Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics

ABSTRACTUndecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C55lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. We selected here a vancomycin (VAN)-resistant derivative ofBacillus subtilisW168 that contains a single-point mutation in the ribosome-binding site of theuppSgene designateduppS1. Genetic reconstruction experiments demonstrate that theuppS1allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS rendersB. subtilisslightly more susceptible to many late-acting cell wall antibiotics, including β-lactams, but significantly more resistant to fosfomycin andd-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that theuppS1allele leads to slightly elevated expression of the σMregulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, theuppS1mutation increases resistance to VAN, fosfomycin, andd-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in asigMmutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics.

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C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Blood ◽

10.1182/blood.v110.11.1727.1727 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1727-1727

Author(s):

Vanja Karamatic Crew ◽

Carole Green ◽

Stephen Parsons ◽

Belinda K. Singleton ◽

Geoff Daniels ◽

...

Keyword(s):

Cell Line ◽

Dna Analysis ◽

Single Point ◽

Jurkat Cells ◽

Erythroid Cell ◽

Single Point Mutation ◽

Wild Type ◽

Membrane Glycoproteins ◽

Hemopoietic Cells ◽

Elevated Expression

Abstract Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS2002;99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

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A Defective Undecaprenyl Pyrophosphate Synthase Induces Growth and Morphological Defects That Are Suppressed by Mutations in the Isoprenoid Pathway ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00255-18 ◽

2018 ◽

Vol 200 (18) ◽

Cited By ~ 7

Author(s):

William J. MacCain ◽

Suresh Kannan ◽

Dannah Z. Jameel ◽

Jerry M. Troutman ◽

Kevin D. Young

Keyword(s):

Cell Wall ◽

Bacterial Survival ◽

Cell Wall Synthesis ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Peptidoglycan Biosynthesis ◽

Bacterial Morphology ◽

Morphological Defects ◽

Undecaprenyl Phosphate

ABSTRACTThe peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. InEscherichia coli, this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS).E. coliMG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genesuppS,ispH, andidi. Normal morphology was restored by overexpressinguppSor by replacing the mutant (uppS31) with the wild-type allele. Importantly, movinguppS31into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions ofispHandidi. Purified UppSW31Rwas enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure.IMPORTANCEBacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream ofuppScan reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape.

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Neisseria gonorrhoeaePBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

Infection and Immunity ◽

10.1128/iai.00833-18 ◽

2018 ◽

Vol 87 (2) ◽

Cited By ~ 3

Author(s):

Ryan E. Schaub ◽

Krizia M. Perez-Medina ◽

Kathleen T. Hackett ◽

Daniel L. Garcia ◽

Joseph P. Dillard

Keyword(s):

Molecular Weight ◽

Cell Wall ◽

Inflammatory Response ◽

Neisseria Gonorrhoeae ◽

Wild Type ◽

Cross Link ◽

Content Type ◽

Lytic Transglycosylases ◽

Penicillin Binding Proteins ◽

Proinflammatory Molecules

ABSTRACTNeisseria gonorrhoeaereleases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in eitherdacB, encoding PBP3, orpbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation indacBcaused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminald-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of bothdacBandpbpGeliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by thedacB pbpGmutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.

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An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum

Frontiers in Neural Circuits ◽

10.3389/fncir.2021.676891 ◽

2021 ◽

Vol 15 ◽

Author(s):

Esther Suk King Lai ◽

Hisako Nakayama ◽

Taisuke Miyazaki ◽

Takanobu Nakazawa ◽

Katsuhiko Tabuchi ◽

...

Keyword(s):

Cell Adhesion Molecule ◽

Synapse Formation ◽

Single Point ◽

Autism Spectrum ◽

Mutant Mice ◽

Single Point Mutation ◽

Synapse Elimination ◽

Wild Type ◽

Post Mortem Brain ◽

And Function

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

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FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽

10.1128/mbio.00908-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

Ana R. Pereira ◽

Jen Hsin ◽

Ewa Król ◽

Andreia C. Tavares ◽

Pierre Flores ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Shape ◽

Single Point ◽

Spherical Shape ◽

Single Point Mutation ◽

Wild Type ◽

Dead End ◽

Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

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A Mutation of RNA Polymerase β′ Subunit (RpoC) Converts Heterogeneously Vancomycin-Intermediate Staphylococcus aureus (hVISA) into “Slow VISA”

