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Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (polyethylene terephthalate [PET] bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and next generation sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

A novel acidophilic actinobacterium, designated strain NEAU-YB345 T , was isolated from a pumpkin root collected from Mudanjiang, Heilongjiang Province, northeast PR China. Based on 16S rRNA gene sequence similarity and chemotaxonomic and morphological properties, the isolate was assigned to the genus Streptacidiphilus , with the high 16S rRNA gene sequence similarities to Streptacidiphilus melanogenes JCM 16224 T (99.2 %), Streptacidiphilus anmyonensis JCM 16223 T (99.1 %) and Streptacidiphilus jiangxiensis JCM 12277 T (98.7 %). Its cell wall contained ll-diaminopimelic acid as the major diamino acid. Rhamnose, ribose, glucose and galactose were the detected sugars from the whole-cell hydrolysates. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The menaquinones were MK-9(H 8 ) and MK-9(H 6 ). Major fatty acids were C 16 : 0 , iso-C 16 : 0 , iso-C 15 : 0 and anteiso-C 15 : 0 . Phylogenetic analysis using 16S rRNA gene and whole-genome sequences placed the strain in distinct clades but within the genus Streptacidiphilus . The DNA G+C content was 71.2 mol%. Based on DNA-DNA relatedness and physiological and biochemical data, the isolate could be distinguished from its closest relatives. Therefore, strain NEAU-YB345 T represents a novel species of the genus Streptacidiphilus , for which the name Streptacidiphilus fuscans sp. nov. is proposed. The type strain is NEAU-YB345 T (=CCTCC AA 2020030 T =JCM 33976 T ).

The use of actinomycetes for improving soil fertility and plant production is an attractive strategy for developing sustainable agricultural systems due to their effectiveness, eco-friendliness, and low production cost. Out of 17 species isolated from the soil rhizosphere of legume crops, 4 bioactive isolates were selected and their impact on 5 legumes: soybean, kidney bean, chickpea, lentil, and pea were evaluated. According to the morphological and molecular identification, these isolates belong to the genus Streptomyces. Here, we showed that these isolates increased soil nutrients and organic matter content and improved soil microbial populations. At the plant level, soil enrichment with actinomycetes increased photosynthetic reactions and eventually increased legume yield. Actinomycetes also increased nitrogen availability in soil and legume tissue and seeds, which induced the activity of key nitrogen metabolizing enzymes, e.g., glutamine synthetase, glutamate synthase, and nitrate reductase. In addition to increased nitrogen-containing amino acids levels, we also report high sugar, organic acids, and fatty acids as well as antioxidant phenolics, mineral, and vitamins levels in actinomycete treated legume seeds, which in turn improved their seed quality. Overall, this study shed the light on the impact of actinomycetes on enhancing the quality and productivity of legume crops by boosting the bioactive primary and secondary metabolites. Moreover, our findings emphasize the positive role of actinomycetes in improving the soil by enriching its microbial population. Therefore, our data reinforce the usage of actinomycetes as biofertilizers to provide sustainable food production and achieve biosafety.

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Non-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) still remain elusive. Here, by re-engineering the existing genome-wide search approach, we discover a family of structurally conserved RNAs that are present ubiquitously across actinobacteria, including mycobacteria. In vitro analysis shows that RNAs belonging to this family bind response-regulator proteins that contain the widely prevalent ANTAR domain. The Mycobacterium tuberculosis ANTAR protein gets phosphorylated by a histidine kinase and interacts with RNA only in its phosphorylated state. These newly identified RNAs reside only in certain transcripts and typically overlap with the ribosome-binding site, regulating translation of these transcripts. In this way, the RNAs directly link signaling pathways to translational control, thus expanding the mechanistic tool kit available for ANTAR-based control of gene expression. In mycobacteria, we find that RNAs targeted by ANTAR proteins majorly encode enzymes of lipid metabolism and associated redox pathways. This now allows us to identify the key genes that mediate ANTAR-dependent control of lipid metabolism. Our study establishes the identity and wide prevalence of ANTAR-target RNAs in mycobacteria, bringing RNA-mediated regulation in these bacteria to the center stage., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca 2+ -mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

Microbial diversity in lava tubes from Canary Islands (Spain) has never been explored thus far offering a unique opportunity to study subsurface microbiology. Abundant yellow coloured mats developing on coralloid speleothems in a lava tube from La Palma Islands were studied by next-generation sequencing and DNA/RNA clone library analyses for investigating both total and metabolically active bacteria. In addition, morphological and mineralogical characterization was performed by field emission scanning electron microscopy (FESEM), micro-computed tomography, X-ray diffraction and infrared spectroscopy to contextualize sequence data. This approach showed that the coralloid speleothems consist of banded siliceous stalactites composed of opal-A and hydrated halloysite. Analytical pyrolysis was also conducted to infer the possible origin of cave wall pigmentation, revealing that lignin degradation compounds can contribute to speleothem colour. Our RNA-based study showed for the first time that members of the phylum Actinobacteria, with 55% of the clones belonging to Euzebyales order, were metabolically active components of yellow mats. In contrast, the DNA clone library revealed that around 45% of clones were affiliated to Proteobacteria. Composition of microbial phyla obtained by NGS reinforced the DNA clone library data at the phylum level, in which Proteobacteria was the most abundant phylum followed by Actinobacteria.

