Characterisation of an Escherichia coli line that completely lacks 1 ribonucleotide reduction yields insights into the evolution of obligate 2 intracellularity. 3

Abstract


Abstract
All life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides.
A handful of obligate intracellular species are known to lack ribonucleotide reduction and are instead dependent on their host for deoxyribonucleotide synthesis.As ribonucleotide reduction has on occasion been lost in obligate intracellular parasites and endosymbionts, we reasoned that it should in principle be possible to knock this process out entirely under conditions where deoxyribonucleotides are present in the growth media.We report here the creation of a strain of E. coli where all three ribonucleotide reductase operons have been fully deleted.Our strain is able to grow in the presence of deoxyribonucleosides and shows slowed but substantial growth.Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly.Finally, we examined whether our lines are able to adapt to limited supplies of deoxyribonucleosides, as might occur in the evolutionary switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis.
Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth.
Genome analysis of replicate lines reveals that several lines carry mutations in deoB and cdd.deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis.Rather than synthesis via this pathway complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism.Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction.We conclude that our experiments recapitulate key evolutionary steps in the adaptation to intracellular life without ribonucleotide reduction.

Introduction
All life on our planet requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides, from ribonucleotides 1,2 .The emergence of ribonucleotide reduction likely drove the transition from RNA to DNA early in the evolution of life [3][4][5][6] and its centrality is reflected in its near ubiquity in organismal genomes 2 .Structural analyses indicate that the catalytic cores of ribonucleotide reductases (RNRs) are structurally homologous 3,7 , despite sequence similarities between them being extremely limited 8 .RNRs catalyse deoxyribonucleotide synthesis via a common mechanism wherein a cysteinyl free radical is generated in the active site.While this similarity in catalysis underscores a common evolutionary origin, RNRs have diverse mechanisms for radical generation 1 .Three broad, evolutionarily distinct, families exist which are also divided on the basis of their radical generation mechanism 1,3,9 .This divergence likely coincides with adaptation to varied environments 9,10 : class I enzymes use a radical generation mechanism based on an oxygen-containing metal ion centre and are strictly aerobic, while class III enzymes generate a stable glycine radical via cleavage of S-adenosyl methionine, and are strictly anaerobic.Class II enzymes generate a 5'deoxyadenosyl radical through cleavage of adenosylcobalamin and operate irrespective of oxygen presence or absence.
To date, there is only one type of environment known where ribonucleotide reduction is dispensable, albeit indirectly: some obligately intracellular species lack genes for ribonucleotide reduction and instead rely on their hosts for deoxyribonucleotides 2 .Examples include a strain of Buchnera aphidicola str.Cc from the Cedar bark aphid, Cinara cedri 11 .Buchnera aphidicola are maternally inherited aphid endosymbionts that synthesise amino acids in short supply in the aphid diet and appear to be on the path to transitioning to an organelle 12,13 .Two obligate intracellular pathogens-Ureaplasma urealyticum 14 and Borrelia burgdorferi 15 -have also lost genes for ribonucleotide reduction 2 .
While ribonucleotide reduction is essential, we reasoned that it should be possible to dispense with genes for ribonucleotide reduction if deoxyribonucleotides or their precursors are available via growth media.We report the successful elimination of all three RNR operons from the bacterium E. coli.The resulting strain is dependent on deoxyribonucleosides in the growth medium but cannot grow if the media are supplemented with deoxyribose plus the four nucleobases (A, G, C, T).To understand the impact of limited deoxyribonucleoside availability, we subjected our knockout line to experimental evolution followed by genome sequencing.We were interested in establishing whether, under such conditions, our lines would utilise the reverse deoxyriboaldolase pathway for deoxyribonucleotide synthesis.This pathway is reversible in vitro 16,17 , and has long been considered a plausible alternative to ribonucleotide reduction 18,19 for dNTP synthesis, with some considering it to be a plausible ancestral route for the origin of DNA 20,21 .Our evolution experiments reveal that this pathway is not coopted for deoxyribonucleotide synthesis following loss of ribonucleotide reduction.Rather, it is a liability under conditions of limited deoxyribonucleotide availability; we observe loss-of-function mutations in deoB, which are predicted to prevent catabolism of 2-deoxy-D-ribose-1-phosphate.Available genome sequence data from obligate intracellular bacteria that lack ribonucleotide reduction indicate that deoB has in fact been lost on multiple occasions, suggesting that recycling of deoxyribose is disadvantageous under conditions where this sugar is in limited supply; deletion of deoB would enable this sugar to be rerouted for deoxyribonucleotide synthesis but would also preclude de novo synthesis via the reverse deoxyriboaldolase reaction.Our results thus illuminate a key adaptive step taken by obligate intracellular species to mitigate the loss of ribonucleotide reduction.

