaricus (DSM 14730(T)). Strain Con a/3(T) was therefore identified as representing a novel species of the genus Bacillus, for which the name Bacillus arsenicus sp. nov. is proposed. The type strain is Con a/3(T) (= MTCC 4380(T) = DSM 15822(T) = JCM 12167(T)).

Janibacter melonis sp. nov., isolated from abnormally spoiled oriental melon in Korea.

Two Gram-positive bacterial strains, CM2104(T) and CM2110, isolated from the inner part of abnormally spoiled oriental melon (Cucumis melo) in Korea, were subjected to a polyphasic taxonomic study. The cell-wall peptidoglycan of strains CM2104(T) and CM2110 contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was MK-8(H(4)). The major fatty acids detected in the two strains were iso-C(16 : 0), C(17 : 1)omega8c and C(18 : 1)omega9c or C(17 : 0). The DNA G+C content of the two strains was 73 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a coherent cluster with a clade comprising two Janibacter species, Janibacter limosus and Janibacter terrae. Strains CM2104(T) and CM2110 exhibited a 16S rRNA gene sequence similarity value of 99.7 % and a mean DNA-DNA relatedness level of 89 %. Strains CM2104(T) and CM2110 showed 16S rRNA gene sequence similarity levels of 97.8-98.4 % to the type strains of J. limosus and J. terrae. DNA-DNA relatedness between strains CM2104(T) and CM2110 and the type strains of these two Janibacter species was 7-11 %. On the basis of the phenotypic and phylogenetic data and genomic distinctiveness, strains CM2104(T) and CM2110 should be placed within the genus Janibacter as members of a novel species, for which the name Janibacter melonis sp. nov. is proposed. The type strain is CM2104(T) (=KCTC 9987(T)=DSM 16063(T)=JCM 12321(T)).

Planotetraspora silvatica sp. nov. and emended description of the genus Planotetraspora.

An actinomycete that developed sporangia containing four spores in a single row at the ends of short sporangiophores on branched aerial hyphae was isolated from subtropical forest soil. The isolate contained menaquinone MK-9(H(4)), glutamic acid, alanine and meso-diaminopimelic acid as cell-wall amino acids and madurose in the whole-cell hydrolysate. The 16S rRNA gene sequence indicated that the isolate formed a monophyletic cluster with Planotetraspora mira. On the basis of morphological and chemotaxonomic characteristics, phylogenetic analysis and DNA-DNA relatedness data, a novel species of the genus Planotetraspora is proposed, Planotetraspora silvatica sp. nov. (type strain, TT 00-51(T)=NBRC 100141(T)=DSM 44746(T)).

Brevibacterium celere sp. nov., isolated from degraded thallus of a brown alga.

Two whitish yellow, Gram-positive, non-motile, aerobic bacteria were isolated from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens. The bacteria studied were chemo-organotrophic, mesophilic and grew well on nutrient media containing up to 15 % (w/v) NaCl. The DNA G+C content was 61 mol%. The two isolates exhibited a conspecific DNA-DNA relatedness value of 98 %, indicating that they belong to the same species. A comparative analysis of 16S rRNA gene sequences revealed that strain KMM 3637(T) formed a distinct phyletic lineage in the genus Brevibacterium (family Brevibacteriaceae, class Actinobacteria) and showed the highest sequence similarity (about 97 %) to Brevibacterium casei. DNA-DNA hybridization experiments demonstrated 45 % binding with the DNA of B. casei DSM 20657(T). Physiological and chemotaxonomic characteristics (meso-diaminopimelic acid in the peptidoglycan, major cellular fatty acids 15 : 0ai and 17 : 0ai) of the bacteria studied were consistent with the genomic and phylogenetic data. On the basis of the results of this study, a novel species, Brevibacterium celere sp. nov., is proposed. The type strain is KMM 3637(T) (=DSM 15453(T)=ATCC BAA-809(T)).

Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce.

Five strains of Gram-positive, endospore-forming, moderately halotolerant bacteria were studied taxonomically. Four were isolated from Vietnamese fish sauce and one from the Gulf of Mexico. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains clustered within the radiation of the genus Bacillus but separately from recognized Bacillus species. DNA G+C composition of the isolates ranged from 43 to 44 mol%. Strains 15-1(T) and NRRL B-14850 showed high levels of DNA-DNA relatedness (82-100 %) to each other and to the other strains isolated here; they displayed low levels of DNA-DNA relatedness (<29 %) to the type strains of selected recognized Bacillus species. They grew in 15 % NaCl and optimally in 1 % NaCl, which is characteristic of moderately halotolerant bacteria. The isolates grew at pH 6.5 to 10.0 but not at pH 6.0. Their cell walls contained meso-diaminopimelic acid. The major isoprenoid quinone was MK-7 and the principal cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). Based on these results, the strains tested were regarded as members of a novel Bacillus species for which the name Bacillus vietnamensis sp. nov. is proposed. The type strain is 15-1(T) (=JCM 11124(T)=NRIC 0531(T)=NRRL 23890(T)).

