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.

Novel insights into the transcriptional regulation of cell division in Corynebacterium glutamicum

Kim Julia Kraxner

In the first part of this doctoral thesis the transcriptional regulation of the odhI gene (cg1630) of Corynebacterium glutamicum was analyzed. OdhI in its unphoshorylated state functions as inhibitor of the 2-oxoglutarate dehydrogenase complex (ODHC) by binding to the OdhA subunit. Phosphorylation of OdhI by serine/threonine protein kinases abolishes this effect. Inhibition of ODHC activity by OdhI was shown to be crucial for overproduction and secretion of L-glutamate, which is used as a flavour enhancer. Since downstream of odhI two genes presumably encoding transcriptional regulators (cg1631 and cg1633) are located, it was speculated that these could be involved in transcriptional regulation of odhI. However, transcriptome analysis of deletion mutants lacking cg1631 or cg1633 and DNA affinity chromatography with the odhI promoter did not support this hypothesis. Furthermore, no other potential transcriptional regulators of odhI could be identified. Thus, there is currently no evidence for transcriptional regulation of odhI. The second part of this thesis addresses the regulation of cytokinesis in C. glutamicum. In contrast to e.g. Escherichia coli and Bacillus subtilis, knowledge about regulators of cytokinesis in Actinobacteria is very limited. In this study, the so far uncharacterized Cg1631 protein was discovered to be a transcriptional regulator of the ftsZ gene in C. glutamicum encoding the key player of bacterial cell division. Therefore, Cg1631 was named FtsR, standing for FtsZ regulator. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology, emphasizing an important function of FtsR in cell division or cell wall synthesis. The wild-type phenotype could be restored by plasmid-based complementation. Chromatin affinity purification with subsequent next generation sequencing (ChAP-Seq) identified a region in the ftsZ promoter as a major FtsR binding site, but revealed also additional potential target genes. With the ChAP-Seq results a putative DNA-binding motif could be identified for FtsR. Transcriptional activation of ftsZ expression by FtsR was underlined by DNA microarray experiments, electrophoretic mobility shift assays (EMSAs), and reporter gene studies. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ftsR mutant is not solely caused by reduced ftsZ expression but involves additional factors. In summary, FtsR was identified as the first transcriptional regulator of ftsZ in C. glutamicum. Furthermore, since FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, it can be assumed that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria. This makes FtsR a promising target for the development of new antimicrobial drugs against pathogenic relatives of C. glutamicum

DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans

Daniel R. McHugh, Elena Koumis, Paul Jacob, Jennifer Goldfarb, Michelle Schlaubitz-Garcia, Safae Bennani, Paul Regan, Prem Patel, and Matthew J. Youngman

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie agedependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegans DAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16- mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

The conserved actinobacterial transcriptional regulator FtsR controls expression of ftsZ and further target genes and influences growth and cell division in Corynebacterium glutamicum

Kim Julia Kraxner, Tino Polen, Meike Baumgart, and Michael Bott

Key mechanisms of cell division and its regulation are well understood in model bacteria such as Escherichia coli and Bacillus subtilis. In contrast, current knowledge on the regulation of cell division in Actinobacteria is rather limited. FtsZ is one of the key players in this process, but nothing is known about its transcriptional regulation in Corynebacterium glutamicum, a model organism of the Corynebacteriales.

In this study, we used DNA affinity chromatography to search for transcriptional regulators of ftsZ in C. glutamicum and identified the Cg1631 protein as candidate, which was named FtsR. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology. Plasmid-based expression of native ftsR or of homologs of the pathogenic relatives Corynebacterium diphtheriae and Mycobacterium tuberculosis in the ΔftsR mutant could at least partially reverse the mutant phenotype. Absence of ftsR caused decreased expression of ftsZ, in line with an activator function of FtsR. In vivo crosslinking followed by affinity purification of FtsR and next generation sequencing of the enriched DNA fragments confirmed the ftsZ promoter as in vivo binding site of FtsR and revealed additional potential target genes and a DNA-binding motif. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ΔftsR mutant is not solely caused by reduced ftsZ expression, but involves further targets.

In this study, we identified and characterized FtsR as the first transcriptional regulator of FtsZ described for C. glutamicum. Both the absence and the overproduction of FtsR had severe effects on growth and cell morphology, underlining the importance of this regulatory protein. FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, which suggests that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria.