Strains and growth conditions
All E. coli strains used in this study are listed in Supplementary Table 1.E. coli B strain REL606 22 was used as the wild type strain for all experiments.Strains were grown in LB (1% tryptone, 1% NaCl and 0.5% yeast extract +/-2% agar) with the addition of 1 mg/mL each of the four deoxyribonucleosides (dNS-deoxyguanine (dG), deoxyadenine (dA), deoxycytosine (dC) and deoxythymine (dT) (AK Scientific)) for mutant strains, unless stated otherwise.For evolution experiments, strains were grown in 1x MOPS media 23 supplemented with dNS, as specified.All experiments were conducted in the presence of 100 µg/mL streptomycin.

Creation of a ribonucleotide reductase mutant
Deletion of RNR operons was carried out using a scarless genome engineering method 24 in series (Supplementary Text 1): the nrdDG operon (class III RNR) was deleted first, followed by nrdHIEF (class IB) then nrdAB (class IA).For each operon, 500 bp upstream and downstream from the start and stop codon was PCR amplified.A second round of overlap PCR combined fragments together and the three resulting product was cloned into pST76a.The resulting construct was transformed into wild type at 30°C, then with genome integration induced by increasing temperature to 42°C.
Helper plasmid (pSTKST) was transformed into the resulting strain, and deletion was induced with chlortetracycline.Successful deletion was confirmed using PCR across the operon (Primers used in this study are listed in Supplementary Table 2).The resulting strain, which we call DRNR, was confirmed by whole genome sequencing to lack all three RNR operons (DnrdAB, DnrdHIEF, DnrdDG).

Total RNA extraction and RT-PCR
To confirm absence of ribonucleotide reductase expression, strains were grown overnight in LB and diluted to 1:100 in 10 mL fresh media after reaching stationary phase.The cultures were then grown for ~3 hours and total RNA was isolated from mid-log phase cultures using TRIzol Max Bacterial Isolation kit (ThermoFisher).RNA was quantified using a Qubit 4.0 fluorometer.Purified RNA was diluted to 300 ng/mL and treated with TURBO DNaseI (Ambion).This DNA-free RNA was then subjected to RT-PCR using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA Polymerase kit (Invitrogen) with primers specific to the gene of interest (Supplementary Table 2).

Growth assays
Bacterial strains were retrieved from -80°C stocks and grown overnight in LB (1 mg/ml dNS was added to media for growth of DRNR lines).Cultures were washed twice in 1x PBS and 10 µL equivalent was added to fresh 24-well plates containing 1 mL of 1x MOPS + 1% glucose.Differing concentrations of dNS were added from a 20 mg/mL stock solution to each individual well.Growth was monitored for 24-48 hours, taking measurements (OD595) every 6 minutes using a FLUOstar Omega Microplate Reader (BMG Labtech).All experiments were performed in triplicate.

Microscopy
Overnight cultures were grown at 37°C in MOPS media (with dNS added as required).
Cultures were twice spun down and washed in 1x PBS. 10 µl of culture was aliquoted onto a microscope slide.Brightfield images were taken using a LEICA ICC50 W microscope (Leica, v.3.2.0) and imported to Photoshop (v 22.4.2) for cell-length measurement.Cell length measurements were determined for each strain by averaging from 20 observations (length of the first 5 cells from top left to right were counted from each of 4 images).
To visualise DNA, we used a modified version of a FITC/DRAQ-5 double-staining protocol (Silva et al., 2010), instead using DAPI in place of DRAQ-5.Coverslips were coated in poly-D-lysine and placed at 37°C overnight.Coverslips were washed twice with water, dried and stored at 4°C until ready for use.Overnight cultures were washed in 1x PBS, resuspended, and placed on a poly-D-lysine coated coverslip at 37°C for 1 hour.Cells were washed with PBS, and fixed using 4% paraformaldehyde at room temperature for 10 minutes.Coverslips were washed with 1x PBS, followed by 1% Triton X-100 for 5 minutes.Following a further wash with 1x PBS, coverslips were then incubated in 1x PBS containing 6 µg/mL FITC for 30 minutes at 37°C.The coverslips were washed again with 1x PBS, then suspended in 1x PBS containing 5 µg/mL DAPI and placed in the dark for 10 minutes.Coverslips were lastly washed twice more in 1x PBS, then were mounted on microscope slides.Strains were visualised on a Nikon Ni-E Fluorescence microscope using fluorescent filter cubes for DAPI and FITC, and a 100x oil objective lens.Images were overlaid using Nikon NIS-Elements software.