Molinate is a thiocarbamate herbicide used in rice crop protection. As other pesticides, molinate is a recognized environmental pollutant and bio-accumulated by some wildlife forms. Gulosibacter molinativorax ON4 T is able to hydrolyse molinate into metabolites which are further degraded by other un-related bacteria. Hence, it can be used in molinate bioremediation processes. The aim of this work was to investigate the possibility of producing G. molinativorax ON4 T microparticles, using different non-toxic biopolymers (arabic gum, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. Several formulations of microparticles were prepared, and their physicochemical structures were analyzed by scanning electron microscopy (SEM), laser granulometry analysis and zeta potential analysis. The obtained microparticles were evaluated considering their ability to degrade molinate, the metabolic activity (by colour development of the tetrazolium violet redox), and also the survival rate and shelf-life/storage stability of microparticles. Based on their molinate degrading activity, the biopolymers calcium alginate and modified chitosan cross-linked with tripolyphosphate appear to be the best options for the microencapsulation of the G. molinativorax ON4 T . However, the microparticles produced with modified chitosan cross-linked with tripolyphosphate present the best combination of physical properties and activity degradation of molinate., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.

Three novel actinobacteria, designated strains NEAU-FSHN1(T), NEAU-hd-3(T) and NEAU-Y6(T), were isolated from a stream base, soil adjacent to the stream and a root of Corydalis yanhusuo L, respectively, collected from Wuchang, Heilongjiang Province, China. A polyphasic study was carried out to establish the taxonomic positions of these strains. The three strains were observed to form scant aerial hyphae that differentiated into spherical spore vesicles. The phylogenetic analysis based on the 16S rRNA gene sequences of strains NEAU-FHSN1(T), NEAU-hd-3(T) and NEAU-Y6(T) showed that the three novel isolates exhibit 99.2 % (NEAU-FHSN1(T)/NEAU-hd-3(T)), 99.2 % (NEAU-FHSN1(T)/NEAU-Y6(T)) and 99.7 % (NEAU-hd-3(T)/NEAU-Y6(T)) 16S rRNA gene sequence similarities with each other and that they are closely related to strains Streptosporangium shengliense NEAU-GH7(T) (sequence similarities 98.72, 98.85, 98.99 %), Streptosporangium roseum DSM 43021(T) (98.65, 98.51, 98.58 %) and Streptosporangium album DSM 43023(T) (98.41, 98.96, 98.89 %). However, the DNA-DNA hybridization values between strains NEAU-FSHN1(T), NEAU-hd-3(T) and NEAU-Y6(T) were 61.2 % (NEAU-FSHN1(T)/NEAU-hd-3(T)), 63.5 % (NEAU-FHSN1(T)/NEAU-Y6(T)) and 65.8 % (NEAU-hd-3(T)/NEAU-Y6(T)), and the values between the three strains and their close phylogenetic relatives were also below 70 %. With reference to phenotypic characteristics, phylogenetic data and DNA-DNA hybridization results, the three strains can be distinguished from each other and their close phylogenetic relatives. Thus, strains NEAU-FHSN1(T), NEAU-hd-3(T) and NEAU-Y6(T) are concluded to represent three novel species of the genus Streptosporangium, for which the names Streptosporangium lutulentum sp. nov., Streptosporangium fenghuangense sp. nov. and Streptosporangium corydalis sp. nov. are proposed. The type strains are NEAU-FHSN1(T) (=CGMCC 4.7141(T) = DSM 46740(T)), NEAU-Y6(T) (=CGMCC 4.7150(T) = DSM 46722(T)) and NEAU-hd3(T) (CGMCC 4.7212(T) = JCM 30058(T)), respectively.

ZJ0273 (propyl 4-(2-(4, 6-demethoxy pyrimidin-2-yloxy) benzylamino) benzoate) is a novel pyrimidynyloxybenzoic-based herbicide developed in China for oilseed crop. This study was aimed to construct new strains capable of degrading naphthalene and ZJ0273 by protoplast fusion between Amycolatopsis sp. M3-1 and Pseudomonas sp. Nai8. Eight recombinant strains were successfully produced, and the strains could simultaneously utilize ZJ0273 and naphthalene as the sole carbon and energy source, respectively. One of recombinant strains, MN6 with higher degrading efficiency, was chosen for further study. Under the condition of pH 7.0, 30 °C, ZJ0273 and naphthalene degradation percent by the recombinant strain MN6 could reach 65.10% (20 days) and 88.46% (48 h), respectively. According to the identified six metabolites (M1-M6) by LC-MS/MS, biodegradation pathway of ZJ0273 was proposed. ZJ0273 biodegradation catalyzed by the recombinant strain MN6 involved continuous biocatalytic reactions such as de-estering, hydrolysis, acylation, C-N cleavage, de-methyl, and ether cleavage reactions.

