Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin

Summary The ¹H, ¹⁵N and ¹³C backbone and ¹H and ¹³C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, ³J_Hn_H coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn¹¹, Ser⁶⁸ and Asn⁷² are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ semiquinone - FMN riboflavin 5-monophosphate; nif, nitrogen fixation - TSP 3-(trimethylsilyl)propionate sodium salt - DSS 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the ¹⁵N, H^N, ¹³C, ¹H, ¹³C, ¹H and ¹³CO chemical shifts.To whom correspondence should be addressed.     

Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy

Summary A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance ¹³C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.     

Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR^® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr?≤?1.65%, R² values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

     

The use of compensatory base change analysis of ITS2 as a tool in the phylogeny of Mucorales, illustrated by the Mucor circinelloides complex

Compensatory base changes (CBCs) in helix II of rDNA ITS2, suggested as a molecular classifier for fungi, were analyzed in Mucor circinelloides and its varieties. Only a few CBCs were found in the complex. Three out of the four accepted formae (f. circinelloides, f. lusitanicus, f. janssenii) did not exhibit CBCs. One CBC was found between strains that form zygospores; consequently, CBC is not always concordant with mating experiments. Strains with two CBC are unable to breed. It is suggested that some strains of the M. circinelloides complex are at the beginning of speciation.     

Phylogenetic Analysis Reveals a High Prevalence of Sporothrix brasiliensis in Feline Sporotrichosis Outbreaks

Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n = 3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.     

Phylogenetic Analysis of Clinically Relevant Fusarium Species in Iran

Mycopathologia - Fungi of the genus Fusarium are well known as major plant pathogens but also cause a broad spectrum of human infections. Sixty-three clinical isolates, collected during...     

Black Yeast Biota in the Mangrove, in Search of the Origin of the Lethargic Crab Disease (LCD)

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis, two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea- or Exophiala-like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae, was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis, which is probably not dependent on the host species, U. cordatus. However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves.     

Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties

Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments where they are concomitantly subjected to several types of stress, such as toxicity, scarce nutrient availability, and high or low temperature extremes. Their ability to assimilate alkylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions, including on railway ties. Samples were collected using cotton swabs, premoistened with physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt extract agar supplemented with chloramphenicol, and incubated at 26 °C for 4 weeks. Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P = 0.02), tested positive for fungi. Exophiala crusticola was found to be the most common species (n = 13), followed by Exophiala phaeomuriformis (n = 7) and Exophiala heteromorpha (n = 4). These results suggest that hydrocarbons, particularly creosote-treated oak woods, support the growth of black yeasts, some of which are opportunists in humans.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.