Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Surgical Resection to Treat a Japanese Patient with Pulmonary Coccidioidomycosis.

Coccidioidomycosis is an endemic disease that is particularly prevalent in the United States. However, its geographic distribution is becoming widespread. Here, we present a Japanese male who resided in the United States for 1 year, where he was diagnosed with pulmonary coccidioidomycosis that was accompanied by cavity formation. He did not tolerate antifungal therapy and consequently underwent partial resection of the upper lobe of his left lung upon his return to Japan. The patient's symptoms improved after surgery. The trend toward global networking and logistics means that a diagnosis of coccidioidomycosis should be considered in routine practice in nonendemic areas. Due to the rarity of surgical treatment for this disease, prolonged follow-up is necessary. During the last follow-up, the patient was symptom-free.

Aureobasidium melanigenum catheter-related bloodstream infection: a case report.

BACKGROUND: Aureobasidium melanigenum is a ubiquitous dematiaceous fungus that rarely causes invasive human infections. Here, we present a case of Aureobasidium melanigenum bloodstream infection in a 20-year-old man with long-term catheter use. CASE PRESENTATION: A 20-year-old man receiving home care with severe disabilities due to cerebral palsy and short bowel syndrome, resulting in long-term central venous catheter use, was referred to our hospital with a fever. After the detection of yeast-like cells in blood cultures on day 3, antifungal therapy was initiated. Two identification tests performed at a clinical microbiological laboratory showed different identification results: Aureobasidium pullulans from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Cryptococcus albidus from a VITEK2 system. Therefore, we changed the antifungal drug to liposomal amphotericin B. The fungus was identified as A. melanigenum by DNA sequence-based analysis. The patient recovered with antifungal therapy and long-term catheter removal. CONCLUSION: It is difficult to correctly identify A. melanigenum by routine microbiological testing. Clinicians must pay attention to the process of identification of yeast-like cells and retain A. melanigenum in cases of refractory fungal infection.

Examination of Cyp51A-Mediated Azole Resistance in Aspergillus lentulus Using CRISPR/Cas9 Genome Editing.

Aspergillus lentulus was first reported in 2005 as a cryptic species of Aspergillus fumigatus, and since then, its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Although it has been reported that P450 14-α sterol demethylase (Cyp51) is involved in azole resistance in A. lentulus, the specific resistance mechanism has not been elucidated. In this study, we successfully introduced the entire A. fumigatus cyp51A gene into the cyp51A locus in A. lentulus using the CRISPR/Cas9 genome-editing system. The A. lentulus strains harboring A. fumigatus cyp51A showed reduced minimum inhibitory concentrations for itraconazole and voriconazole compared with those of the parent strain. This finding suggests that Cyp51A is involved in azole resistance in A. lentulus and may contribute to the elucidation of the mechanism of resistance to azole drugs via Cyp51A and to the development of new antifungal drugs. In addition, our successful application of the CRISPR/Cas9 system to A. lentulus opens the door to examination of other gene functions in this fungus.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

Factors Associated with Breakthrough Fungemia Caused by Candida, Trichosporon, or Fusarium Species in Patients with Hematological Disorders.

