<Emphasis Type="Italic">Streptomyces sudanensis</Emphasis> sp. nov., a new pathogen isolated from patients with actinomycetoma

Nine strains isolated from mycetoma patients and received as Streptomyces somaliensis were the subject of a polyphasic taxonomic study. The organisms shared chemical markers consistent with their classification in the genus Streptomyces and formed two distinct monophyletic subclades in the Streptomyces 16S rRNA gene tree. The first subclade contained four organisms, including the type strain of S. somaliensis, and the second clade the remaining five strains which had almost identical 16S rRNA sequences. Members of the two subclades were sharply separated using DNA:DNA relatedness and phenotypic data which also showed that the subclade 1 strains formed an heterogeneous group. In contrast, the subclade 2 strains were assigned to a single genomic species and had identical phenotypic profiles. It is evident from these data that the subclade 2 strains should be recognised as a new species of Streptomyces. The name proposed for this new species is Streptomyces sudanensis sp. nov. The type strain is SD 504^T (DSM = 41923^T = NRRL B-24575^T). Erika T. Quintana and Katarzyna Wierzbicka contributed equally to this work. The GenBank accession numbers for the 16S rRNA gene sequences of Streptomyces somaliensis DSM 40738^T and Streptomyces sudanensis DSM 41607, DSM 41608, DSM 41609, SD 504^T and SD 509 are EF540897, EF540898, EF540999, EF515876 and EF540900.     

Diversity of the culturable lichen-derived actinobacteria and the taxonomy of Streptomyces parmotrematis sp. nov.

A total of 37 actinobacteria were isolated from eighteen lichen samples collected in Thailand. Based on the 16S rRNA gene sequences, they were identified into five genera including Actinoplanes (1 strain), Actinomadura (1 strain), Pseudosporangium (1 strain), Wangella (1 strain) and Streptomyces (33 strains). Among these isolates, strain Ptm05^T, Ptm01 and Ptm12 showed low 16S rRNA gene similarity and was selected for the further taxonomic study using the polyphasic approach. These strains showed the highest 16S rRNA gene sequence similarity with Streptomyces sparsogenes ATCC 25498^T (97.44–97.72%). Strain Ptm05^T was selected for the type strain. The chemical cell composition of the strain was similar to the members of Streptomyces genus. LL-diaminopimelic acids were detected in the peptidoglycan. Menaquinones were MK-9(H₈) and MK-9(H₆). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid, one unidentified glycolipid and one unidentified lipid were detected as the polar lipids. The predominant cellular fatty acids are anteiso-C_15:0, iso-C_15:0, iso-C_16:0, iso-C_17:0 and C_16:0. The dDNA-DNA hybridization values among strain Ptm05^T and its closely related Streptomyces type strains were 17.2–18.0%. In addition, the ANIb and ANIm between strain Ptm05^T and related Streptomyces type strains were ranged from 75.69 to 76.13% and 85.21 to 85.35%, respectively. Based on phenotypic and genomic evidence, strain Ptm05^T (=?TBRC 14546^T?=?NBRC 115203^T) represents the novel species of the genus Streptomyces for which the name Streptomyces parmotrematis sp. nov. is proposed. This study showed that the lichens are the promising source of the novel actinobacterial taxa.

     

Selective inhibition of the cyanobacterium, <Emphasis Type="Italic">Microcystis</Emphasis>, by a <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

A Streptomyces strain, NT0401, was isolated from soil that selectively inhibited Microcystis strains but did not affect other microorganisms. Based on its morphology, physiology and 16S rDNA sequence, it was identified as Streptomyces grisovariabilis. The active substance produced by NT0401 was a water-soluble compound with a Mr <1 kDa that was stable over a broad pH range and at 100°C for 20 min. This organism should be a potential environment-friendly strain for control of Microcystis blooms.     