Experimental Evolution
All evolution experiments were performed at 37 °C.Original bacterial strains were retrieved from -80 °C stocks and grown overnight in LB (1mg/ml dNS was added to media for growth of DRNR lines).Cultures were then washed twice in 1x PBS and 50 µL equivalent was added to fresh 6-well plates containing 5 mL 1x MOPS media (1% glucose) supplemented with the dNS concentration required for each condition (1 mg/ml, 0.25 mg /ml, 0.01 mg/mL).5 lineages of DRNR and 3 for WT (E. coli REL606 22,25 ) were passaged for each condition until transfer 30.Line 1 of DRNR grown at 0.25 mg/ml (DRNR_T30_250_1) was serially passaged as ten independent lines for an additional 10 transfers in MOPS+1% glucose and 0.01 mg/ml dNS (DRNR_T40_10_1-10).Plates were left to reach stationary with agitation at 120 rpm.Approximately 10 7 -10 8 of wash cells were transferred to a fresh well.A glycerol stock was created for each line every 5 transfers.PCR contamination checks were performed every 5 transfers.

Sequencing and genome assembly
All strains and lineages required for sequencing were streaked for single colonies on LB media, with a single colony being used to inoculate an overnight culture.Genomic material was isolated using 20µg genomic tips (Qiagen) as per manufacturer's instructions and DNA quantified using a Qubit 4.0 fluorometer (ThermoFisher).

Sequence analyses
For creation of the phosphopentomutase alignment, we ran BLASTP with default settings (E-value threshold = 0.1, BLOSUM62, Filtering: none, Gapped: yes, Hits: 1000) against the Uniprot reference genomes plus Swiss-Prot database (https://www.uniprot.org/blast/)using the deoB protein sequence from E. coli (REL606) as query.To create our alignment dataset, we filtered results for Swiss-Prot sequences and removed duplicates.We then added the E. coli REL606 query sequence and the phosphopentomutase from Bacillus cereus, the crystal structure of which has been determined 28 .We generated our alignment with Muscle 29 using the Geneious plugin (v.3.8.425)and default settings.
To screen for presence/absence of ribonucleotide reduction, phosphopentomutase and cytidine deaminases in Buchera, Borrelia and Ureaplasma, we ran blastp searches against the nr_protein database restricted to these taxa using query sequences listed in Supplementary Table 10.Where data indicated gene absence, we confirmed this by examining the genome sequences for assembly issues, tblastn searches, and manual inspection of predicted ORFs from the genome assembly files (https://www.ncbi.nlm.nih.gov/genome/browse/#!/overview/).For cases where gene absence was clearly due to the assembly, these were excluded from further analysis.
Assembly accession numbers and taxids are provided in Supplementary Text 2.

Creation of an E. coli line lacking ribonuclease reduction
E. coli carries genes for three RNRs: aerobic class Ia (encoded by the nrdAB operon), class Ib (nrdHIEF), and anaerobic class III (nrdDG) (Figure 1).Under aerobic growth, ribonucleotide reduction is primarily performed by the class Ia enzyme, with the role of the Ib enzyme being less clear 30 .
We reasoned that, while ribonucleotide reduction is an essential process, it should be possible to compensate for its loss via media supplementation.To eliminate ribonucleotide reduction, we sequentially deleted the RNR operons using a scarless deletion protocol (Methods).We first deleted nrdDG, as this operon is known not to be expressed under aerobic conditions.We next deleted the nrdHIEF operon from the resulting DnrdDG line.Finally, we attempted to delete the nrdAB operon the DnrdHIEF DnrdDG line, grown in the presence of deoxyribonucleosides (dNS).We were initially unsuccessful in deleting nrdAB.This is not unexpected as, while deoxyribonucleosides are known to be taken up, some (dG, dC, dA) are unavailable for DNA synthesis, even when genes in the DERA pathway are mutated, preventing their catabolism 31 .We reasoned that may be because of the absence of a suitable deoxynucleoside kinase activity for converting those deoxyribonucleosides to deoxyribonucleotides. We therefore tried knocking out nrdAB in the presence of a heterologously expressed deoxyadenosine kinase (dAK) gene from Mycoplasma mycoides.Mycoplasmas are known to be dependent on salvage for dNTP production and possess deoxyadenosine kinases genes that permit utilisation of deoxyribonucleosides for DNA synthesis 32 .To establish if resulting lines lacked all three nrd operons, we first PCR screened for evidence of chromosomal nrd operon deletions.Our results indicate that all three operons were successfully deleted (Figure 1).As deletion does not exclude relocation of functional gene copies to another genomic location, we confirmed gene absence via PCR using primers internal to nrd genes and performed RT-PCR to confirm absence of gene expression (Supplementary Figure 1).Finally, knockout status was confirmed with whole genome sequencing.This confirmed deletion of all genes for ribonucleotide reduction, with the nrdAB operon successfully deleted under heterologous expression of dAK from M. mycoides (Supplementary Text 1; Supplementary Figure 2).Of four knockout lines, we selected one isolate (hereafter called DRNR) for all subsequent work.In addition to lacking all nrd genes, genome sequencing revealed 23 SNPs that likely appeared during the creation of DRNR from wild-type progenitor line (REL606) (Supplementary Table 3).