The complete genomic sequence of Streptomyces spectabilis NRRL-2792 and identification of secondary metabolite biosynthetic gene clusters

Arkadeep Sinha, Silvia Phillips-Salemka, Tanu-Adhikari Niraula, Kevin A Short, Narayan P Niraula

This is the first report of a fully annotated genomic sequence of Streptomyces spectabilis NRRL-2792, isolated and identified by The Upjohn Company in 1961. The genome was assembled into a single scaffold for annotation and analysis. The chromosome is linear, 9.5 Mb in size which is one of the largest Streptomyces genomes yet described, has a G+C content of 72%, and encodes for approximately 7943 genes. Antibiotic Secondary Metabolite Analysis Shell (antiSMASH) and Basic Local Alignment Search Tool (BLAST) bioinformatics analyses identified six complete secondary metabolite biosynthetic gene clusters for ectoine, melanin, albaflavenone, spectinomycin, 2-methylisoborneol and coelichelin. Additionally, biosynthetic clusters were identified that shared ≥ 90% gene content with complestatin, hopene, neoaureothin, or undecylprodigiosin. Thirty-one other likely secondary metabolite gene clusters were identified by antiSMASH. BLAST identified two subsets of undecylprodigiosin biosynthetic genes at polar opposites of the chromosome; their duplication was subsequently confirmed by primer walking.

Fermentation Optimization by Understanding the Yeast Gene Transcriptomics in Cellulosic Ethanol Production in Bench-top Bioreactors

Joseph Bridges, Ananda Nanjundaswamy, Lydia Batey, Keerthi Mandyam, Vinayak Kapatral, Benjamin Vaisvil, and Daniel Schmitt

Poster presentation at SIMB Annual Meeting 2019.

SIMB 2019

Poster presentation

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.

Global Profiling of Lysine Acetylation in Borrelia burgdorferi B31 Reveals Its Role in Central Metabolism

Sébastien Bontemps-Gallo,1 Charlotte Gaviard,2,3 Crystal L. Richards,1 Takfarinas Kentache,2,3 Sandra J. Raffel,1 Kevin A. Lawrence,1 Joseph C. Schindler,4 Joseph Lovelace,4 Daniel P. Dulebohn,1 Robert G. Cluss,4 Julie Hardouin,2,3 and Frank C. Gherardini1

1 Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States

2 CNRS UMR 6270 Polymères, Biopolymères, Surfaces Laboratory, Université de Rouen, Mont-Saint-Aignan, France

3 PISSARO Proteomic Facility, Institut de Recherche et d’Innovation Biomédicale, Mont-Saint-Aignan, France

4 Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT, United States

This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

The post-translational modification of proteins has been shown to be extremely important in prokaryotes. Using a highly sensitive mass spectrometry-based proteomics approach, we have characterized the acetylome of B. burgdorferi. As previously reported for other bacteria, a relatively low number (5%) of the potential genome-encoded proteins of B. burgdorferi were acetylated. Of these, the vast majority were involved in central metabolism and cellular information processing (transcription, translation, etc.). Interestingly, these critical cell functions were targeted during both ML (mid-log) and S (stationary) phases of growth. However, acetylation of target proteins in ML phase was limited to single lysine residues while these same proteins were acetylated at multiple sites during S phase. To determine the acetyl donor in B. burgdorferi, we used mutants that targeted the sole acetate metabolic/anabolic pathway in B. burgdorferi (lipid I synthesis). B. burgdorferi strains B31-A3, B31-A3 ΔackA (acetyl-P- and acetyl-CoA-) and B31-A3 Δpta (acetyl-P+ and acetyl-CoA-) were grown to S phase and the acetylation profiles were analyzed. While only two proteins were acetylated in the ΔackA mutant, 140 proteins were acetylated in the Δpta mutant suggesting that acetyl-P was the primary acetyl donor in B. burgdorferi. Using specific enzymatic assays, we were able to demonstrate that hyperacetylation of proteins in S phase appeared to play a role in decreasing the enzymatic activity of at least two glycolytic proteins. Currently, we hypothesize that acetylation is used to modulate enzyme activities during different stages of growth. This strategy would allow the bacteria to post-translationally stimulate the activity of key glycolytic enzymes by deacetylation rather than expending excessive energy synthesizing new proteins. This would be an appealing, low-energy strategy for a bacterium with limited metabolic capabilities. Future work focuses on identifying potential protein deacetylase(s) to complete our understanding of this important biological process.

Genome Sequence and Comparative Pathogenic Determinants of Multidrug Resistant Uropathogenic Escherichia coli O25b:H4, A Clinical Isolate from Saudi Arabia

Essam J. Alyamani, Anamil M. Khiyami, Rayan Y. Booq, Fayez S. Bahwerth, Benjamin Vaisvil, Daniel P. Schmitt and Vinayak Kapatral

Escherichia coli serotype O25b:H4 is involved in human urinary tract infections. In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient suffering from recurring UTI infections in an intensive care unit at Hera General Hospital in Makkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis and drug resistance of this isolate compared to other E. coli strains. We sequenced and analyzed the E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Using the ERGO genome analysis platform, we performed annotations and identified virulence and antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genome was assembled into four contigs representing a total chromosome size of 5.28 Mb, and three contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing the bla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to other uropathogenic E. coli genomes, we identified unique drug resistance and pathogenicity factors. In this work, whole-genome sequencing and targeted comparative analysis of a clinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strain encodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that are expressed constitutively in E. coli ST131. We identified the genes responsible for pathogenesis and drug resistance and performed comparative analyses of the virulence and antibiotic resistance determinants with those of other E. coli UPEC isolates. This is the first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.