Rapid discrimination of potato scab-causing <Emphasis Type="Italic">Streptomyces</Emphasis> species based on the RNase P RNA gene sequences

Scab disease significantly damages potatoes and other root crops. Some Streptomyces species have been reported as potato-scab pathogens. Identification of the phytopathogenic Streptomyces is mainly done on the basis of the 16S rRNA gene, but use of this gene has some limitations for discriminating these strains because they share high similarities of 16S rRNA gene sequences. We tested the RNase P RNA (rnpB) gene as a taxonomic marker to clarify the relationship among closely related scab-causing Streptomyces strains. The rnpB genes were analyzed for 41 strains including 9 isolates from Jeju, Korea. There were 4 highly variable regions including nucleotide gaps in the rnpB genes. Interspecies similarity of the rnpB gene in tested Streptomyces strains was lower than about 97%, while the intraspecies similarity was higher than about 98%. Phylogenetic analysis demonstrated that the rnpB tree has similar topology to the 16S rRNA gene tree, but produces a more divergent phyletic lineage. These results revealed that the rnpB gene could be used as a powerful taxonomic tool for rapid differentiation of closely related Streptomyces species. In addition, it was also suggested that the variable regions marked as α, β, γ, and δ in the rnpB gene could be useful markers for the detection of specific Streptomyces species.     

<Emphasis Type="Italic">Streptomyces fildesensis</Emphasis> sp. nov., a novel streptomycete isolated from Antarctic soil

A novel actinomycete strain, GW25-5^T, was isolated from a soil sample collected from the Fildes Peninsula, King George Island, West Antarctica. The strain was characterized by white to grey aerial mycelia, which were differentiated to straight to flexuous spore chains, with rod-shaped smooth spores. The cell wall of strain GW25-5^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell sugars were galactose and minor amounts of mannose and glucose. The predominant menaquinones were MK-9(H₆) (49%), MK-9(H₈) (24%) and MK-9(H₄) (12%). The phospholipids contained DPG, PE, PI, PIM and PL(s). The major cellular fatty acids were iso-C_16:0 and anteiso-C_15:0. Genomic DNA G+C content of strain GW25-5^T was 70.0 mol%. BLAST result showed that strain GW25-5 has the 16S rRNA gene sequence highest similarity of 97.5% with members of genus Streptomyces and phylogenetic analysis indicated that this strain belongs to the genus Streptomyces. DNA–DNA relatedness values of strain GW25-5^T with the closest species of Streptomyces purpureus LMG 19368^T and Streptomyces beijiangensis YIM 6^T were significantly lower than 70% of the threshold value for the delineation of genomic species. A polyphasic taxonomic investigation based on a judicious combination of genotypic and phenotypic characteristics revealed that the organism represents a novel species of the genus Streptomyces. Thus, we propose strain GW25-5^T as the type strain of this novel species, Streptomyces fildesensis (=CGMCC 4.5735^T = YIM 93602^T = DSM 41987^T = NRRL B 24828^T).     

Novel aromatic polyketides from soil <Emphasis Type="Italic">Streptomyces</Emphasis> spp.: purification,characterization and bioactivity studies

Aromatic polyketides are important therapeutic compounds which include front line antibiotics and anticancer drugs. Since most of the aromatic polyketides are known to be produced by soil dwelling Streptomyces, 54 Streptomyces strains were isolated from the soil samples. Five isolates, R1, B1, R3, R5 and Y8 were found to be potent aromatic polyketide producers and were identified by 16S rRNA gene sequencing as Streptomyces spectabilis, Streptomyces olivaceus, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae respectively. Their sequences have been deposited in the GenBank under the accession numbers KF468818, KF681280, KF395224, KF527511 and KF681281 respectively. The Streptomyces strains were cultivated in the media following critically optimised culture conditions. The resulting broth extracts were fractionated on a silica gel column and preparative TLC to obtain pure compounds. The pure compounds were tested for bioactivity and the most potent compound from each isolate was identified by UV–Vis, IR and NMR spectroscopic methods. Isolated S. spectabilis (R1), yielded one potent compound identified as dihydrodaunomycin with an MIC of 4 µg/ml against Bacillus cereus and an IC₅₀ value of 24 µM against HeLa. S. olivaceus (B1), yielded a comparatively less potent compound, elloramycin. S. purpurascens (R3) yielded three compounds, rhodomycin, epelmycin and obelmycin. The most potent compound was rhodomycin with an MIC of 2 µg/ml against B. cereus and IC₅₀ of 15 µM against HeLa. S. coeruleorubidus (R5), yielded daunomycin showing an IC₅₀ of 10 µM and also exhibiting antimetastatic properties against HeLa. S. lavendofoliae (Y8), yielded a novel aclacinomycin analogue with IC₅₀ value of 2.9 µM and potent antimetastatic properties at 1 µM concentration against HeLa. The study focuses on the characterization of aromatic polyketides from soil Streptomyces spp., which can serve as potential candidates for development of chemotherapeutic drugs in future.     