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the "Pantanal" region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related β-carbolines, and 3 Indoles. Compound 1-vinyl-β-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-β-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungi Phyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83.

This study reports the characterization of the Tetrasphaera duodecadis bacteria and the techniques used therein. In order to evaluate the morphological characteristics of the T. duodecadis bacteria scanning electron microscope (SEM) was used throughout its different growth stages. These microorganisms were grown in vitamin B12 broths with 1% tryptone, 0.2% yeast extract, and 0.1% glucose. The turbidimetric method was employed for the determination of bacterial concentration and growth curve. The SEM results show small agglomerates of 0.8 ± 0.05 µm during the lag phase, and rod-like shapes during the exponential phase with similar shapes in the stationary phase., (© 2014 Wiley Periodicals, Inc.)

A new trehalose analog, lentztrehalose [4-O-(2,3-dihydroxy-3-methylbutyl)trehalose], was isolated from an actinomycete Lentzea sp. Lentztrehalose is only weakly hydrolyzed by the trehalose-hydrolyzing enzyme, trehalase, so can be regarded as an enzyme-stable analog of trehalose. Although lentztrehalose does not show apparent toxicity to mammalian cells and microbes, it has antitumor activity in mice bearing S-180 sarcoma and Ehrlich carcinoma cells. In ovariectomized mice, lentztrehalose displayed a bone reinforcement effect in the femur that was superior to trehalose and induced non-morbid suppression of weight gain comparable with trehalose. These results indicate that enzyme-stable analogs of trehalose, such as lentztrehalose, may be more beneficial for human health and thus have potential as substitutes for trehalose as a sweetener.

A simple and rapid method for the preparation of actinomycete cultures before scanning electron microscopy (SEM) is reported. Streptomyces strains were grown in starch casein broth with sterile glass wool as support. The cultures were dehydrated by lyophilization eliminating the use of chemical fixatives and dehydrating agents., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The charcoalified fragment of the dorsiventrally organized, internally stratified presumed green algal lichen Chlorolichenomycites salopensis from the Lower Devonian Lochkovian strata in the Welsh Borderland carries bacterial colonies on the upper surface, i.e. the cortex, and actinobacterial filaments in the medulla underneath the photobiont layer. Moreover relatively thin hyphae of presumed endolichenic fungi were found. As in extant lichens, which are best regarded as consortia with an unknown number of participants, this internally stratified, fossil thallus fragment of a presumed green algal lichen harbours a diverse microbial community., (Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Termite gut flagellates are colonized by host-specific lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluorescence in situ hybridization and electron microscopy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonympha species from different termites. The flagellates in Trichonympha Cluster I were the only species associated with 'Endomicrobia', which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked 'Endomicrobia' and were associated with endosymbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name 'Candidatus Ancillula trichonymphae', represent a novel, deep-branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Trichonympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplasmic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invaginations of the plasma membrane that were clearly connected to the exterior of the host cell., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

A novel Gram-stain-positive actinobacterium, designated RI148-Li105(T), was isolated from a lichen sample from Rishiri Island, Japan, and its taxonomic position was investigated by a polyphasic approach. 16S rRNA gene sequencing study indicated that strain RI148-Li105(T) was related to the type strain of Luteimicrobium subarcticum, with a similarity of 97.8%. Cells of strain RI148-Li105(T) exhibited a rod-coccus cycle. The diagnostic cell-wall diamino acid of this organism was lysine and the peptidoglycan type was found to be A4α. The predominant menaquinones were MK-8(H(2)) and MK-9(H(2)), and the major fatty acids were iso-C(16:0), C(17:1) ω9c and C(17:0). The DNA G+C content was 73.6 mol%. The major phenotypic characteristics of strain RI148-Li105(T) basically corresponded to those of the genus Luteimicrobium excluding the fatty acid composition. These results suggest that strain RI148-Li105(T) should be affiliated with the genus Luteimicrobium. Meanwhile, DNA-DNA hybridization and some phenotypic characteristics revealed that the strain differs from L. subarcticum. Therefore, strain RI148-Li105(T) represents a novel species of the genus Luteimicrobium, for which the name Luteimicrobium album sp. nov. is proposed. The type strain of Luteimicrobium album is RI148-Li105(T) (=NBRC 106348(T)=DSM 24866(T)).

