Karin Martin

Intimate bacterial–fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans

Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the full-genome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.

Niabella hirudinis and Niabella drilacis sp. nov., isolated from the medicinal leech Hirudo verbana.

Two Gram-negative, rod-shaped bacteria, strains E96(T) and E90(T), were isolated from medicinal leeches (Hirudo verbana) and characterized by a polyphasic taxonomic approach. Phylogenetic analysis based on the nearly full-length 16S rRNA gene sequence showed that the two strains shared 98.1% sequence similarity and were affiliated with the genus Niabella within the phylum Bacteroidetes, with 94.4-97.6% sequence similarity to type strains of species of the genus Niabella and highest sequence similarity to the type strain of Niabella aurantiaca (97.3 and 97.6%, respectively). Niabella-related 16S rRNA gene sequences were recently detected in the bladders of Hirudo verbana; however, no cultured representatives were so far available. Genomic fingerprint analysis using repetitive element primed (rep)- and randomly amplified polymorphic DNA (RAPD)-PCRs and DNA-DNA hybridization experiments clearly showed that the strains were different from each other (DNA-DNA relatedness values of 39.1%, reciprocal 28.0%) and from the type strains of N. aurantiaca (<19.7%) and Niabella tibetensis (<41.1%). Chemotaxonomic analyses confirmed the affiliation to the genus Niabella. Both strains contained MK-7 as the predominant menaquinone. The major fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), which is characteristic for the genus Niabella. Based on genotypic, chemotaxonomic and physiological characterization, we propose two novel species of the genus Niabella, Niabella hirudinis sp. nov., with strain E96(T) (u200a=DSM 25812(T)u200a=CCM 8411(T)u200a=LMG 26956(T)) as the type strain, and Niabella drilacis sp. nov., with strain E90(T) (u200a=DSM 25811(T)u200a=CCM 8410(T)u200a=LMG 26954(T)) as the type strain.

Massilia oculi sp. nov., isolated from a human clinical specimen.

A gram-negative, rod-shaped, non-spore-forming bacterium (strain CCUG 43427A(T)) was isolated from a patient suffering from endophthalmitis and its taxonomic position was studied. 16S rRNA gene sequence analysis indicated that this strain was a member of the genus Massilia. Strain CCUG 43427A(T) was most closely related to the type strains of Massilia timonae (97.4u200a% 16S rRNA gene sequence similarity) and Massilia aurea (97.2u200a%); levels of similarity to the type strains of all other recognized Massilia species were below 97.0u200a%. Chemotaxonomic data [Q-8 as major ubiquinone; phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major polar lipids; and summed feature 3 (C(16u200a:u200a1)ω7c and/or iso-C(15u200a:u200a0) 2-OH), C(16u200a:u200a0), C(18u200a:u200a1)ω7c, C(12u200a:u200a0) and C(10u200a:u200a0) 3-OH as major fatty acids] supported the affiliation of the isolate to the genus Massilia. Levels of DNA-DNA relatedness of strain CCUG 43427A(T) with M. timonae CCUG 45783(T) and M. aurea AP13(T) were 60.6u200a% (reciprocal, 55.8u200a%) and 58.1u200a% (reciprocal, 34.0u200a%), respectively. Strain CCUG 43427A(T) could be differentiated from its closest phylogenetic neighbours based on a range of phenotypic characteristics. Strain CCUG 43427A(T) is therefore considered to represent a novel species of the genus Massilia, for which the name Massilia oculi sp. nov. is proposed. The type strain is CCUG 43427A(T) (u200a=u200aCCM 7900(T)).

Duganella phyllosphaerae sp. nov., isolated from the leaf surface of Trifolium repens and proposal to reclassify Duganella violaceinigra into a novel genus as Pseudoduganella violceinigra gen. nov., comb. nov.