Limited data are available on breakthrough fungemia, defined as fungemia that develops on administration of antifungal agents, in patients with hematological disorders. We reviewed the medical and microbiological records of adult patients with hematological diseases who had breakthrough fungemia between January 2008 and July 2019 at Toranomon Hospital and Toranomon Hospital Kajigaya in Japan. A total of 121 cases of breakthrough fungemia were identified. Of the 121 involved patients, 83, 11, 5, and 22 were receiving micafungin, voriconazole, itraconazole, and liposomal amphotericin B, respectively, when the breakthrough occurred. Of the 121 causative breakthrough fungal strains, 96 were Candida species, and the rest were 13 cases of Trichosporon species, 7 of Fusarium species, 2 of Rhodotorula mucilaginosa, and 1 each of Cryptococcus neoformans, Exophiala dermatitidis, and Magnusiomyces capitatus. The crude 14-day mortality rate of breakthrough fungemia was 36%. Significant independent factors associated with the crude 14-day mortality rate were age of ≥60 years (P = 0.011), chronic renal failure (P = 0.0087), septic shock (P < 0.0001), steroid administration (P = 0.0085), and liposomal amphotericin B breakthrough fungemia (P = 0.0011). An absolute neutrophil count of >500/µL was significantly more common in candidemia in the multivariate analysis (P = 0.0065), neutropenia and nonallogeneic hematopoietic stem cell transplants were significantly more common in Trichosporon fungemia (P = 0.036 and P = 0.033, respectively), and voriconazole breakthrough fungemia and neutropenia were significantly more common in Fusarium fungemia (P = 0.016 and P = 0.016, respectively). The epidemiological and clinical characteristics of breakthrough fungemia of patients with hematological disorders were demonstrated. Some useful factors to predict candidemia, Trichosporon fungemia, and Fusarium fungemia were identified.

Gordonia crocea sp. nov. and Gordonia spumicola sp. nov. isolated from sludge of a wastewater treatment plant.

Two novel actinobacteria, designated NBRC 107696T and NBRC 107697T, were isolated from sludge samples from a wastewater treatment plant and their taxonomic positions were investigated by a polyphasic approach. The cells of the strains were aerobic, rod-shaped, non-motile and non-endospore-forming. The strains contained glutamic acid, alanine and meso-diaminopimelic acid in the peptidoglycan. Galactose and arabinose were detected as cell-wall sugars. The predominant menaquinone was identified as MK-9(H2) and the major fatty acids were C16  :  0, C18 : 1ω9c and C16 : 1ω7c. The DNA G+C contents of NBRC 107696T and NBRC 107697T were 68.07 and 68.99 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that NBRC 107696T and NBRC 107697T were a clade with members of the genus Gordonia. The highest 16S rRNA gene sequence similarity values were obtained with Gordonia araii IFM 10211T (98.9 %) for NBRC 107697T, and Gordonia malaquae IMMIB WWCC-22T, Gordonia neofelifaecis AD-6T and Gordonia humi CC-12301T (98.1 %) for NBRC 107696T, respectively. The digital DNA-DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel separate species. The names proposed to accommodate these two strains are Gordonia spumicola sp. nov. and Gordonia crocea sp. nov., and the type strains are NBRC 107696T (=IFM 10067T=TBRC 11239T) and NBRC 107697T (=IFM 10881T=TBRC 11240T), respectively.

Disseminated cutaneous cryptococcosis in an immunocompetent elderly long-term pigeon fancier.

Cutaneous cryptococcosis is classified as localized cutaneous cryptococcosis and cutaneous manifestations of disseminated cryptococcosis. The former presents as lesions, confined to isolated parts of the skin, which are neither systemically disseminated nor associated with cryptococcal fungemia or antigenemia. The latter presents as lesions through dissemination of Cryptococcus from visceral organs such as the lungs, with most cases being immunosuppressed hosts. We report the case of an immunocompetent elderly long-term pigeon fancier who presented with disseminated cutaneous cryptococcosis caused by Cryptococcus neoformans. Although the patient had been at risk of inhaling the pathogen by keeping pigeons for many years, and had been treated with topical steroids for a localized nodular lesion, the cause of development of multiple skin lesions could not be determined. The patient paradoxically showed no pulmonary or central nervous system symptoms, fungemia or glucuronoxylomannan antigenemia. Treatment with oral itraconazole 200 mg/day was not effective, but combination therapy of 5-fluorocytosine 200 mg/kg per day and fluconazole 100 mg/day resolved the disease.

Identification of a Novel Variant Form of Aspergillus fumigatus CalC and Generation of Anti-CalC Monoclonal Antibodies.

