Integrative characterization of the near-minimal bacterium Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

Efficient computation of co-transcriptional RNA-ligand interaction dynamics.

Riboswitches form an abundant class of cis-regulatory RNA elements that mediate gene expression by binding a small metabolite. For synthetic biology applications, they are becoming cheap and accessible systems for selectively triggering transcription or translation of downstream genes. Many riboswitches are kinetically controlled, hence knowledge of their co-transcriptional mechanisms is essential. We present here an efficient implementation for analyzing co-transcriptional RNA-ligand interaction dynamics. This approach allows for the first time to model concentration-dependent metabolite binding/unbinding kinetics. We exemplify this novel approach by means of the recently studied I-A 2'-deoxyguanosine (2'dG)-sensing riboswitch from Mesoplasma florum.

Cloning and Transplantation of the Mesoplasma florum Genome.

Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by the use of genome donor and recipient cell belonging to different species. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.

Electrostatic confinement and manipulation of DNA molecules for genome analysis.

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Development of oriC-Based Plasmids for Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

Probabilistic inference of lateral gene transfer events.

BACKGROUND: Lateral gene transfer (LGT) is an evolutionary process that has an important role in biology. It challenges the traditional binary tree-like evolution of species and is attracting increasing attention of the molecular biologists due to its involvement in antibiotic resistance. A number of attempts have been made to model LGT in the presence of gene duplication and loss, but reliably placing LGT events in the species tree has remained a challenge. RESULTS: In this paper, we propose probabilistic methods that samples reconciliations of the gene tree with a dated species tree and computes maximum a posteriori probabilities. The MCMC-based method uses the probabilistic model DLTRS, that integrates LGT, gene duplication, gene loss, and sequence evolution under a relaxed molecular clock for substitution rates. We can estimate posterior distributions on gene trees and, in contrast to previous work, the actual placement of potential LGT, which can be used to, e.g., identify "highways" of LGT. CONCLUSIONS: Based on a simulation study, we conclude that the method is able to infer the true LGT events on gene tree and reconcile it to the correct edges on the species tree in most cases. Applied to two biological datasets, containing gene families from Cyanobacteria and Molicutes, we find potential LGTs highways that corroborate other studies as well as previously undetected examples.

Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments.

Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR(-)) mutation-accumulation (MA) lines of the gram-positive model organism Bacillus subtilis. We then analyzed>7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR(-) MA lines, finding a context-dependent mutation pattern that is asymmetric around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of positive and purifying selection.

Noncovalent spin labeling of riboswitch RNAs to obtain long-range structural NMR restraints.

Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2'dG sensing aptamer domain of Mesoplasma florum (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in (15)N-TROSY and (1)H,(1)H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined (1)H-Γ2 rates are in agreement with reported crystal structure data and show that distance restraints up to 25 Å can be obtained from NMR PRE data of RNA.

Molecular evolution of the substrate utilization strategies and putative virulence factors in mosquito-associated Spiroplasma species.

Comparative genomics provides a powerful tool to characterize the genetic differences among species that may be linked to their phenotypic variations. In the case of mosquito-associated Spiroplasma species, such approach is useful for the investigation of their differentiations in substrate utilization strategies and putative virulence factors. Among the four species that have been assessed for pathogenicity by artificial infection experiments, Spiroplasma culicicola and S. taiwanense were found to be pathogenic, whereas S. diminutum and S. sabaudiense were not. Intriguingly, based on the species phylogeny, the association with mosquito hosts and the gain or loss of pathogenicity in these species appears to have evolved independently. Through comparison of their complete genome sequences, we identified the genes and pathways that are shared by all or specific to one of these four species. Notably, we found that a glycerol-3-phosphate oxidase gene (glpO) is present in S. culicicola and S. taiwanense but not in S. diminutum or S. sabaudiense. Because this gene is involved in the production of reactive oxygen species and has been demonstrated as a major virulence factor in Mycoplasma, this distribution pattern suggests that it may be linked to the observed differences in pathogenicity among these species as well. Moreover, through comparative analysis with other Spiroplasma, Mycoplasma, and Mesoplasma species, we found that the absence of glpO in S. diminutum and S. sabaudiense is best explained by independent losses. Finally, our phylogenetic analyses revealed possible recombination of glpO between distantly related lineages and local rearrangements of adjacent genes.

Mutation rates: how low can you go?

Two recent reports combine mutation accumulation and whole-genome sequencing to measure mutation rates in microbes with unusual genome sizes and life cycles. The results are broadly consistent with the hypothesis that genetic drift plays a role in shaping genomic mutation rates across a wide range of taxa.

Drift-barrier hypothesis and mutation-rate evolution.