DRNR is dependent on deoxyribonucleoside supplementation
Our DRNR line was created in the presence of dNS supplementation, but this does not mean it is dependent on dNS supplementation for growth.We therefore sought to understand what supplementation, if any, our DRNR line requires.To determine the lowest concentration of dNS that permits growth, we generated a series of growth curves for differing dNS concentrations.At high dNS concentrations (1 mg/mL), DRNR grows favourably, though shows a clear lag compared to wild-type on equivalent media (Figure 2A).At lower dNS concentrations, DRNR growth is impaired, but there is still discernible growth at 0.05 mg/mL (Figure 2C).When this is dropped to 0.01 mg/mL we observed only marginal growth of DRNR (Figure 2D).As expected, in the absence of dNS supplementation no growth is observed, while wild-type lines are unaffected (Figure 2E).Finally, we tried growing DRNR on deoxyribose (dR) plus each of the four bases (A, G, C, T).No growth was observed (Figure 2F).These results indicate that deletion of the three nrd operons from DRNR has completely eliminated the capacity for de novo deoxyribonucleotide synthesis; no other genes appear able to compensate for this deficiency.
We next sought to establish whether DRNR requires supplementation of all four dNSs, or whether any are dispensable.Strains were once again grown in 1x MOPS+1% glucose, with individual deoxyribonucleosides (dA, dG, dT or dC) added at a concentration of 0.25 mg/mL.This revealed that DRNR is unable to grow in the presence of dA, dG, or dT (either alone (Figure 3A-C), or in combination (Figure 3E), even after 44 hours of monitoring (data not shown).However, DRNR can grow on dC alone (Figure 3D).Together these data indicate deoxycytidine (dC) is the sole deoxyribonucleoside required for DRNR growth in minimal media.

DRNR exhibits a filamentous cell morphology when grown under limiting dNS
During our growth assays we observed a 'clumping' phenotype when DRNR is grown in liquid media at concentrations of dNS that limit growth.This contrasts with the uniform cloudy appearance of wild-type E. coli (Figure 4C, top left panel).
Examination of cells at 100x magnification revealed that DRNR cells are elongated and filamentous at very low levels of dNS, whereas DRNR cells have a similar morphology to wild-type at higher dNS concentrations (Figure 4A).Some cells reached lengths several times that of wild-type (Supplementary Table 4).This phenotype appears most pronounced at low dNS concentrations.We therefore sought to establish if this phenotype is the result of environmental conditions.We transferred a population of DRNR cells exhibiting the filamentous cell phenotype from low dNS (0.01 mg/mL) media to a less restrictive environment (dNS = 1 mg/mL).
Following transfer to higher [dNS], the filamentous phenotype is heavily diminished (Figure 4C).This demonstrates that the phenotype is environmental and can be reversed if cells are moved to a high dNS environment.
One possibility is that, under low [dNS], cells are growing but unable to complete cell division.If so, this might be reflected by the presence of multiple DNA-dense regions across the length of the cells.To visualise DNA within filamentous cells, we stained DRNR cells grown in low dNS (0.01 mg/mL) with FITC and DAPI.This revealed the presence of multiple DAPI-stained regions across the length of the cells (Figure 4B), suggestive of the presence of multiple DNA nucleoids in DRNR cells grown at low [dNS].