The discovery of Penicillin in 1928 and that of Streptomycin in 1943, has been pivotal to the exploration of nature as a source of new lead molecules. Globally, the microbiologist today is acknowledged as a crucial player in the drug discovery program. The microbial products, especially those from actinomycetes have been a phenomenal success for the past seven decades. Bioprospecting for new leads are often compounded by the recurrence of known antibiotics in newer microbial isolates. Despite all these deterrents, actinomycetes have proved to be a sustained mine of novel antibiotics, which selectively destroys the pathogens without affecting the host tissues. Each of these antibiotics is unique in their mode of action. Their versatility and immense economic value is something, which is extremely noteworthy. The anti-infective turn-over of over 79 billion US dollars in 2009, includes about 166 antibiotics and derivatives such as the Beta-lactam peptide antibiotics, the macrolide polyketide erythromycin, tetracyclines, aminoglycosides, daptomycin, tigecycline, most of which are produced by actinomycetes (1). Actinomycetes continue to play a highly significant role in drug discovery and development. Among the bioactive compounds that have been obtained so far from microbes, 45 % are produced by actinomycetes, 38 % by fungi and 17 % by unicellular eubacteria (2). Further many chemically synthesized drugs owe their origin to natural sources. In this review article, we highlight the recent antibiotics from actinomycetes with emphasis on their source, structures, activity, mechanism of action and current status.

The peroxisome is an organelle found in most eukaryotes that is crucial for lipid metabolism. The ability of peroxisomes to divide themselves and transport post-translational proteins suggests that the peroxisome may have had an endosymbiotic origin. However, the localization of peroxisomal proteins to the endoplasmic reticulum (ER) and the similarity of some peroxisomal proteins to those localized in the ER suggest an alternative hypothesis: that the peroxisome was developed from the ER. To study the evolutionary distance between the peroxisome, the ER and prokaryotes, we conducted a phylogenetic analysis of cell division control 48 (CDC48) and its homologs, including the ER-localized CDC48, the CDC48 homologs in prokaryotes and the peroxisome-localized PEX1 and PEX6. We also conducted a similarity search of peroxisomal protein sequences against prokaryotic protein sequences using BLAST at several E-value thresholds. We provide several lines of evidence supporting an actinobacteria symbiotic origin for the peroxisome: (1) PEX1 and PEX6 are more closely related to the CDC48 homologs in actinobacteria than to the ER-localized CDC48; (2) actinobacterial proteins show higher levels of similarity to those of the peroxisome than to those of other prokaryotes.

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.

Microbial biodiversity provides an increasingly important source of medically and industrially useful compounds. We have isolated 14 actinomycete species from a collection of approximately 300 plant stem samples from the upper Amazonian rainforest in Peru. All of the cultured isolates produce substances with inhibitory activity directed at a range of potential fungal and bacterial pathogens. For some organisms, this activity is very broad in spectrum while other organisms show specific activity against a limited number of organisms. Two of these organisms preferentially inhibit bacterial test organisms over eukaryotic organisms. rDNA sequence analysis indicates that these organisms are not equivalent to any other cultured deposits in GenBank. Our results provide evidence of the untapped biodiversity in the form of biologically active microbes present within the tissues of higher plants.

We compared the influences of grazing by the bacterivorous nanoflagellate Poterioochromonas sp. strain DS on ultramicrobacterial Actinobacteria affiliated with the Luna-2 cluster and ultramicrobacterial Betaproteobacteria of the species Polynucleobacter cosmopolitanus. These bacteria were almost identical in size (<0.1 microm(3)) and shape. Predation on a Polynucleobacter strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of >or=39 days. The differences in predation mortality between the actinobacterial strains and the Polynucleobacter strain clearly demonstrated size-independent grazing resistance for the investigated Actinobacteria. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait.

Polymetallic/ferromanganese nodules (Mn-nodules) provide a rich source for manganese. It is not yet known if the nodules have a biogenic or an abiogenic origin. Here we applied the technique of high-resolution scanning electron microscopy, in combination with energy dispersive X-ray spectroscopical (EDX) analysis, to trace the existence of microbial biofilms. Two spatially separated assemblies exist, the exolithobiontic- and endolithobiontic colonizations. The exolithobiontic colonization is seen in the micro-canals, which traverse the outer surface layer of the nodules and are formed by elongated filamentous organisms, which show no signs of mineralization. In the center of the nodules three types of endolithobiontic microbial biofilms exist: first, cone-like microorganisms forming biofilms, second stone/pillar-like microorganisms and finally paving stone-like, hexagonal microorganisms. All are covered by brick-like mineral deposits. By EDX analysis we could measured the highest relative level of carbon (C) with respect to manganese (Mn) and sodium on the microorganisms. Our data are in perfect agreement with the assumption that the Mn deposits in the nodules are of biogenic origin. In a first approach, DNA from microorganisms from the interior of those Mn-nodules was isolated by PCR, and sequenced with respect to the 16S ribosomal RNA gene. Sequence comparison revealed that the sequence from the Mn-nodule, studied here, shares highest similarity to a bacterium living in soil, rich in iron and manganese. We propose that the microorganisms form a biofilm within the nodules onto which Mn is deposited due to an oxidation from Mn(II) to Mn(III)/Mn(IV).

