Comparison of Yamuna (India) and Mississippi River (United States of America) bacterial communities reveals greater diversity below the Yamunotri Glacier

Osvaldo Martinez, Silas R. Bergen, Jacob B. Gareis

Abstract

The Yamuna River in India and the Mississippi River in the United States hold significant commercial, cultural, and ecological importance. This preliminary survey compares the bacterial communities sampled in surface waters at 11 sites (Yamuna headwaters, Mississippi headwaters, Yamuna River Yamunotri Town, Mississippi River at Winona, Tons River, Yamuna River at Paonta Sahib, Yamuna River Delhi-1, Yamuna River Delhi-2, Yamuna River before Sangam, Sangam, Ganga River before Sangam). Bacterial 16S rDNA analyses demonstrate dominance of Proteobacteria and Bacteroidetes phyla. Actinobacteria were also dominant at sites near Sangam in India and sites in Minnesota. A dominance of Epsilonbacteraeota were found in Delhi, India. Principal component analysis (PCA) using unique operational taxonomic units (OTUs) resulted in the identification of 3 groups that included the Yamuna River locations in Delhi (Delhi locations), Yamuna headwaters and Yamuna River at Yamunotri (Yamuna River locations below the Glacier) and Mississippi, Ganga, Tons, and other Yamuna River locations. Diversity indices were significantly higher at the Yamuna River locations below the Glacier (Simpson D = 0.986 and Shannon H = 5.06) as compared (p value <0.001) to the Delhi locations (D = 0.951 and H = 4.23) and as compared (p value < 0.001) to Mississippi, Ganga, Tons, and other Yamuna River locations (D = 0.943 and H = 3.96). To our knowledge, this is the first survey to compare Mississippi and Yamuna River bacterial communities. We demonstrate higher diversity in the bacterial communities below the Yamunotri glacier in India.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

Binod Rayamajhee, Mark Willcox, Savitri Sharma, Ronnie Mooney, Constantinos Petsoglou, Paul R Badenoch, Samendra Sherchan, Fiona L Henriquez, Nicole Carnt

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II

Tom G. Schwan, Sandra J. Raffel, Stacy M. Ricklefs, Daniel P. Bruno, and Craig Martens

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined.

Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined.

Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86–95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3′ end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined.

Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

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.

Whole-genome sequence and annotation of Penstemon davidsonii

Kate L Ostevik, Magdy Alabady, Mengrui Zhang, Mark D Rausher

Penstemon is the most speciose flowering plant genus endemic to North America. Penstemon species’ diverse morphology and adaptation to various environments have made them a valuable model system for studying evolution. Here, we report the first full reference genome assembly and annotation for Penstemon davidsonii. Using PacBio long-read sequencing and Hi-C scaffolding technology, we constructed a de novo reference genome of 437,568,744 bases, with a contig N50 of 40 Mb and L50 of 5. The annotation includes 18,199 gene models, and both the genome and transcriptome assembly contain over 95% complete eudicot BUSCOs. This genome assembly will serve as a valuable reference for studying the evolutionary history and genetic diversity of the Penstemon genus.

De novo assembly of Roseomonas mucosa isolated from patients with atopic dermatitis

Kent Barbian, Daniel Bruno, Lydia Sykora, Stacy Ricklefs, Prem Prashant Chaudhary, Paul A. Beare, Ian A. Myles, Craig M. Martens

ABSTRACT

Roseomonas mucosa is associated with the normal skin microflora. Here, we present de novo sequence assemblies from R. mucosa isolates obtained from the skin lesions of three atopic dermatitis patients.

ANNOUNCEMENT

Roseomonas mucosa is a Gram-negative coccobacillus found in aquatic environments and from various clinical samples (1 – 5). R. mucosa is commonly isolated from the microbiota of human skin (4, 6). Atopic dermatitis (AD), an inflammatory skin disease, causes susceptibility to Staphylococcus aureus infection, immune dysregulation function, and an impaired skin barrier. Improved AD clinical outcome was observed when R. mucosa was used to topically treat patients with AD (2, 4, 7). This treatment had an improved outcome in mouse models of AD. Interestingly, this mouse model treated with R. mucosa from AD patients, had no impact or a worse clinical outcome (2). We present the sequences from three AD-sourced R. mucosa isolates (2).