Evolution of ∆RNR lines under restricted dNS supplementation
The loss of ribonucleotide reduction in obligate intracellular lifestyles presumably resulted from relaxed selection on deoxyribonucleotide production when deoxyribonucleotides are available from the environment.However, for both parasitism and endosymbiosis, the host dNTP pool must be shared.We were therefore interested to assess if the ∆RNR line adapts to a reduction in dNS availability, as might occur in the evolutionary switch from de novo synthesis to dependency on host production.
In order to allow the strains to adapt to a lower concentration of dNS supplementation in the growth media, we initiated evolution experiments at dNS concentrations where ∆RNR lines grow, but not as well as wildtype (1 mg/mL and 0.25 mg/ml).Five independent lines of ∆RNR were serially passaged in each condition by growing the culture to stationary phase, then transferring 50 µL into a new 6-well plate containing 5 mL of fresh MOPS media+ 1% glucose and dNS (Figure 5).We also established three control lines of the wild-type progenitor strain (REL606) at each dNS concentration.The initial experiment was run for a total of 30 transfers.Glycerol stocks were created every 5 transfers for each line, with contamination checks performed concurrently.

Elongate cell morphology in ∆RNR lines diminishes over the course of the evolution experiment
Our initial observations of unevolved ∆RNR (henceforth ∆RNR_T0) revealed an elongated filamentous cellular phenotype at lower concentrations of dNS (Figure 4).
We therefore monitored cell morphology during our evolution experiment.Every 5 transfers, we examined length of ∆RNR and REL606 cells from each condition (1 mg/mL or 0.25 mg/mL dNS; cells from well position A1 (∆RNR) and A1 or B1 (REL606) were used for all measurements, and mean length calculated).At the beginning of the experiment, ∆RNR cells are substantially longer than wild-type, particularly at 0.25 mg/mL dNS (Figure 6).We recorded cell lengths of 20 µM at this lower concentration (average of 6 µM), a staggering 10-20 times the length of wildtype cells.By the time the experiment had reached 30 transfers, average cell length was comparable to wild type at both dNS concentrations (Figure 6A).These changes in gross morphology suggest cells had begun to adapt to restricted dNS availability.

∆RNR lines exhibit improved growth following evolution under restricted dNS availability
After 30 transfers, we sought to determine if ∆RNR lines had improved their capacity to grow under restricted (either 1 mg/mL or 0.25 mg/mL) dNS availability.We use the following nomenclature for our evolution lines: ∆RNR_[dNS] _transfer#_line#, so ∆RNR1000_T30_L1 is replicate line #1 of ∆RNR evolved at 1 mg/mL (i.e.1000 µg/mL) dNS for 30 transfers.At 1 mg/mL, ∆RNR1000_T30_L1 through L5 all showed improved growth relative to ∆RNR_T0 (Figure 7A).Moreover, compared to ∆RNR_T0, which exhibited almost no growth at 0.01 mg/ml dNS (Figure 2D), all evolved lines (∆RNR1000_T30_L1-5) showed improved growth at this low concentration (Figure 7D).A similar overall pattern of improved was also seen for the five ∆RNR replicate lines evolved in 0.25 mg/mL (∆RNR250_T30_L1-5) (Supplementary Figure 3).Note however that elongate cell morphology and clumping preclude use of OD measurements for accurate estimation of doubling time or cell counts 33 (reflected in the large standard error relative to REL606 controls), so can only be used to give a general indication of growth.All lines failed to grow in the absence of dNS supplementation (Figure 7E, Supplementary Figure 3), indicating they remain reliant on media supplementation.
Evolution of ∆RNR at 0.25 mg/mL dNS results in A:T à G:C mutational skew.
Following 30 transfers, we sequenced the genomes of ∆RNR_T0, our lines evolved in 0.25 mg/mL dNS (∆RNR250_T30_L1-5) and 1 mg/mL dNS (∆RNR1000_T30_L1-5), and each set of three wild-type control lines (REL606_1000_T30_L1-3 and REL606_250_T30_L1-3).The filamentous phenotype of our ∆RNR lines under restricted dNS supplement precluded accurate estimation of the number of generations per transfer, which in turn precluded reliable calculation of mutation rates.We therefore report total observed mutations (single nucleotide substitutions plus indels) for each experimental line (Figure 8, Supplementary Table 5).Our evolved knockout lines (∆RNR250_T30_L1-5 & ∆RNR1000_T30_L1-5) all accumulated substantially more mutations than wild-type controls evolved at the same dNS concentration (Figure 9A & Supplementary Table 5).Our ∆RNR250_T30 lines accumulated the greatest numbers of mutations (range: 39-118 SNPs).∆RNR1000_T30 lines did exhibit a small but significant increase in total SNPs relative to wild type (Supplementary Figure 4 & Supplementary Table 5; p=0.01, unpaired t-test).There was no significant difference in total mutations between wild type lines evolved in either 1 mg/ml or 0.25 mg/ml dNS (p=0.10;unpaired t-test) It is well documented that ribonucleotide reductases keep deoxyribonucleotide pool sizes balanced through allosteric regulation 10 .We were therefore interested to see whether loss of ribonucleotide reduction resulted in mutational skew in E. coli.Our null expectation was that there would not be any detectable skew as our lines are supplemented with equal concentrations of all four deoxyribonucleosides.If there was skew, we expected this to be most evident in our ∆RNR250_T30 lines, as these accumulated the greatest number of mutations.Indeed, 88% of SNPs in our ∆RNR250_T30 lines are of one type: A:TàG:C (Figure 8, Supplementary Figure 4).