Published Nov 5. 2016. DOI: 10.22207/JPAM.10.4.01

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

Hannah K. Wilder, Sandra J. Raffel, Alan G. Barbour, Stephen F. Porcella, Daniel E. Sturdevant, Benjamin Vaisvil, Vinayak Kapatral, Daniel P. Schmitt, Tom G. Schwan, Job E. Lopez

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Published: February 4, 2016 DOI: 10.1371/journal.pone.0147707

The genome of Shigella dysenteriae strain Sd1617 comparison to representative strains in evaluating pathogenesis

Ajchara A. Vongsawan , Vinayak Kapatral , Benjamin Vaisvil , Henry Burd , Oralak Serichantalergs , Malabi M. Venkatesan , Carl J. Mason

We sequenced and analyzed Shigella dysenteriae strain Sd1617 serotype 1 that is widely used as model strain for vaccine design, trials and research. A combination of next-generation sequencing platforms and assembly yielded two contigs representing a chromosome size of 4.34 Mb and the large virulence plasmid of 177 kb. This genome sequence is compared with other Shigella genomes in order to understand gene complexity and pathogenic factors.

FEMS Microbiology Letters, 362, 2015, fnv011 doi: 10.1093/femsle/fnv011

Genomic Avenue to Avian Colisepticemia

Sagi Huja, Yaara Oren, Eva Trost, Elzbieta Brzuszkiewicz, Dvora Biran,a Jochen Blom, Alexander Goesmann, Gerhard Gottschalk, Jörg Hacker, Eliora Z. Ron, Ulrich Dobrindt

ABSTRACT Here we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenic Escherichia coli (APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. coli type 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of the Yersinia high-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum. IMPORTANCE Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.

The consequence of an additional NADH dehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504

D. Kostner, B. Luchterhand, A. Junker, S. Volland, R. Daniel, J. Büchs, W. Liebl & A. Ehrenreich

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

Complete Genome Sequence of Flavobacterium psychrophilum Strain CSF259-93, Used To Select Rainbow Trout for Increased Genetic Resistance against Bacterial Cold Water Disease.

Wiens GD, LaPatra SE, Welch TJ, Rexroad C 3rd, Call DR, Cain KD, LaFrentz BR, Vaisvil B, Schmitt DP, Kapatral V.

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease.

Genome Announc. 2014 Sep 18;2(5). pii: e00889-14. doi: 10.1128/genomeA.00889-14.

Draft Genome Sequence of a New Homofermentative, Lactic AcidProducing Enterococcus faecalis Isolate, CBRD01

Lew P. Christopher, Vinayak Kapatral, Benjamin Vaisvil, Ginger Emel,b and Linda C. DeVeauxc

We report here the draft genome sequence of the novel homofermentative Enterococcus faecalis isolate CBRD01, which is capable of high lactic acid productivity and yields, with minimal nutritional requirements. The genome is 2.8 Mbp, with 37% G+C, and contains genes for two lactate dehydrogenase (LDH) enzymes found in related organisms.

Genome Announc. 2014 Mar-Apr; 2(2): e00147-14.
Published online 2014 Mar 27. doi:  10.1128/genomeA.00147-14

Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

Yong-Yeol Ahn, Deok-Sun Lee, Henry Burd, William Blank, Vinayak Kapatral 

The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.

Published: January 15, 2014DOI: 10.1371/journal.pone.0085195

Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum

Dirk Oehler, Anja Poehlein, Andreas Leimbach, Nicolai Müller, Rolf Daniel, Gerhard Gottschalk & Bernhard Schink

Background

Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life.

Results

The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome.

Conclusions

The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.

Comparative Genomics and Transcriptomics of Propionibacterium acnes

Elzbieta Brzuszkiewicz, January Weiner, Antje Wollherr, Andrea Thürmer,Jennifer Hüpeden, Hans B. Lomholt,Mogens Kilian, Gerhard Gottschalk, Rolf Daniel, Hans-Joachim Mollenkopf, Thomas F. Meyer, Holger Brüggemann

The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with maintenance of skin homeostasis. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits differ between phylotypes, as judged from PCR analysis of a collection of P. acnes strains. Comparative transcriptome analysis of strains KPA171202 (type I-2) and 266 during exponential growth revealed inter-strain differences in gene expression of transport systems and metabolic pathways. In addition, transcript levels of genes encoding possible virulence factors such as dermatan-sulphate adhesin, polyunsaturated fatty acid isomerase, iron acquisition protein HtaA and lipase GehA were upregulated in strain 266. We investigated differential gene expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by upregulation of genes involved in stress responses and amino acid metabolism. Our data highlight the genomic basis for strain diversity and identify, for the first time, the actively transcribed part of the genome, underlining the important role growth status plays in the inflammation-inducing activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by the phylotype-specific genome content but also by variable gene expression.