Pyrindicin,a New Alkaloid from a Streptomyces Strain

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.

After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD₅₀ of the hydrochloride (ip, in mice) was 87 mg/kg.     

Presence and Diversity of <Emphasis Type="Italic">Streptomyces</Emphasis> in <Emphasis Type="Italic">Dendroctonus</Emphasis> and Sympatric Bark Beetle Galleries Across North America

Recent studies have revealed several examples of intimate associations between insects and Actinobacteria, including the Southern Pine Beetle Dendroctonus frontalis and the Spruce Beetle Dendroctonus rufipennis. Here, we surveyed Streptomyces Actinobacteria co-occurring with 10 species of Dendroctonus bark beetles across the United States, using both phylogenetic and community ecology approaches. From these 10 species, and 19 other scolytine beetles that occur in the same trees, we obtained 154 Streptomyces-like isolates and generated 16S sequences from 134 of those. Confirmed 16S sequences of Streptomyces were binned into 36 distinct strains using a threshold of 0.2% sequence divergence. The 16S rDNA phylogeny of all isolates does not correlate with the distribution of strains among beetle species, localities, or parts of the beetles or their galleries. However, we identified three Streptomyces strains occurring repeatedly on Dendroctonus beetles and in their galleries. Identity of these isolates was corroborated using a house-keeping gene sequence (efTu). These strains are not confined to a certain species of beetle, locality, or part of the beetle or their galleries. However, their role as residents in the woodboring insect niche is supported by the repeated association of their 16S and efTu from across the continent, and also having been reported in studies of other subcortical insects.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

<Emphasis Type="Italic">Streptomyces nanningensis</Emphasis> sp. nov. a novel Streptomycete from forest soil

A strain YIM 33098^T (= CCTCC AA001027^T = DSM 41831^T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098^T was clustered in the same subclade with Streptomyces tendae ATCC19812^T and Streptomyces eurythermus ATCC14975^T. Similarities of strain YIM 33098^T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098^T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

A single amino acid substitution in the MurF UDP‐MurNAc‐pentapeptide synthetase renders Streptococcus pneumoniae dependent on CO2 and temperature

The respiratory tract pathogen Streptococcus pneumoniae encounters different levels of environmental CO₂ during transmission, host colonization and disease. About 8% of all pneumococcal isolates are capnophiles that require CO₂‐enriched growth conditions. The underlying molecular mechanism for caphnophilic behaviour, as well as its biological function is unknown. Here, we found that capnophilic S. pneumoniae isolates from clonal complex (CC) 156 (i.e. Spain^9V‐3 ancestry) and CC344 (i.e. Norway^NT‐42 ancestry) have a valine at position 179 in the MurF UDP‐MurNAc‐pentapeptide synthetase. At ≤ 30°C, the growth characteristics of capnophilic and non‐capnophilic CC156 strains were equal, but at > 30°C growth and survival of MurF^V179 strains was dependent on > 0.1% CO₂‐enriched conditions. Expression of MurF^V179 in S. pneumoniae R6 and G54 rendered these, otherwise non‐capnophilic strains, capnophilic. Time‐lapse microscopy revealed that a capnophilic CC156 strain undergoes rapid autolysis upon exposure to CO₂‐poor conditions at 37°C, and staining with fluorescently labelled vancomycin showed a defect in de novo cell wall synthesis. In summary, in capnophilic S. pneumoniae strains from CC156 and CC344 cell wall synthesis is placed under control of environmental CO₂ levels and temperature. This mechanism might represent a novel strategy of the pneumococcus to rapidly adapt and colonize its host under changing environmental conditions.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