A bright yellow pigmented bacterium was isolated from the leaf surface of Trifolium repens in Germany. Comparative analysis of 16S rRNA gene sequences showed that this bacterium is most closely related to Duganella zoogloeoides IAM 12670(T), with a similarity of 99.3%, but revealed only a moderate similarity (96.8%) to the second Duganella species, Duganella violaceinigra YIM 31327(T). Strain T54(T) is clearly different from D. zoogloeoides IAM 12670(T) in that DNA-DNA hybridization revealed a similarity value of 46% (reciprocal 42%). Ubiquinone (Q-8) was the respiratory quinone and the predominant polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids and one aminolipid. Strain T54(T) can be distinguished from D. zoogloeoides by the carbon substrate utilization tests of d-trehalose, cis-aconitate, trans-aconitate, glutarate and dl-3-hydroxybutyrate, and 4-hydroxybenzoate in addition to a different polar lipid profile. The name Duganella phyllosphaerae sp. nov. is proposed for this novel species, with the type strain T54(T) (=LMG 25994 = CCM 7824(T)) [corrected]. In addition, it is proposed to reclassify D. violaceinigra into a novel genus Pseudoduganella gen. nov. as the novel species Pseudoduganella violaceinigra comb. nov. because of the low 16S rRNA gene sequence similarities to the other Duganella species (<97%) and striking differences in chemotaxonomic (lipid profiles and fatty acid patterns) and other phenotypic features, including the colony pigmentation.

Lysinibacillus contaminans sp. nov., isolated from surface water.

A Gram-positive-staining, aerobic, endospore-forming bacterium, isolated as a contamination from an enrichment of enteric bacteria from surface water, was studied using a polyphasic taxonomic approach. 16S rRNA gene sequence similarity comparisons revealed that strain FSt3A(T) was grouped in the genus Lysinibacillus, most closely related to Lysinibacillus xylanilyticus XDB9(T) (98.1%), Lysinibacillus parviboronicapiens BAM-582(T) and Lysinibacillus sphaericus DSM 28(T) (both 98.0%). The 16S rRNA gene sequence similarity to other species of the genus Lysinibacillus was <97.5%. The allocation to the genus Lysinibacillus was supported by a detailed chemotaxonomic characterization revealing a cell wall containing alanine, glutamic acid, aspartic acid and the diagnostic diamino acid lysine in a molar ratio of 1.6:1:0.9:0.8 (peptidoglycan type A4α), the major menaquinones MK-7 and MK-6, and polar lipids consisting of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unknown phospholipids, one unknown aminophospholipid and one unidentified aminolipid. The major fatty acids were iso- and anteiso-branched fatty acids. DNA-DNA hybridizations with the type strains of the most closely related species, L. parviboronicapiens DSM 25242(T), L. xylanilyticus DSM 23493(T) and L. sphaericus DSM 28(T), in addition to the results of physiological and biochemical tests, allowed genotypic and phenotypic differentiation of strain FSt3A(T) from these related species. Thus, FSt3A(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus contaminans sp. nov. is proposed, with FSt3A(T) (u200a=CCM 8383(T)u200a=DSM 25560(T)u200a=CIP 110362(T)) as the type strain.

Genomics-Guided Discovery of Endophenazines from Kitasatospora sp. HKI 714

In this study we report on the genomics-guided exploration of the metabolic potential of the newly discovered strain Kitasatospora sp. HKI 714. The bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the biosynthesis of formerly unknown endophenazine derivatives. A 200 L cultivation combined with bioactivity-guided isolation techniques revealed four new natural products belonging to the endophenazines and the 5,10-dihydrophenazines. Detailed descriptions of their biological effects, mainly focused on antimicrobial properties against several mycobacteria, are given.

Proposal of Novosphingobium rhizosphaerae sp. nov., isolated from the rhizosphere

A yellow, Gram-stain-negative, rod-shaped, non-spore-forming bacterium (strain JM-1(T)) was isolated from the rhizosphere of a field-grown Zea mays plant in Auburn, AL, USA. 16S rRNA gene sequence analysis of strain JM-1(T) showed high sequence similarity to the type strains of Novosphingobium capsulatum (98.9%), Novosphingobium aromaticivorans (97.4%), Novosphingobium subterraneum (97.3%) and Novosphingobium taihuense (97.1%); sequence similarities to all other type strains of species of the genus Novosphingobium were below 97.0%. DNA-DNA hybridizations of strain JM-1(T) and N. capsulatum DSM 30196(T), N. aromaticivorans SMCC F199(T) and N. subterraneum SMCC B0478(T) showed low similarity values of 33% (reciprocal: 21%), 14% (reciprocal 16%) and 36% (reciprocal 38%), respectively. Ubiquinone Q-10 was detected as the major respiratory quinone. The predominant fatty acid was C18:1ω7c (71.0%) and the typical 2-hydroxy fatty acid C14:0 2-OH (11.7%) was detected. The polar lipid profile contained the diagnostic lipids diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine. Characterization by 16S rRNA gene sequence analysis, physiological parameters, pigment analysis, and ubiquinone, polar lipid and fatty acid composition revealed that strain JM-1(T) represents a novel species of the genus Novosphingobium. For this species we propose the name Novosphingobium rhizosphaerae sp. nov. with the type strain JM-1(T) (u200a=u200aLMG 28479(T)u200a=CCM 8547(T)).