R. mucosa from lesions of three AD patients was isolated using a FloqSwab moistened in phosphate-buffered saline. Swabs were placed in Reasoner’s 2A (R2A) broth containing amphotericin B and vancomycin and incubated for 48–72 h at 32°C. Cultures were spread on R2A agar and incubated as above to form single colonies (4). Bacteria, stored at −80°C, were spread on R2A agar and a single colony used to inoculate 100 mls of R2A broth, and grown at 32°C for 20–25 h (8). Bacteria were washed, resuspended, and heated to 65°C, followed by separate incubations with proteinase k, lysozyme, and SDS. Each sample was incubated with RNaseI for 30 min at 37°C, then combined with a 3/4 vol of saturated phenol solution, mixed for 10 min and centrifugated at 12,800 × g for 10 min. The aqueous phase was removed and treated with phenol:chloroform:isoamyl alcohol (25:24:1) until the white precipitate interface was absent. A chloroform-only extraction was performed, and aqueous solution was collected. DNA was precipitated with 3 M sodium acetate, washed 2× with 70% EtOH, and resuspended in Qiagen’s EB buffer.

PacBio and Illumina sequencing libraries were generated from the same DNA using the 10 kb Template Preparation and Sequencing kit with Covaris g-tubes (sheared to >8,700 bp) and Illumina TruSeq Nano DNA kit using a Covaris M220 (sheared to ~550 bp), respectively (PacBio protocol 100-092-800-06 and TruSeq protocol 15041110 rev. D). Libraries were sequenced with PacBio Sequel and Illumina MiSeq (2 × 300 bp read length) platforms. Illumina reads were adapter trimmed using cutadapt (v.1.12) and trimmed and filtered for quality using FASTX-Toolkit (v.0.0.14). Bioinformatic analysis and construction of the genomes were performed using the following programs: CANU (v.1.0), Bowtie2 (v.2.2.4), pilon (v.1.16), and MIRA (v.4.0). All software was used with default parameters. The PacBio data were used to construct DNA scaffolds which were optimized using Illumina data. Igenbio Inc. annotated the genomes, while public annotation was compiled by the NCBI Prokaryotic Genome Annotation Pipeline (9).

The R. mucosa genomes consist of a single large circular chromosome (4.01–4.28 Mb) and five to six autonomously replicating plasmid sequences (8.9–506.3 kb) (Table 1). Genome properties and annotation statistics are listed in Table 1. These genomes may help determine the genes that are important for successful treatment of AD.

ACKNOWLEDGMENTS

We would like to thank Igenbio Inc. their sequencing services and data analysis.

This work was supported by funding from the Intramural Research Program of the NIH, NIAID 67 (Z01-147170). The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

Beyond rational—biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes

Philipp T. Baumann, Michael Dal Molin, Hannah Aring, Karin Krumbach, Moritz-Fabian Müller, Bas Vroling, Philana V. van Summeren-Wesenhagen, Stephan Noack, and Jan Marienhagen

Background

In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum l-histidine producer. Since all known genetic targets contributing to l-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to l-histidine biosynthesis.

Results

In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and l-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10–80% more l-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled l-histidine titer (29 mM) and product yield (0.13 C-mol C-mol−1) in comparison to the starting variant.

Conclusions

This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12915-023-01688-x.

Keywords: Biosensor, Fluorescence-activated cell sorting, High-throughput screening, Genome sequencing, Comparative genome analysis, Single-nucleotide polymorphism, Metabolic engineering, l-histidine, Corynebacterium glutamicum

Transcriptomics elucidates metabolic regulation and functional promoters in the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938

Emma E. Tobin, Joseph H. Collins, Celeste B. Marsan, Gillian T. Nadeau, Kim Mori, Anna Lipzen, Stephen Mondo, Igor V. Grigoriev, Eric M. Young

Genomics has become the primary way to explore microbial diversity, because genetic tools are currently difficult to develop in non-model organisms. Here, we demonstrate that -omics can be leveraged to accelerate genetic tool development for the basidiomycete yeast Xanthophyllomyces dendrorhous CBS 6938, the sole biotechnologically relevant organism in the Tremellomycete family. First, we sequence the genome. Then, we perform transcriptomics under a variety of conditions, focusing on light and oxidative stress. This data not only reveals novel photobiology and metabolic regulation, it also allows derivation of constitutive and regulated gene expression parts. Our analysis of X. dendrorhous photobiology shows for the first time that a complex system of white-collar and cryptochrome homologs mediate response to ultraviolet light (UV). Our analysis of metabolic regulation shows that UV activates DNA repair, aromatic amino acid and carotenoid biosynthesis and represses central carbon metabolism and the fungal-like apoptotic pathway. Thus, X. dendrorhous shows a dynamic response toward biosynthetic pathways for light-absorbing compounds and survival and away from energy production. We then define a modular cloning system, including antibiotic selections, integration sites, and reporter genes, and use the transcriptomics to derive strong constitutive and regulated promoters. Notably, we discover a novel promoter from a hypothetical gene that has 9-fold activation upon UV exposure. Thus, -omics-to-parts workflows can simultaneously provide useful genomic data and advance genetic tools for non-model microbes, particularly those without a closely related model organism. This approach will be broadly useful in current efforts to engineer diverse microbes.