<Emphasis Type="Italic">Streptomyces shenzhenensis</Emphasis> sp. nov., a novel actinomycete isolated from mangrove sediment

An actinomycete strain, which was designated 172115^T, was isolated from mangrove soil in Shenzhen, China. Strain 172115^T fell within the genus Streptomyces in the 16S rRNA gene tree and could be grouped into this genus based on its chemotaxonomic and morphological data. The strain shared the highest 16S rRNA gene sequence similarities with Streptomyces lanatus NBRC 12787^T (AB184845) (98.29%) and Streptomyces lucensis NBRC 13056^T (AB184280) (98.26%). The DNA–DNA hybridization values between strain 172115^T and the two most closely related type strains were low enough to justify the assignment of the strain to a novel species. On the basis of these phenotypic, phylogenetic and chemotaxonomic characteristics, 172115^T represents a novel species of the genus Streptomyces, for which, the name Streptomyces shenzhenensis sp. nov. is proposed for strain 172115^T (=CCTCC AA 2011001^T=DSM 42034^T).     

n-Alkane uptake and utilisation by Streptomyces strains

Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-¹⁴C as sole carbon and energy source, radioactive CO₂ was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

The <Emphasis Type="Italic">Streptomyces violaceusniger</Emphasis> clade: a home for streptomycetes with rugose ornamented spores

The taxonomic status of 16 strains received as Streptomyces hygroscopicus, Streptomyces melanosporofaciens, Streptomyces sparsogenes, Streptomyces sporoclivatus and Streptomyces violaceusniger was evaluated in a polyphasic study. Eleven of the organisms formed a distinct clade in the Streptomyces 16S rRNA gene tree with the type strains of Streptomyces asiaticus, Streptomyces cangkringensis, Streptomyces indonesiensis, Streptomyces javensis, Streptomyces malaysiensis, Streptomyces rhizosphaericus, Streptomyces yatensis and Streptomyces yogyakartensis, the members of this group produced rugose ornamented spores in spiral spore chains. The eleven strains were assigned to three established and four novel species, namely Streptomyces albiflaviniger sp. nov., Streptomyces demainii sp. nov., Streptomyces geldanamycininus sp. nov., Streptomyces griseiniger sp. nov., and Streptomyces hygroscopicus, Streptomyces melanosporofaciens and Streptomyces violaceusniger. It is also proposed that S. sporoclivatus becomes a subjective synonym of S. melanosporofaciens. S. sparsogenes NRRL 2940^T, which produced ridged ornamented spores in spiral spore chains, formed a distinct phyletic line in the Streptomyces 16S rRNA gene tree and was readily distinguished from the other strains using a range of phenotypic properties. S. violaceusniger strains NRRL 8097, NRRL B-5799, NRRL 2834 and ISP 5182 fell outside the S. violaceusniger 16S rRNA gene clade and formed either smooth or ridged ornamented spores in either flexuous or spiral spore chains. These organisms were distinguished from one another and from their closest phylogenetic neighbors and were considered to merit species status as Streptomyces auratus sp. nov., Streptomyces phaeoluteichromatogenes sp. nov., Streptomyces phaeogriseichromatogenes sp. nov., and Streptomyces phaeoluteigriseus sp. nov., respectively. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the tested strains are S. albiflaviniger NRRL B-1356^T (AJ391812), S. auratus NRRL 8097^T (AJ391816), S. geldanamycininus NRRL 3602^T (DQ334781), S. griseiniger NRRL B-1865^T (AJ391818), S. hygroscopicus NRRL 2387^T (AJ391820), NRRL 2339 (AJ391821) and NRRL B-1477 (AJ391819), S. demainii NRRL B-1478^T (DQ334782), S. melanosporofaciens NRRL B-12234^T (AJ391837), S. phaeogriseichromatogenes NRRL 2834^T (AJ391813), S. phaeoluteichromatogenes NRRL B-5799^T (AJ391814), S. phaeoluteigriseus ISP 5182^T (AJ391815), S. sparsogenes NRRL 2940^T (AJ391817), S. sporoclivatus NRRL B-24330^T (AJ 781369), S. violaceusniger ISP 5563^T (AJ 391823) and NRRL B-1476^T (AJ 391822).     