A novel bacterial strain, designated ANL-iso2(T), was obtained from an enrichment culture inoculated with a mixture of soda lake sediments by using isobutyronitrile (iBN) as the carbon, energy and nitrogen source at pH 10. The enrichment resulted in a stable binary culture containing iBN-degrading Gram-positive rods and a satellite Gram-negative gammaproteobacterium Marinospirillum sp. strain (ANL-isoa) scavenging the products of nitrile hydrolysis. Cells of the iBN-degrading strain, ANL-iso2(T), were short, non-motile, non-spore-forming rods. Strain ANL-iso2(T) was capable of utilizing propionitrile (C(3)), butyronitrile (C(4)), isobutyronitrile (C(4)), valeronitrile (C(5)) and capronitrile (C(6)) as the only growth substrate. Growth on nitriles was biphasic with fast initial hydrolysis of nitriles to the corresponding amides, carboxylic acids and ammonia and slow further utilization of these products resulting in biomass growth. Cells of strain ANL-iso2(T) grown with iBN were capable of extremely active hydration of a wide range of nitriles into the corresponding amides and much slower hydrolysis of these amides to the corresponding carboxylic acids. This indicated the presence of the nitrile hydratase/amidase pathway of nitrile degradation in the novel bacterium. Strain ANL-iso2(T) showed obligately alkaliphilic growth on iBN within the pH range 8.4-10.6, with optimum growth at 9.0-9.5. It was moderately salt-tolerant, with a salt range for growth of 0.1-2.0 M Na(+) and an optimum salt concentration for growth of 0.2-0.3 M. The dominant fatty acids in the polar lipids were C(16 : 0), iso-C(14), C(14 : 0), iso-C(16) and C(16 : 1)omega7. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic analysis placed strain ANL-iso2(T) within the class Actinobacteria as an independent lineage with only uncultured bacteria from soda lakes as its nearest relatives. On the basis of its unique phenotype and distinct phylogeny, strain ANL-iso2(T) is considered to represent a novel species of a new genus, for which the name Nitriliruptor alkaliphilus gen. nov., sp. nov. is proposed. The type strain of the type species, Nitriliruptor alkaliphilus, is ANL-iso2(T) (=DSM 45188(T)=NCCB 100119(T)=UNIQEM U239(T)). Phylogenetic data suggest that the novel bacterium forms the basis of a new family Nitriliruptoraceae fam. nov. and a novel order Nitriliruptorales ord. nov. within the class Actinobacteria.

Culture broth of an actinomycete isolate, Nocardia sp. CS682 showed specifically higher antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA). Purified substance from the organism, CS-682, which is active against MRSA and Micrococcus leuteus, is a C(28)H(37)NO(8) (M+H(+), observed: 516.83) and identified as an unusual macrolide antibiotic, nargenicin. The chemical structure of CS-682 was identified by FT-IR, (1)H-NMR, (13)C-NMR, and ((1)H-(1)H and (1)H-(13)H) COSY. The anti-MRSA activity of CS-682 was stronger than that of oxacillin, vancomycin, monensin, erythromycin, and spiramycin. Phylogenetic analysis showed that strain CS682 is closely related to Nocardia tenerifensis DSM 44704(T) (98.7% sequence similarity), followed by N. brasiliensis ATCC 19296(T) (98.4% sequence similarity). The ability of Nocardia sp. CS682 to produce nargenicin was unique.

Objective: Endophytic actinomycete Lj20 with antifungal activity was isolated from the roots of capsicum plants. We identified Lj20 and synthesized its antifungal substances., Methods: Morphological, biological and biochemical characteristics, chemotaxonomy analysis and 16S rDNA sequences were used to identify Lj20. According to GC-MS extrapolation result, one of antifungal substances in the metabolites of Lj20 was chemically synthesized. The bioactivities were determined by mycelium growth inhibition method., Results: Lj20 belonged to Streptomyces sp. and was similar to Streptomyces rochei. The metabolites contained butylated hydroxytoluene and 3, 5-di-tert-butyl-4-hydroxybenzyl methyl ether. The median effective concentration (EC50) of these two compounds to Botrytis cinerea Pers. were 237.04 mg/L of the water and 186.48 mg/L of the water, respectively., Conclusion: Lj20 was classified as Streptomyces rochei. Butylated hydroxytoluene and 3, 5-di-tert-butyl-4-hydroxybenzyl methyl ether had significant inhibition to the pathogen.

The inventory of microorganisms responsible for biological deterioration of ancient paintings has become an integral part of restoration activities. Here, the microbial agent of rosy discoloration on medieval frescoes in the Crypt of the Original Sin (Matera, Italy) was investigated by a combination of microscopic, molecular and spectroscopic approaches. The bacterial community from three rosy-discoloured painting sites was characterized by 16S rRNA gene-based techniques. The eubacterial population was prevalently composed of Actinobacteria, among which Rubrobacter radiotolerans-related bacteria accounted for 63-87% of the 16S rRNA gene pool per sampled site. Archaea, with prevalence of Haloarchaea-related species, were detected in one of the three sites where they accounted for < 0.1% of the total 16S rRNA gene pool. Raman spectroscopy confirmed the identity between R. radiotolerans carotenoids (bacterioruberins) and pigments responsible for colour alteration of frescoes. This investigation provides the first evidence of a causal relationship between heavy contamination by Rubrobacter-related bacterioruberin-producing bacteria and rosy discoloration of ancient wall paintings.