The cell wall of Micromonospora purpurea contains a high conductance channel.

In this communication it is demonstrated that the cell wall of the gram-positive bacterium Micromonospora purpurea contains a cell wall channel for the passage of hydrophilic solutes. The channel-forming protein was identified in sucrose step-density-gradient fractions of the cell envelope and in whole cell extracts using either organic solvent or detergent and the lipid bilayer technique. The fractions of the sucrose step-density centrifugation were assayed for NADH-oxidase activity and for the formation of ion-permeable channels in lipid bilayers. The highest NADH-oxidase activity and the highest channel-forming ability were found in different fractions. The cell wall fraction was identified by the presence of meso-diaminopimelic acid and contained an ion-permeable channel with the extremely high single-channel conductance of about 14 nS in 1 M KCl. The channel-forming unit was purified to homogeneity by FPLC on a HiTrap-Q column. It was identified as a heat- and SDS-resistant 200-kDa band on SDS-PAGE and formed the same general diffusion pores in lipid bilayer membranes as those formed by detergent extracts of the cell wall fraction of the sucrose step-density centrifugation. The channels were slightly selective for potassium ions over chloride, possibly caused by an excess of negative charges in or near the channel.

New LL-diaminopimelic acid-containing actinomycetes from hypersaline, heliothermal and meromictic Antarctic Ekho Lake: Nocardioides aquaticus sp. nov. and Friedmanniella [correction of Friedmannielly] lacustris sp. nov.

Two Gram-positive, non-motile and aerobic bacteria were isolated from a water sample of the hypersaline Ekho Lake, Antarctica. The cocci or short rods grew well on oligotrophic PYGV agar of pH 7.5 and at 26 degrees C. Strains EL-17KT and EL-17AT both required thiamine and biotin, strain EL-17AT also required nicotinic acid. Carbon sources utilized by both strains were acetate, pyruvate, alpha-D-glucose, glutamate and (weakly) citrate, but succinate, malate or butyrate were utilized only by EL-17KT. Gelatin, starch and DNA were hydrolyzed, NH, was formed from peptone, and nitrate was reduced aerobically by both strains. The isolates had the same temperature tolerance for growth in the range tested (below 3 to above 33.5 degrees C) and pH range (<5.5 to >9.5) and were sensitive to chloramphenicol and penicillin G. Their cell walls contained LL-diaminopimelic acid and had a single glycine residue as interpeptide bridge. Strain EL-17AT contained glycine at position 1 of the peptide subunit (peptidoglycan type A 3gamma'). Isolates EL-17KT and EL-17AT differed in their maximum NaCl tolerance, which was 15% or 6-8%, respectively. The major fatty acid of EL-17KT was C18:1 and that of EL-17AT was ai-C15:0. The major respiratory quinones of EL-17KT and EL-17AT were MK-8(H4) and MK-9(H4), respectively. The former isolate had 69 mol% G+C, the latter had 73 mol% G+C. Comparative 16S rRNA gene sequencing revealed phylogenetic relationships of isolate EL-17KT with the genus Nocardioides, with N. pyridinolyticus and N. plantarum as the closest relatives. Phenotypic and genotypic characteristics support the description of a new species, Nocardioides aquaticus sp. nov., with EL-17KT as the type strain (= DSM 11439T). Isolate EL-17AT is related to the genus Friedmanniella, with E antarctica and E spumicola as the closest relatives. The differentiating characteristics support the description of a new species, Friedmanniella lacustris sp. nov., with EL-17AT as the type strain (= DSM 11465T).

Measurement of total and separate stereoisomers of diaminopimelic acid in rumen bacteria by high-performance liquid chromatography.