Mutation dictates the tempo and mode of evolution, and like all traits, the mutation rate is subject to evolutionary modification. Here, we report refined estimates of the mutation rate for a prokaryote with an exceptionally small genome and for a unicellular eukaryote with a large genome. Combined with prior results, these estimates provide the basis for a potentially unifying explanation for the wide range in mutation rates that exists among organisms. Natural selection appears to reduce the mutation rate of a species to a level that scales negatively with both the effective population size (N(e)), which imposes a drift barrier to the evolution of molecular refinements, and the genomic content of coding DNA, which is proportional to the target size for deleterious mutations. As a consequence of an expansion in genome size, some microbial eukaryotes with large N(e) appear to have evolved mutation rates that are lower than those known to occur in prokaryotes, but multicellular eukaryotes have experienced elevations in the genome-wide deleterious mutation rate because of substantial reductions in N(e).

Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches.

Riboswitches are elements in the 5'-untranslated region of mRNAs that regulate gene expression by directly interacting with metabolites related to their own gene products. A remarkable feature of this gene regulation mechanism is the high specificity of riboswitches for their cognate ligands. In this study, we used a combination of static and time-resolved NMR-spectroscopic methods to investigate the mechanisms for ligand specificity in purine riboswitches. We investigate the xpt-aptamer domain from a guanine-responsive riboswitch and the mfl-aptamer domain from a 2'-deoxyguanosine-responsive riboswitch. The xpt-aptamer binds the purine nucleobases guanine/hypoxanthine with high affinity, but, unexpectedly, also the nucleoside 2'-deoxyguanosine. On the other hand, the mfl-aptamer is highly specific for its cognate ligand 2'-deoxyguanosine, and does not bind purine ligands. We addressed the question of aptamer`s ligand specificity by real-time NMR spectroscopy. Our studies of ligand binding and subsequently induced aptamer folding revealed that the xpt-aptamer discriminates against non-cognate ligands by enhanced life-times of the cognate complex compared with non-cognate complexes, whereas the mfl-aptamer rejects non-cognate ligands at the level of ligand association, employing a kinetic proofreading mechanism.

Structural principles of nucleoside selectivity in a 2'-deoxyguanosine riboswitch.

Purine riboswitches have an essential role in genetic regulation of bacterial metabolism. This family includes the 2'-deoxyguanosine (dG) riboswitch, which is involved in feedback control of deoxyguanosine biosynthesis. To understand the principles that define dG selectivity, we determined crystal structures of the natural Mesoplasma florum riboswitch bound to cognate dG as well as to noncognate guanosine, deoxyguanosine monophosphate and guanosine monophosphate. Comparison with related purine riboswitch structures reveals that the dG riboswitch achieves its specificity through modification of key interactions involving the nucleobase and rearrangement of the ligand-binding pocket to accommodate the additional sugar moiety. In addition, we observe new conformational changes beyond the junctional binding pocket extending as far as peripheral loop-loop interactions. It appears that re-engineering riboswitch scaffolds will require consideration of selectivity features dispersed throughout the riboswitch tertiary fold, and structure-guided drug design efforts targeted to junctional RNA scaffolds need to be addressed within such an expanded framework.

Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy.

The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2'-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2'-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2'-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg(2+) is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.

Guanine riboswitch variants from Mesoplasma florum selectively recognize 2'-deoxyguanosine.

Several mRNA aptamers have been identified in Mesoplasma florum that have sequence and structural features resembling those of guanine and adenine riboswitches. Two features distinguish these RNAs from established purine-sensing riboswitches. All possess shortened hairpin-loop sequences expected to alter tertiary contacts known to be critical for aptamer folding. The RNAs also carry nucleotide changes in the core of each aptamer that otherwise is strictly conserved in guanine and adenine riboswitches. Some aptamers retain the ability to selectively bind guanine or adenine despite these mutations. However, one variant type exhibits selective and high-affinity binding of 2'-deoxyguanosine, which is consistent with its occurrence in the 5' untranslated region of an operon containing ribonucleotide reductase genes. The identification of riboswitch variants that bind nucleosides and reject nucleobases reveals that natural metabolite-sensing RNA motifs can accrue mutations that expand the diversity of ligand detection in bacteria.

Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data.

Genomic DNA sequence data for the 16S rRNA gene and the gyrB gene of Mesoplasma pleciae PS-1(T) (=ATCC 49582(T)=NBRC 100476(T)) demonstrate a much closer relationship to Acholeplasma laidlawii and Acholeplasma oculi than to other species in the order Entomoplasmatales. In addition, the preferred use of UGG rather than UGA as the codon for tryptophan in the gyrB sequence probably places the organism outside the order Entomoplasmatales. It is proposed that M. pleciae be reclassified in the genus Acholeplasma, as Acholeplasma pleciae comb. nov.