Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes enzootic pneumonia in pigs but it is still largely unknown which host-pathogen interactions enable persistent infection and cause disease. In this study, we analyzed the host and bacterial transcriptomes during infection using RNA sequencing. Comparison of the transcriptome of lung lesion tissue from infected pigs with lung tissue from non-infected animals, identified 424 differentially expressed genes (FDR < 0.01 and fold change > 1.5LOG2). These genes were part of the following major pathways of the immune system: interleukin signaling (type 4, 10, 13, and 18), regulation of Toll-like receptors by endogenous ligand and activation of C3 and C5 in the complement system. Besides analyzing the lung transcriptome, a sampling protocol was developed to obtain enough bacterial mRNA from infected lung tissue for RNA sequencing. This was done by flushing infected lobes in the lung, and subsequently enriching for bacterial RNA. On average, 2.2 million bacterial reads were obtained per biological replicate to analyze the bacterial in vivo transcriptome. We compared the in vivo bacterial transcriptome with the transcriptome of bacteria grown in vitro and identified 22 up-regulated and 30 down-regulated genes (FDR < 0.01 and fold change > 2LOG2). Six out of seven genes in the operon encoding the mycoplasma specific F1-like ATPase (MHP_RS02445-MHP_RS02475) and all genes in the operon MHP_RS01965-MHP_RS01990 with functions related to nucleotide metabolism, spermidine transport and glycerol-3-phoshate transport were up-regulated in vivo. Down-regulated in vivo were genes related to glycerol uptake, cilium adhesion (P102), cell division and myo-inositol metabolism. In addition to providing a novel method to isolate bacterial mRNA from infected lung, this study provided insights into changes in gene expression during infection, which could help development of novel treatment strategies against enzootic pneumonia caused by M. hyopneumoniae.

Risk-Based Bioengineering Strategies for Reliable Bacterial Vaccine Production.

Design of a reliable process for bacterial antigen production requires understanding of and control over critical process parameters. Current methods for process design use extensive screening experiments for determining ranges of critical process parameters yet fail to give clear insights into how they influence antigen potency. To address this gap, we propose to apply constraint-based, genome-scale metabolic models to reduce the need of experimental screening for strain selection and to optimize strains based on model driven iterative Design-Build-Test-Learn (DBTL) cycles. Application of these systematic methods has not only increased the understanding of how metabolic network properties influence antigen potency, but also allows identification of novel critical process parameters that need to be controlled to achieve high process reliability.

Metabolic modeling of energy balances in Mycoplasma hyopneumoniae shows that pyruvate addition increases growth rate.

Mycoplasma hyopneumoniae is cultured on large-scale to produce antigen for inactivated whole-cell vaccines against respiratory disease in pigs. However, the fastidious nutrient requirements of this minimal bacterium and the low growth rate make it challenging to reach sufficient biomass yield for antigen production. In this study, we sequenced the genome of M. hyopneumoniae strain 11 and constructed a high quality constraint-based genome-scale metabolic model of 284 chemical reactions and 298 metabolites. We validated the model with time-series data of duplicate fermentation cultures to aim for an integrated model describing the dynamic profiles measured in fermentations. The model predicted that 84% of cellular energy in a standard M. hyopneumoniae cultivation was used for non-growth associated maintenance and only 16% of cellular energy was used for growth and growth associated maintenance. Following a cycle of model-driven experimentation in dedicated fermentation experiments, we were able to increase the fraction of cellular energy used for growth through pyruvate addition to the medium. This increase in turn led to an increase in growth rate and a 2.3 times increase in the total biomass concentration reached after 3-4 days of fermentation, enhancing the productivity of the overall process. The model presented provides a solid basis to understand and further improve M. hyopneumoniae fermentation processes. Biotechnol. Bioeng. 2017;114: 2339-2347. © 2017 Wiley Periodicals, Inc.

Persistence of Functional Protein Domains in Mycoplasma Species and their Role in Host Specificity and Synthetic Minimal Life.

Mycoplasmas are the smallest self-replicating organisms and obligate parasites of a specific vertebrate host. An in-depth analysis of the functional capabilities of mycoplasma species is fundamental to understand how some of simplest forms of life on Earth succeeded in subverting complex hosts with highly sophisticated immune systems. In this study we present a genome-scale comparison, focused on identification of functional protein domains, of 80 publically available mycoplasma genomes which were consistently re-annotated using a standardized annotation pipeline embedded in a semantic framework to keep track of the data provenance. We examined the pan- and core-domainome and studied predicted functional capability in relation to host specificity and phylogenetic distance. We show that the pan- and core-domainome of mycoplasma species is closed. A comparison with the proteome of the "minimal" synthetic bacterium JCVI-Syn3.0 allowed us to classify domains and proteins essential for minimal life. Many of those essential protein domains, essential Domains of Unknown Function (DUFs) and essential hypothetical proteins are not persistent across mycoplasma genomes suggesting that mycoplasma species support alternative domain configurations that bypass their essentiality. Based on the protein domain composition, we could separate mycoplasma species infecting blood and tissue. For selected genomes of tissue infecting mycoplasmas, we could also predict whether the host is ruminant, pig or human. Functionally closely related mycoplasma species, which have a highly similar protein domain repertoire, but different hosts could not be separated. This study provides a concise overview of the functional capabilities of mycoplasma species, which can be used as a basis to further understand host-pathogen interaction or to design synthetic minimal life.

Bacterial antisense RNAs are mainly the product of transcriptional noise.

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.