Aspergillus fumigatus is a critical human fungal pathogen that infects the host via inhalation of airborne conidia. These conidia then germinate to form filamentous hyphae, which secrete various elements to survive in the host lung.Elements such as proteins secreted by A. fumigatus can act as virulence factors in host tissues. Among secreted proteins, we were interested in the thaumatin-like proteins of A. fumigatus. In our analysis of the function of thaumatin-like proteins, we found that, like CalA and CalB, CalC has a secreted form. Originally, CalC was predicted to be a GPI-anchored protein, as documented in the Aspergillus Genome Database. Here, we report on a novel secreted form of CalC. Furthermore, we established two novel hybridomas, C103 and C306, which recognized CalC. Monoclonal antibodies produced by these hybridomas responded to recombinant CalC produced by the mammalian cell line HEK293T and to the supernatant of cultured A. fumigatus.Taken together, our data suggest that calC can be spliced to give rise to a novel secretory form of CalC, which is present in the supernatant of cultured A. fumigatus. The hybridomas that we established will be helpful in understanding the biological role of A. fumigatus CalC.

Insight into the draft whole-genome sequence of the dermatophyte Arthroderma vanbreuseghemii.

Next-generation technologies have prompted efforts towards generating a large repertoire of whole-genome sequences. The dermatophyte Arthroderma vanbreuseghemii has been considered as a good model in which to conduct molecular biological studies on this fungal group. Despite the considerable repertoire of molecular tools developed for this fungus, the lack of genomic data has represented a major limitation, preventing effective implementation of those tools. Herein, the authors report the first draft whole-genome sequence of this dermatophytic species. The size of the draft genome was 23 Mb, exhibiting a GC content of 48.1%. Given the significance of secreted proteases in tissue invasion, a comparative analysis of genes encoding extracellular proteases was performed between A. vanbreuseghemii and other dermatophytes. Furthermore, genes that might be involved in DNA repair also were compared among dermatophytes. Moreover, the complete mitochondrial genome of A. vanbreuseghemii was obtained and shown to consist of 24,287 bp with a GC content of 24%. In conclusion, the availability of genomic data for A. vanbreuseghemii is expected to facilitate the implementation of the molecular tools established for this fungus, enhancing our understanding of the biology of dermatophytes.

CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus.

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea.

OBJECTIVE: Streptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified. METHODS: Effects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography. RESULTS: Assam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation. CONCLUSIONS: The higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.

Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Cloning and nucleotide sequence analyses of 11 genome segments of two American and one British equine rotavirus strains.

Group A equine rotavirus (ERV) is the main cause of diarrhea in foals and causes severe economic loss due to morbidity and mortality on stud farming worldwide. Molecular evolution of equine rotaviruses remains understudies. In this study, whole-genomic analysis of 2 group A ERV, FI-14 (G3P[12]), H-2 (G3P[12]) isolated from American, and FI23 (G14P[12]) from British was carried out and genotype constellations were determined as G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 for FI-14; G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7 for FI23; and G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 for H-2, respectively. With the exception of the VP7 and VP6 gene, 2 G3P[12] strains (FI-14 and H-2) and one G14P[12] strain (FI23) were highly related genetically. Of note, the VP6 genotype of H-2 strain was previously reported to be I2, however, sequence and phylogenetic analyses demonstrated that it was I6. Therefore, it showed that G3P[12] ERV strains and G14P[12] ERV strains bore a distinct VP6 genotype: I6 for G3P[12] strains and I2 for G14P[12] strains. Moreover, it demonstrated that T-cell epitope 299P-300P/Q residues (PP/Q) of VP6 may be considered as I2 ERV typical molecular marker, which facilitates the analysis of the molecular evolution of equine rotaviruses.

Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 µg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 µg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.

The spike protein VP4 defines the endocytic pathway used by rotavirus to enter MA104 cells.

Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway.

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.

Complete genome sequence of the motile actinomycete Actinoplanes missouriensis 431(T) (= NBRC 102363(T)).

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds. Here, we describe the features of A. missouriensis 431(T), together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

Identification of nocobactin NA biosynthetic gene clusters in Nocardia farcinica.

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.