RNA-seq analysis reveals that an ECF σ factor, AcsS, regulates achromobactin biosynthesis in Pseudomonas syringae pv. syringae B728a.

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a.

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant.

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.

Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum.

Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.

Temporal and spatial regulation of gene expression during asexual development of Neurospora crassa.

In this study we profiled spatial and temporal transcriptional changes during asexual sporulation in the filamentous fungus Neurospora crassa. Aerial tissue was separated from the mycelium to allow detection of genes specific to each tissue. We identified 2641 genes that were differentially expressed during development, which represents ∼25% of the predicted genes in the genome of this model fungus. On the basis of the distribution of functional annotations of 1102 of these genes, we identified gene expression patterns that define key physiological events during conidial development. Not surprisingly, genes encoding transcription factors, cell wall remodeling proteins, and proteins involved in signal transduction were differentially regulated during asexual development. Among the genes differentially expressed in aerial tissues the majority were unclassified and tended to be unique to ascomycete genomes. This finding is consistent with the view that these genes evolved for asexual development in the Pezizomycotina. Strains containing deletions of several differentially expressed genes encoding transcription factors exhibited asexual development-associated phenotypes. Gene expression patterns during asexual development suggested that cAMP signaling plays a critical role in the transition from aerial growth to proconidial chain formation. This observation prompted us to characterize a deletion of the gene encoding a high-affinity cAMP phosphodiesterase (NCU00478). NCU00478 was determined to be allelic to aconidiate-2, a previously identified genetic locus controlling conidiation.

Comparison of loline alkaloid gene clusters across fungal endophytes: predicting the co-regulatory sequence motifs and the evolutionary history.

LOL, a fungal secondary metabolite gene cluster found in Epichloë and Neotyphodium species, is responsible for production of insecticidal loline alkaloids. To analyze the genetic architecture and to predict the evolutionary history of LOL, we compared five clusters from four fungal species (single clusters from Epichloë festucae, Neotyphodium sp. PauTG-1, Neotyphodium coenophialum, and two clusters we previously characterized in Neotyphodium uncinatum). Using PhyloCon to compare putative lol gene promoter regions, we have identified four motifs conserved across the lol genes in all five clusters. Each motif has significant similarity to known fungal transcription factor binding sites in the TRANSFAC database. Conservation of these motifs is further support for the hypothesis that the lol genes are co-regulated. Interestingly, the history of asexual Neotyphodium spp. includes multiple interspecific hybridization events. Comparing clusters from three Neotyphodium species and E. festucae allowed us to determine which Epichloë ancestors are the most likely contributors of LOL in these asexual species. For example, while no present day Epichloë typhina isolates are known to produce lolines, our data support the hypothesis that the E. typhina ancestor(s) of three asexual endophyte species contained a LOL gene cluster. Thus, these data support a model of evolution in which the polymorphism in loline alkaloid production phenotypes among endophyte species is likely due to the loss of the trait over time.

Button osteoma: its etiology and pathophysiology.

The present study investigates a circumscribed bony overgrowth on the cranial vault, known as button osteoma (BtO) and referred to here as button lesion (BtL). We discuss its anthropological implications. Data on its histology, location, and population distribution (by age, race, and gender) are provided. Microscopically, BtL is composed of well-organized dense lamellated bone which is poorly vascularized and with very few osteocytes. It forms a dome-shaped roof over an underlying diploeized area which includes the ectocranial table. The frequency of BtL is similar in modern (37.6%) and archaeological (41.1%) populations, in blacks, whites, males, and females, and correlates with age. It is rare in nonhuman primates. Fifty-five percent of the human skulls studied by us had BtL only on the parietal, 23.6% on the frontal, and 3.6% on the occipital bones. Fifteen percent had BtL on both the frontal and parietal bones. No lateral preference was found. Most skulls with BtL (64.1%) had only one lesion, 20.4% had two BtL, and 15.4% demonstrated multiple BtL. The average number of button osteomas on an affected skull was 1.97. The frequency of large osteomas (0.5-1.0 cm) was similar in young and old age groups. The demographic characteristics of BtL, mainly its high frequency among ancient and modern populations, its independence of sex and race, its scarcity in other primates, and the fact that its macro- and microstruture are indicative of an hamartoma (and not an osteoma or exostosis) suggest an evolutionary background to the phenomenon.

Serpens endocrania symmetrica (SES): a new term and a possible clue for identifying intrathoracic disease in skeletal populations.

This paper describes a phenomenon in the endocranial plate, which we have termed "serpens endocrania symmetrica" (SES), and discusses its value as a diagnostic tool. The affected discolored bone area exhibits disruption of the endocranial surface, lending it a maze-like appearance. Histological sections demonstrate that the process is limited to the most superficial portion of the endocranium, with no diploic and ectocranial involvement (sinus areas excepted). Adult skulls (n = 1,884) from the Hamann-Todd collection (HTH), housed at the Cleveland Museum of Natural History, were utilized for the present study. SES was recognized in 32 of the 1,884 skulls studied (1.7%). The frequency of SES among individuals reported to have died from tuberculosis (TB) was 4.4%. The rate of SES in the non-TB sample was only 0.53%. The locations were as follows: limited to sinus area, 28.1%; calvarium (excluding the sinuses), 46.9%; sinus + calvarium, 25.0%. SES was bilateral in 90.9% of cases. Twenty-five of the 32 individuals (78.1%) with SES in the HTH collection had tuberculosis specifically listed as the cause of death. Six of the other 7 individuals had infections other than TB. In 29 of the 32 individuals with SES, infection involved structures within the thorax. As SES was also associated with another osteological phenomenon known to represent pulmonary disease, i.e., hypertrophic osteoarthropathy (HOA; 68.0% of SES individuals also had HOA), SES may be of diagnostic value in paleopathology for the recognition of intrathoracic disease, and perhaps tuberculosis.