Aims: Identification of a new actinomycete strain Sg3, belonging to the genus Streptosporangium and partial characterization of the produced antibacterial activities., Methods and Results: The strain Sg3 was isolated from an Algerian Saharan soil and identified by morphological, chemotaxonomic and phylogenetic analyses to the genus Streptosporangium. The comparison of its physiological characteristics with those of known species of Streptosporangium showed significant differences with the nearest species Streptosporangium carneum. Analysis of the 16S rDNA sequence of strain Sg3 showed a similarity level ranging between 97% and 98.8% within Streptosporangium species, with S. carneum the most closely related. Strain Sg3 showed a red coloured antibacterial activity against gram-positive bacteria on several culture media. The purification of the red pigment by chromatographic methods led to the isolation of three active products. The (1)H nuclear magnetic resonance (NMR), mass, infrared (IR) and ultraviolet-visible (UV-VIS) data of these molecules strongly suggested that they belonged to the quinone-anthracycline group with three or more rings., Conclusions: Strain Sg3 represents a distinct phyletic line suggesting a new genomic species. It produces antibacterial activities identified as quinone-anthracycline aromatics., Significance and Impact of the Study: The quinone-anthracycline antibiotics are known for their antimicrobial and antineoplastic activities and are used in chemotherapy for the treatment of many cancer diseases. The present work constitutes the first stage of a whole series of studies to be realized on these antibiotics before arriving at a possible application.

The presence of Tropheryma whippelii was demonstrated in the PAS-negative mesenteric granulomatous lymph nodes of a patient affected by Whipple disease. Ultrastructurally a few bacteria, enclosed by a membrane characteristic of Tropheryma whippelii, were found in the extracellular spaces and remnants of bacteria were found in the phagocytic vacuoles of macrophages. The scarce number of bacilli, probably due to the fact that the disease was at an initial phase, could explain the absence of PAS positivity. This case confirms the role of the electron microscopy in the diagnosis of Whipple disease, especially for extra-intestinal lesions and at the initial phase of the disease, when the characteristic PAS-positive macrophages can be absent.

In the screening of antitumor compounds from microbial secondary metabolites, myxochelin A was isolated from a culture broth of Nonomuraea pusilla TP-A0861. The absolute configuration was determined to be S by synthesizing both enantiomers from an L- or D-lysine derivative and comparing their specific rotations. Both enantiomers of myxochelin A showed remarkable inhibitory effects on the invasion of murine colon 26-L5 carcinoma cells at non-cytotoxic concentrations.

The in situ physiology of the filamentous bacterium Skermania piniformis frequently seen in activated sludge foams in Australia was investigated. An oligonucleotide probe, Spin1449, targeting the 16S rRNA of S. piniformis was designed for its identification by fluorescence in situ hybridization (FISH), validated with pure cultures and applied successfully to foam samples from two geographically distant Australian plants. While filaments of this bacterium appeared to be comparatively hydrophobic, the organism had no clear preference for hydrophobic or hydrophilic substrates. In both foams examined using microautoradiography (MAR), filaments selectively took up substrates under aerobic and anoxic (NO(3) (-)) but not anaerobic or anoxic (NO(2) (-)) conditions. Skermania piniformis assimilated oleic acid, palmitic acid, glycerol and glycine. Ectoenzyme activities detected suggest that S. piniformis has an ability to assimilate a greater range of substrates than might be concluded from the MAR data obtained here. Based on the substrate uptake data presented here, an anaerobic selector may work for controlling S. piniformis in activated sludge systems.

The aim of this research was to determine the accelerating effects of microorganisms on the biodeterioration of stone under air pollution and continental-cold climatic region in Erzurum, Turkey. Studies have been carried out on specimens of the Rustempasa Bazaar, the Lalapasa Mosque, the Erzurum Castle Mosque, the Double Minarets-Madrasah, the Great Mosque and the Haji Mehmet Fountain aged from 441 to 823 years old. The results showed that vegetative and reproductive (generative) forms of the microorganisms could develop during the winter months when the night time average temperature was even -25 degrees C. Also the reproductive forms had developed and the whole stone surface was covered with a biofilm caused by the microorganisms. Silicon, aluminum, calcium, potassium, titanium, magnesium, zinc, sulfur, iron, sodium, and niobium were found in the stones of the historical buildings with varying amounts through the SEM-EDS analysis. Some of these elements could be used as an energy resource for the microorganisms together with the air pollutants such as sulfur dioxide, nitrogen oxides and particles on the stone surfaces. Of 21 isolates, 15 species from 6 bacterium genera and 5 species from 5 fungi genera plus 1 fungi genera were identified on the deteriorated stone surfaces even during the coldest months by microbial identification system (MIS) and these findings were tested by SEM investigations.

