Microbial Methylotrophic Metabolism: Recent Metabolic Modeling Efforts and Their Applications In Industrial Biotechnology

Developing methylotrophic bacteria into cell factories that meet the chemical demand of the future could be both economical and environmentally friendly. Methane is not only an abundant, low‐cost resource but also a potent greenhouse gas, the capture of which could help to reduce greenhouse gas emissions. Rational strain design workflows rely on the availability of carefully combined knowledge often in the form of genome‐scale metabolic models to construct high‐producer organisms. In this review, the authors present the most recent genome‐scale metabolic models in aerobic methylotrophy and their applications. Further, the authors present models for the study of anaerobic methanotrophy through reverse methanogenesis and suggest organisms that may be of interest for expanding one‐carbon industrial biotechnology. Metabolic models of methylotrophs are scarce, yet they are important first steps toward rational strain‐design in these organisms.     

It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

Ubiquitination, the covalent binding of the small protein modifier ubiquitin to a target protein, is an important and frequently studied posttranslational protein modification. Multiple reports provide useful insights into the plant ubiquitinome, but mostly at the protein level without comprehensive site identification. Here, we implemented ubiquitin combined fractional diagonal chromatography (COFRADIC) for proteome-wide ubiquitination site mapping on Arabidopsis thaliana cell cultures. We identified 3009 sites on 1607 proteins, thereby greatly increasing the number of known ubiquitination sites in this model plant. Finally, The Ubiquitination Site tool (http://bioinformatics.psb.ugent.be/webtools/ubiquitin_viewer/) gives access to the obtained ubiquitination sites, not only to consult the ubiquitination status of a given protein, but also to conduct intricate experiments aiming to study the roles of specific ubiquitination events. Together with the antibodies recognizing the ubiquitin remnant motif, ubiquitin COFRADIC represents a powerful tool to resolve the ubiquitination maps of numerous cellular processes in plants.     

Dynamic investigation and modeling of the nitrogen cometabolism in Methylococcus capsulatus ( Bath)

The methanotrophic bacterium Methylococcus capsulatus is capable of assimilating methane and oxygen into protein-rich biomass, however, the diverse metabolism of the microorganism also allows for several undesired cometabolic side-reactions to occur. In this study, the ammonia cometabolism in Methylococcus capsulatus is investigated using pulse experiments. Surprisingly Methylococcus capsulatus oxidizes ammonia to nitrate through a yet unknown mechanism and fixes molecular nitrogen even at a high dissolved oxygen tension. The observed phenomena can be modeled using 14 ordinary differential equations and 18 kinetic parameters, of which 6 were revealed by Morris screening to be identifiable from the experimental data. Monte Carlo simulations showed that the model was robust and accurate even with uncertainty in the parameter values as confirmed by statistical error analysis.     

Detection of Pseudomonas aeruginosa in sputum headspace through volatile organic compound analysis

Introduction

Chronic pulmonary infection is the hallmark of Cystic Fibrosis lung disease. Searching for faster and easier screening may lead to faster diagnosis and treatment of Pseudomonas aeruginosa (P. aeruginosa). Our aim was to analyze and build a model to predict the presence of P. aeruginosa in sputa.

Methods

Sputa from 28 bronchiectatic patients were used for bacterial culturing and analysis of volatile compounds by gas chromatography–mass spectrometry. Data analysis and model building were done by Partial Least Squares Regression Discriminant analysis (PLS-DA). Two analysis were performed: one comparing P. aeruginosa positive with negative cultures at study visit (PA model) and one comparing chronic colonization according to the Leeds criteria with P. aeruginosa negative patients (PACC model).

Results

The PA model prediction of P. aeruginosa presence was rather poor, with a high number of false positives and false negatives. On the other hand, the PACC model was stable and explained chronic P. aeruginosa presence for 95% with 4 PLS-DA factors, with a sensitivity of 100%, a positive predictive value of 86% and a negative predictive value of 100%.

Conclusion

Our study shows the potential for building a prediction model for the presence of chronic P. aeruginosa based on volatiles from sputum.     

Cyclin‐dependent kinase activity maintains the shoot apical meristem cells in an undifferentiated state

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter‐dependence of cell‐cycle machinery and meristem‐organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell‐cycle machinery in the shoot apex by expression of a dominant negative allele of the A‐type cyclin‐dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo‐reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell‐cycle machinery on meristem organization is determined by the level of CDK activity.     

