The quorum sensing peptide BlpC regulates the transcription of genes outside its associated gene cluster and impacts the growth of Streptococcus thermophilus

Michael J. McAnulty, Giselle K. Guron, Adam M. Oest, Amanda L. Miller, and John A. Renye, Jr.

Bacteriocin production in Streptococcus thermophilus is regulated by cell density-dependent signaling molecules, including BlpC, which regulates transcription from within the bacteriocin-like peptide (blp) gene cluster. In some strains, such as S. thermophilus ST106, this signaling system does not function properly, and BlpC must be supplied exogenously to induce bacteriocin production. In other strains, such as S. thermophilus B59671, bacteriocin (thermophilin 110 in strain B59671) production occurs naturally. Here, transcriptomic analyses were used to compare global gene expression within ST106 in the presence or absence of synthetic BlpC and within B59671 to determine if BlpC regulates the expression of genes outside the blp cluster. Real-time semi-quantitative PCR was used to find genes differentially expressed in the absence of chromosomal blpC in the B59671 background. Growth curve experiments and bacteriocin activity assays were performed with knockout mutants and BlpC supplementation to identify effects on growth and bacteriocin production. In addition to the genes involved in bacteriocin production, BlpC affected the expression of several transcription regulators outside the blp gene cluster, including a putative YtrA-subfamily transcriptional repressor. In strain B59671, BlpC not only regulated the expression of thermophilin 110 but also suppressed the production of another bacteriocin, thermophilin 13, and induced the same YtrA-subfamily transcriptional repressor identified in ST106. Additionally, it was shown that the broad-spectrum antimicrobial activity associated with strain B59671 was due to the production of thermophilin 110, while thermophilin 13 appears to be a redundant system for suppressing intraspecies growth. BlpC production or induction negatively affected the growth of strains B59671 and ST106, revealing selective pressure to not produce bacteriocins that may explain bacteriocin production phenotype differences between S. thermophilus strains. This study identifies additional genes regulated by BlpC and assists in defining conditions to optimize the production of bacteriocins for applications in agriculture or human and animal health.

Safety evaluation of Fy Protein™ (Nutritional Fungi Protein), a macroingredient for human consumption

Brian Furey, Kathleen Slingerland, Mark R. Bauter, Celeste Dunn, Richard E. Goodman, Sophia Koo

Abstract

Fy Protein™ (Nutritional Fungi Protein) is a macro-ingredient produced from the fermentation of the fungal microorganism Fusarium strain flavolapis, isolated from springs in Yellowstone National Park. Fy Protein contains all of the essential amino acids plus fiber, fat, carbohydrates, vitamins, and minerals and is developed as an alternative to animal-based protein foods such as meat and dairy.

Fy Protein's nutritional, digestibility, genotoxicity, allergenicity, toxicity, secondary metabolites, and pathogenicity were evaluated. Fy Protein did not show mutagenic or genotoxic potential in in vitro tests. In an allergenicity review, Fy Protein was found to be of low allergenic potential. In a 90-day sub chronic dietary study in rats, administration of Fy Protein did not produce any significant toxicologic manifestations, and the No Observed Adverse Effect Level (NOAEL) was the highest-level fed of 150,000 ppm (15% in the diet). Regulated secondary metabolites from fungi (termed mycotoxins) were non-detectable and below regulated levels using quantitative analytical techniques. A literature review was completed to identify the potential human pathogenicity of Fusarium sp., showing that Fusarium rarely infects humans, with infections seldom developing even in immunocompromised individuals.

The results of these studies confirm that Fy Protein from fermented F. str. flavolapis has low toxicological, genotoxic, pathogenic, and allergenic potential under the conditions tested and anticipated use.

New genomic resources and comparative analyses reveal differences in floral gene expression in selfing and outcrossing Collinsia sister species

Lauren J. Frazee, Joanna Rifkin 2 Dinusha C. Maheepala, Alannie-Grace Grant, Stephen Wright , Susan Kalisz, Amy Litt, and Rachel Spigler