Novosphingobium gossypii sp. nov., isolated from Gossypium hirsutum.

A Gram-stain-negative, rod-shaped, non-spore-forming bacterium (strain JM-1396(T)) producing a yellow pigment, was isolated from the healthy internal stem tissue of post-harvest cotton (Gossypium hirsutum, cultivar DES-119) grown at the Plant Breeding Unit at the E.V. Smith Research Center in Tallassee (Macon county), AL, USA. 16S rRNA gene sequence analysis of strain JM-1396(T) showed high sequence similarity values to the type strains of Novosphingobium mathurense, Novosphingobium panipatense (both 98.6%) and Novosphingobium barchaimii (98.5%); sequence similarities to all other type strains of species of the genus Novosphingobium were below 98.3%. DNA-DNA pairing experiments of the DNA of strain JM-1396(T) and N. mathurense SM117(T), N. panipatense SM16(T) and N. barchaimii DSM 25411(T) showed low relatedness values of 8% (reciprocal 7%), 24% (reciprocal 26%) and 19% (reciprocal 25%), respectively. Ubiquinone Q-10 was detected as the dominant quinone; the fatty acids C18 : 1ω7c (71.0%) and the typical 2-hydroxy fatty acid, C14 : 0 2-OH (11.7%), were detected as typical components. The polar lipid profile contained the diagnostic lipids diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine. The polyamine pattern contained the major compound spermidine and only minor amounts of other polyamines. All these data revealed that strain JM-1396(T) represents a novel species of the genus Novosphingobium. For this reason we propose the name Novosphingobium gossypii sp. nov. with the type strain JM-1396(T) (u2009= LMG 28605(T) = CCM 8569(T) = CIP 110884(T)).

Resuscitation-promoting factors: distribution among actinobacteria, synthesis during life-cycle and biological activity

We have identified new members of the resuscitation-promoting factor (Rpf) protein family in numerous as yet unsequenced actinobacteria suggesting that Rpfs are common in this bacterial group. Formation and final delivery of the Rpf proteins varied from strain to strain and were dependent on growth conditions. Rpfs were found to act complementary to resuscitate other actinobacteria and might therefore be useful tools for the isolation of new actinobacterial species, especially from soil samples in which lots of microorganisms may have adopted dormant states.

Kytococcus aerolatus sp. nov., isolated from indoor air in a room colonized with moulds

A Gram-positive, coccoid bacterial isolate (02-St-019/1(T)), forming beige pigmented colonies was obtained from an indoor air sample. Based on 16S rRNA gene sequence similarity studies it was determined that this isolate 02-St-019/1(T) belonged to the genus Kytococcus, showing sequence similarties of 98.6% to Kytococcus schroeteri DSM 13884(T) and 98.3% to Kytococcus sedentarius DSM 20547(T), respectively. The diagnostic diaminoacid of the peptidoglycan was lysine, cell wall sugars were ribose and xylose. The major menaquinones detected were MK-7 and MK-8. The polar lipid profile consisted of the major phospholipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidylinositol mannoside. Fatty acid patterns were composed of major amounts of the iso- and anteiso-branched fatty acids anteiso C(17:0), iso C(15:0) and iso C(17:0) and unsaturated fatty acids (C(17:1) omega8c, iso C(17:1) omega9c, and C(17:1) omega8c) with smaller amounts of the straight-chain fatty acids C(15:0), C(16:0) and C(17:0). The results of DNA-DNA hybridizations and physiological and biochemical tests clearly allowed a genotypic and phenotypic differentiation of strain 02-St-019/1(T) from the two described Kytococcus species. On the basis of these results a novel species to be named Kytococcus aerolatus sp. nov., is proposed, with the type strain 02-St-019/1(T) (=DSM 22179(T)=CCM 7639(T)).