Correlation between Genotypic Diversity,Lipooligosaccharide Gene Locus Class Variation,and Caco-2 Cell Invasion Potential of Campylobacter jejuni Isolates from Chicken Meat and Humans: Contribution to Virulotyping

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jejuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C. jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C. jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host.Campylobacter jejuni is an important human enteric pathogen worldwide (3, 7, 26). Infected humans exhibit a range of clinical spectra, from mild, watery diarrhea to severe inflammatory diarrhea (28). Factors influencing the virulence of C. jejuni include motility, chemotaxis, the ability to adhere to and invade intestinal cells, intracellular survival, and toxin production (28, 30, 52). Besides its role in human enteric illnesses, C. jejuni is a predominant infectious trigger of acute postinfectious neuropathies, such as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) (1). Significant interest in studying the structure and biosynthesis of the core lipooligosaccharide (LOS) of C. jejuni has resulted from its potential role in these paralytic disorders. Many studies have now provided convincing evidence that molecular mimicry between C. jejuni LOS and gangliosides in human peripheral nerve tissue plays an important causal role in the pathogenesis of GBS/MFS (16, 17, 19, 21).Initial comparative studies of C. jejuni LOS structure and the corresponding DNA sequences of the LOS biosynthesis loci identified eight different LOS locus classes. Three of these classes, A, B, and C, harbor sialyltransferase genes involved in incorporating sialic acid into the LOS (42). Sialylation of the LOS core was found to be associated with ganglioside mimicry and also to affect immunogenicity and serum resistance (21). Recently, Parker et al. (43) identified 11 additional LOS classes on the basis of the sequence at the LOS biosynthesis locus. Their investigation also suggested that the LOS loci of C. jejuni strains are hot spots for genetic exchange, which can lead to mosaicism.Despite evidence on locus variation within C. jejuni LOS classes, PCR-based screening of a collection of 123 clinical and environmental strains showed that almost 60% of C. jejuni strains belong to class A, B, or C (42). Additionally, Godschalk et al. (16) found that 53% (9/17) of GBS-associated C. jejuni strains possessed LOS of class A, while 64% (35/55) of the non-GBS-associated isolates possessed LOS of class A, B, or C, and 62% (13/21) of enteritis-associated Campylobacter strains expressed LOS of class A, B, or C, as well. This relative representation of sialylated LOS classes A, B, and C was hypothesized to be advantageous for C. jejuni in the colonization and infection of various hosts (42, 49). Recently, Louwen et al. (34) demonstrated that C. jejuni strains possessing sialylated LOS (class A, B, or C) invade Caco-2 cells significantly better than nonsialylated strains (with class D or E). Knockout mutagenesis of the LOS sialyltransferase Cst-II in three C. jejuni strains revealed a significant reduction in the invasion potentials of the mutant strains (34). The possible role of LOS in adhesion and invasion was previously highlighted in the work of Perera et al. (44) and Kanipes et al. (29), where a C. jejuni waaF mutant strain showed significant reductions in levels of adherence to and invasion of INT-407 cells.LOS class diversity in C. jejuni strains isolated from chicken meat, an important source of human campylobacteriosis (6, 7, 26), has hardly been studied at all. In addition, the role of LOS class variation in the invasion potential of C. jejuni strains from chicken meat still needs to be explored. The epidemiological relevance of C. jejuni LOS gene screening can be further elaborated by correlating its results with results from other molecular-typing tools (e.g., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]). In the present study, we screened a diverse collection of C. jejuni isolates, from consumer-packaged chicken meats and from sporadic human cases of diarrhea, by PCR for five LOS classes (A, B, C, D, and E). Then we correlated the LOS classes assigned by PCR screening with the genotypes assigned by PFGE and MLST. Finally, we tested the invasion potentials of a representative subset of C. jejuni isolates in relation to their LOS classes and genotypic diversity.     

Synthesis and characterization of topologically linked single-stranded DNA rings

We investigated the synthesis of linked-ring DNAs by two DNA-ligation-based methods. In the first method, two DNA oligonucleotides were associated through a duplex segment of more than a full helical turn. Circularization of the entwined oligonucleotides by T4 DNA ligase resulted in two linked-ring DNAs with a total yield of approximately 40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of approximately 10% linked-ring product. The circular nature of linked-ring DNAs was verified with exonuclease digestion and the existence of topological linkages was demonstrated by analyzing the electrophoretic mobility pattern of DNA products obtained from the digestion of each linked-ring DNA using specific restriction endonucleases. A linked-ring DNA library in which one of the two rings contained random-sequence nucleotides was also constructed and tested for compatibility with in vitro selection.     

Impact of agricultural practices on the Zea mays L. endophytic community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.