Two actinomycete strains, designated YIM 30006(T) and YIM 31075(T), were isolated from soil samples in Yunnan, China and subjected to a polyphasic taxonomic study. Morphological and chemotaxonomic analysis revealed that the two isolates should be consistent with the nocardioform actinomycetes. Comparative 16S rDNA sequences confirmed that the two unknown isolates to be members of the genus Kribbella. Based on the results of phenotypic characteristics, phylogenetic studies and DNA-DNA hybridization results, strains YIM 30006(T) and YIM 31075(T) should be classified as two novel species of the genus Kribbella, for which the names Kribbella yunnanensis sp. nov. and Kribbella alba sp. nov. are proposed. The type strains for them are YIM 30006(T) (=CCTCC AA001019(T)=DSM 15499(T)) and YIM 31075(T) (=CCTCC AA 001020(T)=DSM 15500(T)), respectively. The 16S rDNA sequences of strains YIM 30006(T), YIM 31075(T) have been deposited in GenBank under the accession numbers AY 082061 and AY 082062, respectively.

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.

The taxonomic position of three actinomycete strains isolated from Malaysian soil was established by using a polyphasic approach. The isolates formed chains composed of four spores on the tip of sporophores branching from the aerial mycelium, and their chemotaxonomic properties were common to those of members of the family Streptosporangiaceae. These phenotypic properties as well as a phylogenetic analysis based on 16S rRNA gene sequences indicated that they should be classified in the genus Microtetraspora. The three isolates showed a unique pattern of cultural, physiological and biochemical properties that distinguished them from previously described species of the genus Microtetraspora. The isolates showed more than 72% DNA relatedness to each other, but only 58% or less relatedness to any previously described species. On the basis of the data presented, a new species of the genus Microtetraspora, Microtetraspora malaysiensis, is proposed. The type strain of the new species is strain H47-7(T) (=JCM 11278(T)=DSM 44579(T)).

The tip growth of filamentary actinomycetes is investigated within the framework of large deformation membrane theory in which the cell wall is represented as a growing elastic membrane with geometry-dependent elastic properties. The model exhibits realistic hyphal shapes and indicates a self-similar tip growth mechanism consistent with that observed in experiments. It also demonstrates a simple mechanism for hyphal swelling and beading that is observed in the presence of a lysing agent.

We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 microm(3)) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 microm(3) and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.

Sediment samples from Krishna River at Nagayalanka of Andhra Pradesh, India were investigated as a source of actinomycetes to screen for the production of novel bioactive compounds. During our investigation on fresh water actinomycetes from 5 different river sediment samples, a total of 80 actinomycetes were isolated. Out of these 80 isolates, 30 isolates which showed distinct macromorphological characteristics were selected. The antimicrobial and enzymatic activities were studied for all the 30 isolates. The preliminary study for antimicrobial activity by cross streak method indicated that 16 isolates (53.3%) have excellent antagonistic properties. All these 16 isolates were subjected to detailed submerged fermentation studies. It was observed that 12 isolates (40.0%) exhibited antibacterial activity, 9 isolates (30.0%) showed antifungal activity while 5 isolates (16.6%) showed both antibacterial and antifungal activities. All the 30 isolates were also subjected for the determination of enzymatic activities 25 isolates (83.3%) exhibited amylolytic activity while 27 isolates (90.0%) showed proteolytic activity. Among these isolates, six promising isolates were selected for detailed morphological, cultural, physiological and biochemical studies. It was established that these isolates belong to the Streptomyces genus by virtue of their cell wall composition pattern and were identified as strains of different Streptomyces species like S. rochei, S. alanosinicus, S. erumpens, S. griseoplanus, S. gancidicus and S. nigrogriseolus.

The amount of wastewater treatment plants (WWTP) dealing with solid separation problems has significantly increased since the new requirements of the EU Directive 271/91 on nutrient removal. In Flanders a number of the nutrient removal WWTP are affected by solid separation problems mostly attributed to Microthrix parvicella being the most common dominant species. The effect of dosing polyaluminium chloride (PAX-14) on activated sludge is illustrated for WWTP solids separation problems, in particular because of Microthrix parvicella. The effects of the addition of PAX-14 on the microbiology and the morphology of Microthrix parvicella were studied in 9 full-scale WWTP. PAX-14 succeeded in reducing high SVI-values and controlled foaming problems whenever caused by Microthrix parvicella. Laboratory trials have shown that the dosage of PAX-14 should be less than 150 microL/L or 7 g Al3+/kg MLSS. At a dosage higher than 250 microL/L, an increase of free bacteria and a decrease of the protozoa activity are observed. In full-scale, PAX-14 is dosed at a concentration of 1.5 to 4.5 g Al3+/kg MLSS. Before addition, the mixed liquor scum layer--if present--should be removed. In our experience, the dosing should last for at least 3 weeks. During the first week, no drastic changes occur. At the end of the first week, an increase of SS and SVI is possible. The SVI and scum start to decrease after 10 to 15 days. The amount of filaments is reduced after 3 to 3 1/2 weeks. The morphological properties of Microthrix parvicella change, while other filaments such as Nostocoida limicola and Nocardia spp. are not affected. This study proves that PAX-14 is effective in controlling bulking and foaming problems at WWTPs when they are due to Microthrix parvicella. Prediction of when the SVI will decrease and when addition should be stopped is possible.