Discovery of novel amino acid production traits by evolution of synthetic co-cultures

Rico Zuchowski, Simone Schito, Friederike Neuheuser, Philipp Menke, Daniel Berger, Niels Hollmann, Srushti Gujar, Lea Sundermeyer, Christina Mack, Astrid Wirtz, Oliver H. Weiergräber, Tino Polen, Michael Bott, Stephan Noack & Meike Baumgart

Background

Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities.

Results

Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na+/H+-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of KD = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain.

Conclusion

Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Differential Gene Expression of Ca2+ Channel-Mediated Signaling Pathways in DPSCs under Hypoxic Condition

JOHN T. TRAN
Dental pulp stem cell (DPSC) survival, differentiation into odontoblast and deposition of matrix for mineralization is essential for vital pulp therapy (VPT) and regenerative endodontic therapy (RET). Calcium homeostasis in DPSC plays a critical role for its survival, longevity, and differentiation processes. Cells use this external source of signal calcium ions by activating various entry channels with different properties. The objective of this research is to investigate key gene candidates involved with calcium homeostasis in human dental pulp tissue and DPSCs.

DPSCs isolated from human dental pulp were cultured in hypoxic chamber (3%) for 21 days at 37°C in differentiation media and controls were cultured under normal conditions. Later, DPSCs collected were extracted for mRNA isolation. The mRNA was sequenced using NextSeq Illunima and was analyzed using the ERGOTM transcription tool using statistical methods such as Limma (Linear Model) and DESeq2 (Negative Binomial).

There were a total of 24 biomarkers identified with a 6-fold higher over expressed in hypoxic conditions compared to normoxia. These include key genes such as CALM2, MYLK, FGR1, ASPH, PDGFRA, ATP2B4, ATP2B1, PDGFRB, CALM1, and PPP3CA. The genes and pathways were identified in KEGG pathways overlaying expression values.

The release of calcium ions from internal stores and the influx of calcium ions from the environment cause a dramatic and rapid increase in cytoplasmic calcium concentration, which has been exploited for signal transduction. In combination with power statistical analysis such as DESeq2 and Limma, key pathways can be identified that are responsible for calcium homeostasis and calcium channel signaling as key diagnostic markers for VPT and RET.

Investigation of Apoptotic Biomarkers in Dental Pulp Stem Cells under Hypoxic Condition using RNA-Seq

Philip William Cannizzaro

Immature teeth can have halted root development through pathology associated with caries, trauma, or dental anomalies such as dens evaginatus. Pulp tissue under stress induces ischemia and subsequent necrosis. Through regenerative endodontic procedures, proper procedural protocol often leads to revitalization following blood clot formation. The aim of this study is focused on understanding the roles of caspase proteins within the inflammasome reaction leading to pulp necrosis by evaluating expression profile levels through RNA sequencing technology. Human dental pulp tissue was extracted from healthy adult third molars extracted prophylactically and healthy adult premolars extracted for orthodontics collected with IRB exemption. Tissue samples purified for mRNA allows expression analysis evaluation. DESeq2 and Limma analysis promoted statistical analysis of expression profile to determine whether or not significant differences in transcription and likely subsequent translation between diseased and healthy tissue. The study identified 27 genes above six-fold higher expression under hypoxia conditions, these markers include the following: TUBA1B, ACTB, NFKB, CTSD, CTSB, MCL1, JUN, TUBA1C, and others.

IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes

Katelyn J. McCann, Stephen M. Christensen, Devon H. Colby, Peter J. McGuire, Ian A. Myles, Christa S. Zerbe, Clifton L. Dalgard, Gauthaman Sukumar, Warren J. Leonard, Beth A. McCormick, Steven M. Holland

Interferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.

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.

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

Aditya Bhalla, Jessie Arce, Bryan Ubanwa, Gursharan Singh, Rajesh K. Sani and Venkatesh Balan

A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost.

The arginine deaminase system plays distinct roles in Borrelia burgdorferi and Borrelia hermsii

Crystal L. Richards, Sandra J. Raffel, Sebastien Bontemps-Gallo,Daniel P. Dulebohn, Tessa C. Herbert, Frank C. Gherardini