Screening of terrestrial <Emphasis Type="Italic">Streptomyces</Emphasis> leading to identification of new stereoisomeric anthracyclines

Thirty different Streptomyces strains were isolated from soil samples collected from different places in Egypt. The strains were isolated using Oatmeal agar and ISP medium 2 agar at pH 7.5. The isolate Streptomyces sp. Eg23 was selected for further working up to yield three new streoisomers of anthracycline metabolites. The structures were elucidated as (7R, 9R, 10S)-e-Rhodomycinone (1), (7R, 9R, 10S) Aklavinone (2), and (9R, 10S) 7-Desoxy-z-Rhodomycinone (3) by interpretation of their 1D and 2D NMR spectra. The absolute stereochemistry of 1 was confirmed by modified Mosher’s method. The (7R, 9R, 10S)-e-Rhodomycinone (1) showed activity against human lung cancer cell H460, murine Lewis lung cancer cell LL/2 and human breast cancer cell MCF-7. Compounds 1–3 were known synthetically, but until now have not been isolated as natural products from a microorganism. The results showed that the Streptomyces sp. Eg23 seems to be an interesting target for studying the regio-and stereochemistry of the cyclization step catalysed by polyketide cyclases to produce new anthracyclines through combinatorial biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Solid-state fermentation production of tetracycline by <Emphasis Type="Italic">Streptomyces</Emphasis> strains using some agricultural wastes as substrate

The ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273 S. aureofaciens NRRL B2183 and S. vendagensis ATCC 25507 to produce tetracycline using some local agricultural wastes as solid state media, were assessed. The wastes employed include peanut (groundnut) shells, corncob, corn pomace and cassava peels. Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity. All the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the carbohydrate source. Streptomyces sp. OXC1 had the highest ability for tetracycline production with peanut shells as the substrate in solid fermentation (13.18 mg/g), followed by S. vendagensis ATCC 25507 (11.08 mg/g), S.rimosus NRRL B1679 (8.46 mg/g), S. alboflavus NRRL B1273 (7.59 mg/g), S. rimosus NRRL B2234 (6.37 mg/g), S. aureofaciens NRRL B2183 (4.27 mg/g). Peanut (groundnut) shells were the most effective substrate (4.36 mg/g) followed by corncob (2.64 mg/g), cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g). The composition of the peanut (groundnut) shell medium optimal for tetracycline production were peanut shells 100 g, organic nitrogen (peanut meal) 10 g, (NH ₄)₂ SO₄ 1 g, KH₂ PO ₄ 0.5 g, CaCO₃ > 0.5 g, NaCl 0.5 g, MgSO₄ · 7H₂ O 0.5 g, soluble starch 10 g, peanut oil 0.25 ml with initial moisture content of 65–68%, and initial pH 5.3–6.3. Substrate (1 g dry weight) was inoculated with 1.0 × 10 ⁸ conidia per ml and incubated at 28–31 °C for 5–7 days, producing 13.18 mg/g of total tetracycline. Tetracycline detection started on day 3 and attained its maximum level on day 5.     

Phylogenetic analysis of the <Emphasis Type="Italic">lux</Emphasis> operon distinguishes two evolutionarily distinct clades of <Emphasis Type="Italic">Photobacterium leiognathi</Emphasis>

The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561^T and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon (luxABFE), whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene (luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554).Electronic Supplementary Material Supplementary material is available for this article at .