Whipple disease is a rare, chronic multisystem disease associated with the recently characterized organism Tropheryma whippelii. Extraintestinal manifestation involving the central nervous system, heart, and joints occasionally occurs. Involvement of the abdominal lymph nodes, especially the mesenteric and periaortic nodes, is not uncommon. However, peripheral lymphadenopathy as the sole clinical manifestation of Whipple disease is rare. We describe 2 patients with Whipple disease whose initial manifestation was lymphadenopathy. Lymph nodes from both patients showed infiltration of the sinuses by macrophages containing periodic acid-Schiff-positive, diastase-resistant, sickle-like structures. Electron microscopic evaluation confirmed the presence of rod-like organisms. DNA from each sample was amplified by the polymerase chain reaction using a specific set of oligonucleotide primers developed against the 16S ribosomal RNA coding sequence of T. whippelii. The histopathologic features and differential diagnosis of lipogranulomatous lymphadenopathy secondary to Whipple disease, as well as use of molecular-based assays, are discussed.

16S rRNA targeted probes, designed using sequence data from pure cultures of the three morphotypes of the filamentous bulking bacteria Nostocoida limicola I, II and III and their successful application to the in situ identification of these bacteria in activated sludge biomass samples are described here. Two probes were required to detect all the sequenced N. limicola II isolates. Results from fluorescent in situ hybridization suggest that the morphotypes N. limicola I and II contain at least two phylogenetically unrelated bacteria. The N. limicola II filaments that did not respond to the probes designed in this study fluoresced instead with the probes previously designed for the alpha-Proteobacteria. The data also suggest that both N. limicola I and III can exist in activated sludge as single, paired or clumped cells and thus in a form not recognizable microscopically as this morphotype. Some N. limicola II filaments which responded to the probes designed here were much thinner than the filaments conventionally 'identified' as this morphotype and better fitted the descriptions often used in the literature for N. limicola I.

A detailed characterization was performed of the Whipple's disease bacillus, strain Twist-MarseilleT, isolated from the cardiac valve of a patient with Whipple's disease bacillus endocarditis. This strain was isolated and maintained on human embryonic lung fibroblast monolayers, but could not be cultivated in the absence of living eukaryotic cells. Two morphological forms were observed, with differing staining properties; an intracellular form with intact and degenerating bacteria within vacuoles of infected cells and an extracellular form with masses of bacteria embedded in an extracellular matrix. Determination of the DNA G+C content confirmed that it belongs to the high-G+C gram-positive bacteria. Strain Twist-MarseilleT (= CNCM I-2202T) is proposed as the type strain of a new species within a new genus, Tropheryma whipplei gen. nov., sp. nov., that was provisionally created solely on the basis of 16S rRNA gene sequence data.

Isolates of eight bacterial filaments fitting the published morphological description of 'Nostocoida limicola' I were obtained from the mixed liquor of four different Australian and one Czech Republic activated sludge plants by micromanipulation. On the basis of their near complete (Ben 200 and Ben 201), or partial (Ben 77, Ben 78, Ben 202, Ben 203, Ben 204 and Ben 205) 16S rRNA gene sequences, six of these isolates were 99.3-100% similar to Lactosphaera pasteurii and Trichococcus flocculiformis, a bulking filament only reported previously in Germany. The other two (Ben 203 and Ben 204) were 99.9% similar to Streptococcus suis. Hence, all are in the low mol % G+C gram-positive bacteria division of the Bacteria. On this evidence 'N. limicola' I is phylogenetically unrelated to 'Nostocoida limicola' II, which is now known to be in the Actinobacteria, even though these two filamentous bacteria appearing in activated sludge systems have been considered to be closely related to each other historically.

Psychrophilic actinobacterial isolates from permanently cold groundwater in Finland were characterized using a polyphasic approach. Growth on agar plates was observed at temperatures down to -2 degrees C, with an optimum at 15-17 degrees C, but no growth was observed at 30 degrees C. The peptidoglycan type was B2y and the characteristic diamino acid was diaminobutyric acid. The cell wall sugars of strain K265T were rhamnose, ribose, xylose and mannose and those of strain K300T were glucose, rhamnose and xylose. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxyhexadecane were present as diagnostic markers. The predominant menaquinones were MK-9 and MK-10. The G+C content of the DNA of strains K265T and K300T was 64.4 and 67.8 mol%, respectively. Comparison of 16S rRNA gene sequences revealed that strains K265T and K300T represent a new lineage among the type-B-peptidoglycan actinomycetes. The closest relatives were Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. On the basis of 16S rDNA sequence, G+C content and chemotaxonomical and physiological characteristics, K265T and K300T clearly represent a new genus. The genus Subtercola gen. nov. is described, together with two species, namely Subtercola boreus sp. nov. (type strain K300T = DSM 13056T = CCUG 43135T) and Subtercola frigoramans sp. nov (type strain K265T = DSM 13057T = CCUG 43136T).