New high-performance liquid chromatographies were examined and applied for the analyses of the total (with one peak) and separate three stereoisomers of 2,6-diaminopimelic acid (DAP) in hydrolysed mixed rumen bacteria. The methods start with the reaction of DAP with 1-fluoro-2,4-dinitrophenylalanine amide for derivatisation. A mixture of 0.05 M triethylamine phosphate (pH 3.0) and acetonitrile (72:28, v/v) was used as an isocratic mobile phase for the total DAP determination (method 1), and a mixture of both solutions (78.5:21.5, v/v) for the determination of the separate three stereoisomers of DAP (method 2). The flow-rate was 1 ml/min; column, Merck LiChrospher 100 RP-18 (250 x 4 mm I.D.) of 5 microns particle size; column temperature, 40 degrees C; wavelength of detector, 325 nm. The retention times were 17.5 min for total DAP (method 1), and 39.4, 63.1 and 77.7 for meso-, LL- and DD-DAP, respectively (method 2). Lysine can also be determined by method 2 and the retention time was 122.8 min. The minimum detectable limit was 2.5 microM. The average analytical recoveries were 98.6% for total DAP and 99.1%, 100.4% and 100.2% for meso-, LL- and DD-DAP, respectively. The content of total DAP of the hydrolysed rumen bacteria collected from three goats fed lucerne cube and concentrate ranged from 25.55 to 27.36 mumol/g bacterial DM (method 1). The contents of the separate stereoisomers of DAP in the hydrolysed rumen bacteria from the three goats ranged from 19.64 to 22.06 and from 4.98 to 5.21 mumol/g bacterial DM for meso- and LL-DAP, respectively (method 2). DD-DAP was not detected.

Purification and properties of diaminopimelic acid epimerase from Escherichia coli.

Diaminopimelic acid epimerase was purified from Escherichia coli. The enzyme is a monomer of Mr = 34,000. Diaminopimelic acid epimerase is not a pyridoxal phosphate-dependent enzyme: there is no evidence for pyridoxal phosphate in the ultraviolet spectrum of the purified enzyme, and the epimerase is not inactivated by carbonyl reagents such as hydroxylamine and sodium borohydride. Exchange of the alpha-protons of the substrates, DL- and LL-diaminopimelic acid, with solvent accompanies epimerization; and exchange of 3H from solvent into diaminopimelic acid gives 3H primarily (80-90%) in the product isomer, regardless of whether the DL- or LL-isomer is substrate. From these results it is concluded that the epimerase utilizes a two-base mechanism for proton translocation. In these major aspects of its mechanism, diaminopimelic acid epimerase resembles proline racemase. It is argued that the relative values of the isotope fractionation factors for the two proton acceptor sites on the enzyme can be estimated from the isotope effects for the DL- and LL-isomers of diaminopimelic acid. The observed difference in the isotope effects predicts that one, but not both, of the proton acceptor sites is a thiol, and it is demonstrated that diaminopimelic acid epimerase has a single thiol which is necessary for activity and which reacts with iodoacetamide.

Studies on a new immunoactive peptide, FK-156. II. Fermentation, extraction and chemical and biological characterization.

An interesting immunoactive peptide, FK-156, has been isolated from the fermentation broth of Streptomyces olivaceogriseus sp. nov. and Streptomyces violaceus. The compound was purified by column chromatography with activated carbon, ion exchange Sephadex and cellulose powder. FK-156, obtained as white powder, exhibits a wide variety of immunostimulatory activity in vivo and in vitro with experimental animals. Pretreatment of mice or rats with this peptide protected the animals against lethal infection with Escherichia coli and resulted in prolongation of life span of tumor bearing animals.

Isolation of N-(2,6-diamino-6-hydroxymethylpimelyl)-L-alanine from Micromonospora chalcea.

A novel dipeptide, N-(2,6-diamino-6-hydroxymethylpimelyl)-L-alanine, was isolated from the culture broth of a microorganism identified as Micromonospora chalcea. The dipeptide exhibits antimicrobial activity against Escherichia coli on a synthetic medium, and the activity is synergistically enhanced by several cell wall synthesis-inhibitors.

Use of auxotrophic mutants to isolate LL- or DD-isomers of 2,6-diaminopimelic acid.

Pseudomonas aeruginosa PAC7 (a mutant deficient in diaminopimelate epimerase), excreted diaminopimelate (solely LL-isomer) after growth in a minimal medium plus lysine with succinate as carbon source. More diaminopimelate was excreted when bacteria were transferred at the end of the exponential phase of growth into fresh minimal medium without lysine but supplemented with pyrivate and additional (NH4)2SO4. The excreted LL-isomer was isolated from the culture filtrate by ion-exchange chromatography and purified by crystallization (1.7 g/9 1 culture). A diaminopimelate-requiring mutant of Bacillus megaterium NCIB7581 grew on LL- and/or meso-diaminopimelate but not on the DD-isomer. This mutant was used to isolate the DD-idomer from a mixture of synthetic LL- and DD-diaminopimelate. It was grown in a minimal medium containing glycerol as carbon source and LL- plus DD-diaminopimelate at a growth-limiting concentration (300 mg l-1); when growth stopped, the DD-diaminopimelate that remained in the culture was isolated and crystallized (1.0 g/II l culture).