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00135-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4215-4225 ◽

Cited By ~ 13

Author(s):

Miki Matsuo ◽

Tomomi Hishinuma ◽

Yuki Katayama ◽

Keiichi Hiramatsu

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Rna Polymerase ◽

Stringent Response ◽

Whole Genome ◽

Multicopy Plasmid ◽

Β Subunit ◽

Whole Genome Analysis ◽

Content Type ◽

Peptidoglycan Biosynthesis

ABSTRACTVarious mutations in therpoBgene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediateStaphylococcus aureus(VISA) and heterogeneously VISA (hVISA) strains. We reported thatrpoBmutations are also linked to the expression of the recently found “slow VISA” (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024–5035, 2014,http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of therpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, inrpoC. Introduction of the wild-type (WT)rpoCgene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that therpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: therpoCgene itself as regulatory mutations, peptidoglycan biosynthesis genes, andrelQ, which is involved in the stringent response. It appears that therpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

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A single point mutation in the embB gene is responsible for resistance to ethambutol in Mycobacterium smegmatis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2629 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2629-2633 ◽

Cited By ~ 39

Author(s):

M A Lety ◽

S Nair ◽

P Berche ◽

V Escuyer

Keyword(s):

Cell Wall ◽

Mycobacterium Smegmatis ◽

Single Point ◽

Mycobacterium Leprae ◽

Single Point Mutation ◽

Missense Mutations ◽

Mycobacterial Species ◽

Mechanisms Of Resistance ◽

Embb Gene ◽

Mycobacterial Cell Wall

Ethambutol [EMB; dextro-2,2'-(ethylenediimino)-di-1-butanol] is an effective drug when used in combination with isoniazid for the treatment of tuberculosis. It inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall. Recent studies have shown that arabinosyltransferases could be targets of EMB. These enzymes are encoded by the emb locus that was identified in Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium avium, and Mycobacterium tuberculosis. We demonstrate that a missense mutation in the M. smegmatis embB gene, one of the genes of the emb locus, confers resistance to EMB. The level of resistance is not dependent on the number of copies of the mutated embB gene, indicating that this is a true mechanism of resistance. The mutation is located in a region of the EmbB protein that is highly conserved among the different mycobacterial species. We also identified in this region two other independent mutations that confer EMB resistance. Furthermore, mutations have recently been described in the same region of the EmbB protein from clinical EMB-resistant M. tuberculosis isolates. Together, these data strongly suggest that one of the mechanisms of resistance to EMB consists of missense mutations in a particular region of the EmbB protein that could be directly involved in the interaction with the EMB molecule.

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The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

Infection and Immunity ◽

10.1128/iai.00005-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 6

Author(s):

Elodie Cuenot ◽

Transito Garcia-Garcia ◽

Thibaut Douche ◽

Olivier Gorgette ◽

Pascal Courtin ◽

...

Keyword(s):

Cell Wall ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

Hamster Model ◽

Content Type ◽

Physiological Processes ◽

Signal Transduction Networks ◽

Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

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Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis

mBio ◽

10.1128/mbio.02401-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Ti-Yu Lin ◽

William S. Gross ◽

George K. Auer ◽

Douglas B. Weibel

Keyword(s):

Cell Wall ◽

Rhodobacter Sphaeroides ◽

Cell Morphology ◽

Cell Shape ◽

Molecular Mechanisms ◽

Wild Type ◽

Anionic Phospholipid ◽

Content Type ◽

Lipid Ii ◽

Topological Features

ABSTRACT Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG—a glycosyltransferase that performs the last step of lipid II biosynthesis—interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall. IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides. The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

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Definitive demonstration by synthesis of genome annotation completeness

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905990116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24206-24213 ◽

Cited By ~ 7

Author(s):

Paul R. Jaschke ◽

Gabrielle A. Dotson ◽

Kay S. Hung ◽

Diane Liu ◽

Drew Endy

Keyword(s):

Essential Gene ◽

Single Point ◽

Viable Cell ◽

Essential Genes ◽

Single Point Mutation ◽

Evolutionary Adaptation ◽

Wild Type ◽

Bioinformatic Tools ◽

Design And Synthesis ◽

New Genes

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available—23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

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