The evolutionary transition from outcross- to self-fertilization is one of the most common in angiosperms and is often associated with a parallel shift in floral morphological and developmental traits, such as reduced flower size and pollen to ovule ratios, known as the “selfing syndrome.” How these convergent phenotypes arise, the extent to which they are shaped by selection, and the nature of their underlying genetic basis are unsettled questions in evolutionary biology. The genus Collinsia (Plantaginaceae) includes seven independent transitions from outcrossing or mixed mating to high selfing rates accompanied by selfing syndrome traits. Accordingly, Collinsia represents an ideal system for investigating this parallelism, but requires genomic resource development. We present a high quality de novo genome assembly for the highly selfing species Collinsia rattanii. To begin addressing the basis of selfing syndrome developmental shifts, we evaluate and contrast patterns of gene expression from floral transcriptomes across three stages of bud development for C. rattanii and its outcrossing sister species Collinsia linearis. Relative to C. linearis, total gene expression is less variable among individuals and bud stages in C. rattanii. In addition, there is a common pattern among differentially expressed genes: lower expression levels that are more constant across bud development in C. rattanii relative to C. linearis. Transcriptional regulation of enzymes involved in pollen formation specifically in early bud development may influence floral traits that distinguish selfing and outcrossing Collinsia species through pleiotropic functions. Future work will include additional Collinsia outcrossing-selfing species pairs to identify genomic signatures of parallel evolution. Keywords: Collinsia; RNA-seq; selfing syndrome; pollen; floral development; differential gene expression; DESeq2; dichogamy; evolutionary genomics; Hi-C scaffolding; parallel evolution

Evaluating potential risks of food allergy of novel food sources based on comparison of proteins predicted from genomes and compared to www.AllergenOnline.org

Mohamed Abdelmoteleb, Chi Zhang, Brian Furey, Mark Kozubal, Hywel Griffiths, Marion Champeaud, Richard E. Goodman

Potential proteins from three novel food sources (Chlorella variabilis, Galdieria sulphuraria, and Fusarium strain flavolapis) were predicted from genomic sequences and were evaluated for potential risks of allergic cross-reactivity by comparing the predicted amino acid sequences against the allergens in the www.AllergenOnline.org (AOL) database. The preliminary analysis used CODEX Alimentarius limits of >35% identity over 80 amino acids to evaluate the predicted proteins which include many evolutionarily conserved proteins. Regulators might expect clinical serum IgE tests based on identity matches above the criteria if the proteins were introduced in genetically engineered crops. Some regulators have the same expectations for proteins in novel foods. To address the inequality of extensively conserved sequences, we compared the predicted proteins from curated genomes of 23 highly diverse allergenic species from animals, plants and arthropods as well as humans to AOL sequences and compiled identities. Identity matches greater than CODEX limits (>35% ID over 80 AA) are common for many proteins that are conserved through extensive evolution but are not predictive of published allergy risks based on observed taxonomic cross-reactivity. Therefore, we recommend changes in the allergen databases or methods of identifying matches for risk evaluation of new food sources. Our results provide critical data for redefining allergens in AOL or for providing guidance on more predictive sequence identity matches for risk assessment of possible risks of food allergy.

Organic farming practices utilizing spent microbial biomass from an industrial fermentation facility promote transition to copiotrophic soil communities

Mathew Halter, Benjamin Vaisvil, Vinayak Kapatral & James Zahn

Organic farming has become more prevalent in recent years as consumer demand for organic food and fiber has rapidly grown. Until recently, organic fertilizers and soil amendments have largely been based on the practices of returning crop residues, manures and related agricultural wastes back to crop production areas. One rapidly growing segment in commercial organic fertilizer development is the use of spent microbial biomass (SMB) from industrial fermentation processes. While SMB is widely accepted in many organic farming systems (OFS), little is known concerning the effectiveness, environmental impact, and influence on prokaryotic communities in soils receiving this treatment. In this study, a comparative analysis of bacterial communities associated with OFS and conventional farming systems was performed over a growing season for a field containing yellow dent corn (Zea mays). A statistically significant increase in microbial population α-diversity, along with a strong recruitment of Proteobacteria and Actinobacteria populations, was observed in soils treated with SMB when compared to areas in the field that utilized conventional farmer practices. These phyla are members of the copiotrophic subgroup, and considered a signature for the use of traditional organic fertilizers. These results provide valuable new information that SMB functions similarly to traditional organic fertilizers in promoting a high level of functional prokaryotic diversity and plant growth-promoting bacteria, but in contrast do not contribute directly to viable microorganisms in the soil due to the sterilization of SMB prior to land application.