Borrelia species are amino acid auxotrophs that utilize di- and tri- peptides obtained through their oligopeptide transport system to supply amino acids for replicative growth during their enzootic cycles. However, Borrelia species from both the Lyme disease (LD) and relapsing fever (RF) groups harbor an amino acid transport and catabolism system, the Arginine Dei- minase System (ADI), that could potentially augment intracellular L-arginine required for growth. RF spirochetes contain a “complete”, four gene ADI (arcA, B, D, and C) while LD spirochetes harbor arcA, B, and sometimes D but lack arcC (encoding carbamate kinase). In this study, we evaluated the role of the ADI system in bacterial survival and virulence and discovered important differences in RF and LD ADIs. Both in vitro and in a murine model of infection, B. hermsii cells significantly reduced extracellular L-arginine levels and that reduc- tion was dependent on arginine deiminase expression. Conversely, B. burgdorferi did not reduce the concentration of L-arginine during in vitro growth experiments nor during infec- tion of the mammalian host, suggesting a fundamental difference in the ability to directly uti- lize L-arginine compared to B. hermsii. Further experiments using a panel of mutants generated in both B. burgdorferi and B. hermsii, identified important differences in growth characteristics and ADI transcription and protein expression. We also found that the ADI system plays a key role in blood and spleen colonization in RF spirochetes. In this study we have identified divergent metabolic strategies in two closely related human pathogens, that ultimately impacts the host-pathogen interface during infection.

Metabolic engineering of Corynebacterium glutamicum for production of scyllo-inositol, a drug candidate against Alzheimer's disease

Paul Ramp, Alexander Lehnert, Susana Matamouros, Astrid Wirtz, Meike Baumgart, Michael Bott

Abstract

Scyllo-inositol has been identified as a potential drug for the treatment of Alzheimer's disease. Therefore, cost-efficient processes for the production of this compound are desirable. In this study, we analyzed and engineered Corynebacterium glutamicum with the aim to develop competitive scyllo-inositol producer strains. Initial studies revealed that C. glutamicum naturally produces scyllo-inositol when cultured with myo-inositol as carbon source. The conversion involves NAD+-dependent oxidation of myo-inositol to 2-keto-myo-inositol followed by NADPH-dependent reduction to scyllo-inositol. Use of myo-inositol for biomass formation was prevented by deletion of a cluster of 16 genes involved in myo-inositol catabolism (strain MB001(DE3)Δiol1). Deletion of a second cluster of four genes (oxiC-cg3390-oxiD-oxiE) related to inositol metabolism prevented conversion of 2-keto-myo-inositol to undesired products causing brown coloration (strain MB001(DE3)Δiol1Δiol2). The two chassis strains were used for plasmid-based overproduction of myo-inositol dehydrogenase (IolG) and scyllo-inositol dehydrogenase (IolW). In BHI medium containing glucose and myo-inositol, a complete conversion of the consumed myo-inositol into scyllo-inositol was achieved with the Δiol1Δiol2 strain. To enable scyllo-inositol production from cheap carbon sources, myo-inositol 1-phosphate synthase (Ino1) and myo-inositol 1-phosphatase (ImpA), which convert glucose 6-phosphate into myo-inositol, were overproduced in addition to IolG and IolW using plasmid pSI. Strain MB001(DE3)Δiol1Δiol2 (pSI) produced 1.8 g/L scyllo-inositol from 20 g/L glucose and even 4.4 g/L scyllo-inositol from 20 g/L sucrose within 72 h. Our results demonstrate that C. glutamicum is an attractive host for the biotechnological production of scyllo-inositol and potentially further myo-inositol-derived products.

IFNγ Mediated Monocyte Metabolic Reprogramming

KATELYN JOSEPHINE MCCANN

IFNγ is an essential and pleiotropic activator of monocytes, but little is known about the effects IFNγ on cellular metabolism. Therefore, we sought to characterize and elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. First, we identified a critical role for IFNγ in the induction of immunoresponsive gene 1 (IRG1) and its product, itaconate. The immunometabolite, itaconate, has been reported to have antibacterial, anti-inflammatory and antioxidant activity. Irg1-/- mice, lacking itaconate, are highly susceptible and phenotypically similar to IFNγ knock out (GKO) mice upon infection with Mycobacterium tuberculosis. Therefore, we assessed the role of IRG1/itaconate in the context of non-tuberculous mycobacterial (NTM) infection, the most common type of infection in patients with immunodeficiencies caused by defects in IFNγ signaling. Our data suggest that impaired induction of itaconate in the context of mycobacterial infection may contribute to mycobacterial susceptibility and immune dysregulation in patients with defects in IFNγ signaling. Next, we evaluated the metabolic phenotype of IFNγ-stimulated human monocytes and found that IFNγ increased oxygen consumption rates (OCR), indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling of human macrophages revealed that this oxidative phenotype was dependent on IFNγ-induced, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for complete induction of the respiratory burst in IFNγ stimulated human monocytes.

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

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism

Viviënne Mol, Martyn Bennett, Benjamín J. Sánchez, Beata K. Lisowska, Markus J. Herrgård, Alex Toftgaard Nielsen, David J. Leak, Nikolaus Sonnenschein

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.

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.