Yeast Transcriptomics During Bioprocessing: A Powerful Tool in Cellulosic Ethanol Production and Process Optimization

Ananda Nanjundaswamy, Alcorn State University Co-Author(s): Keerthi Mandyam, Department of Agriculture, Alcorn State University; Vinayak Kapatral and Benjamin, Vaisvil, and Daniel Schmitt, Igenbio, Inc

Yeast, saccharomyces cerevisiae, is very critical for ethanol production and its performance determines whether a process is economical or not. At first glance, ethanol production appears simple with dosing of yeast to a sugar rich medium where yeast produces ethanol through a fermentation process. But a closer look reveals a much complex biochemical processes involved in ethanol production. During fermentation, yeast will be growing in a high solid medium in presence of inhibitors such as ethanol and furfurals. Also there will be mass transfer limitations such as depletion of oxygen which creates stress for the yeast. In order to optimize the ideal conditions for the yeast growth and ethanol production, it is critical to understand the yeast behavior at transcriptional level. The present study employed leading cellulosic feedstock miscanthus for ethanol production in a benchtop bioreactor. Samples were collected at 24, 48 and 72h and analyzed for yeast behavior during fermentation. Transcriptional analysis of yeast indicated that yeast behaves differently in different me points. Transcriptional information of important biochemical pathways as influenced by the fermentation conditions will be discussed.

Genomic Comparisons of Endophytic Periconia from North American and European Grasslands

Keerthi Mandyam, Alcorn State University Co-Author(s): Anna Kazarina and Ananda Nanjundaswamy, Dept of Agriculture, Alcorn State University; Benjamin Vaisvil, Daniel Schmitt, and Vinayak Kapatral, Igenbio Inc

Plants are associated with a suite of microbial symbioses, with roots offering a unique niche for fungal endophytes. Among root fungal symbionts, dark septate endophytic (DSE) fungi are common, sometimes abundant but enigmatic with poor clarity on their functional roles. Biogeographical distinctions likely exist in DSE communities from forests and grasslands, with North American and European grasslands predominantly represented by Periconia macrospinosa. To understand their endophyc roles, the genome of dark septate P. macrospinosa and Cadophora isolated from Festuca vaginata from semi-arid European grassland were sequenced. To further comprehend DSE funconal roles, the objecve of this study was to compare the North American P. macrospinosa genome with that of the European P. macrospinosa. Periconia was isolated from a stand of Freedom Giant Miscanthus culvated in Lorman, MS and was confirmed to be a DSE. We hypothesized that despite the geographical disncons and diverse grass hosts, P. macrospinosa associated with grasses would have similar funconal roles. Periconia genome was sequenced using Illumina and PacBio plaorms. Our Periconia genome was determined to be ~ 53.5 MB in size with 45% GC content. At least 12,059 ORFs with 9,086 ORFs with introns were idenfied and nearly 35% of the ORFs were assigned funcons. As expected, several plant cell wall degrading enzymes (PCWDEs) like cellulases (12 ORFs), amylases (2 ORFs), pecn esterase (1 ORF), tannase (2 ORFs), laccase (6 ORFs) were idenfied along with several sugar transport systems such as maltose, lactate, sucrose, maltose, xylose isomaltose, palanose, etc. However, ORFs for lignin peroxidase, manganese peroxidase, glyoxal oxidase were not observed. As hypothesized, the Periconia genomes were comparable. Periconia macrospinosa genomics data will be discussed to draw big picture inferences regarding DSE symbiosis.

Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

Wiens GD, Rockey DD, Wu Z, Chang J, Levy R, Crane S, Chen DS, Capri GR, Burnett JR, Sudheesh PS, Schipma MJ, Burd H, Bhattacharyya A, Rhodes LD, Kaul R, Strom MS.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

J Bacteriol. 2008 Nov;190(21):6970-82. 
doi: 10.1128/JB.00721-08. Epub 2008 Aug 22.

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

Gabriel DW, Allen C, Schell M, Denny TP, Greenberg JT, Duan YP, Flores-Cruz Z, Huang Q, Clifford JM, Presting G, González ET, Reddy J, Elphinstone J, Swanson J, Yao J, Mulholland V, Liu L, Farmerie W, Patnaikuni M, Balogh B, Norman D, Alvarez A, Castillo JA, Jones J, Saddler G, Walunas T, Zhukov A, Mikhailova N.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Mol Plant Microbe Interact. 2006 Jan;19(1):69-79.
http://dx.doi.org/10.1094/MPMI-19-0069

Draft Sequencing and Comparative Genomics of Xylella fastidiosa Strains Reveal Novel Biological Insights.

Anamitra Bhattacharyya, Stephanie Stilwagen, Gary Reznik, Helene Feil, William S. Feil, Iain Anderson, Axel Bernal, Mark D'Souza, Natalia Ivanova, Vinayak Kapatral, Niels Larsen, Tamara Los, Athanasios Lykidis, Eugene Selkov, Jr., Theresa L. Walunas, Alexander Purcell, Rob A. Edwards, Trevor Hawkins, Robert Haselkorn, Ross Overbeek, Nikos C. Kyrpides, and Paul F. Predki

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

Genome Res. 2002 Oct; 12(10): 1556–1563.
doi:  10.1101/gr.370702