∆RNR lines lose cytidine deaminase.
Analysis of our genome data from lines at transfer 30 revealed that one gene, cdd, which codes for cytidine deaminase, was mutated in 8 of 10 lines from transfer 30 (Supplementary Tables 6 & 7).Cytidine deaminase catalyses deamination of cytidine and deoxycytidine to uridine and deoxyuridine.Analysis of the data revealed that 7 of 8 lines carried an identical deletion (Supplementary Tables 6 & 7; Supplementary Figure 5), while one line possessed a SNP that was predicted to result in a truncation.
As the deletions are identical, we suspected that this deletion may have occurred in the ancestral population, but was not fixed at the time we initiated our evolution experiment.These genome data were derived from colony isolates, meaning it was not possible to determine if this was the case.Thus, the significance of this mutation was unclear in the context of our evolution experiment.To determine whether cdd is lost in response to the experimental conditions, we screened our original ∆RNR isolates to establish if any carried intact cdd.We undertook PCR screening of glycerol stocks for two knockout lines (∆RNR31, ∆RNR34).This revealed that the glycerol stock that we used to establish our evolution experiments (∆RNR31) was a mixed population, with both intact and cdd deletion present (Supplementary Figure 6).Moreover, it appears that, after thirty transfers, all ten lines (∆RNR_T30_1000_L1-5, ∆RNR_T30_250_L1-5) were polymorphic for the deleted locus.Together, this indicates the deletion is very likely to have occurred in the ancestral population, and that our lines were derived from a genetically heterogeneous population, despite the knockout lines being established from individual colonies following scarless deletion of nrdAB (Methods).Screening of ∆RNR34 glycerol stock however revealed no evidence of a deletion (Supplementary Figure 6).We therefore established a short evolution experiment with five independent ∆RNR34 lines, in 0.25 mg/mL dNS, mimicking the early stages of our evolution experiment with the ∆RNR31 isolate.After two transfers, we observed the emergence of a cdd deletion in several replicate lines (Supplementary Figure 6).In similar experiments not reported here and using these lines, we repeatedly see loss of the cdd locus under similar growth conditions (data not shown).Finally, that ∆RNR_T30_250_L5 carried an inactivating mutation in cdd but not the upstream locus (yohk) suggests that it is deletion of the cdd locus is selected under these conditions.∆RNR lines evolved in 0.01 mg/ml dNS mutate the deoxyriboaldolase (DERA) salvage pathway.
By transfer 30, one of our lines (∆RNR_T30_250_L1) exhibited growth similar to wildtype when grown in 0.25 mg/ml dNS (Supplementary Figure 3).Using this line as progenitor, we initiated a second evolution experiment using ten parallel lines, grown in 0.01 mg/ml dNS (Figure 5).After ten transfers, we sequenced these lines (∆RNR_T40_10_L1.1-1.10).All exhibited a marked accumulation of mutations, with L1.7 accumulating 220 SNPs (Supplementary Figure 7).As with the lines from our first 30 transfers, the majority of mutations were A:TàG:C substitutions.
Genome analysis of T40 lines revealed 648 mutations (Supplementary Table 8), of which 80 were found in more than one line.Of these 59 were present in two lines, 16 in three lines and 4 in four lines (Supplementary Table 9).
One gene, deoB, was mutated in eight lines (Table 1), and is particularly noteworthy in the context of our experiment in that it codes for phosphopentomutase.This enzyme catalyzes the transfer of a phosphate group between the C1 and C5 carbon atoms of deoxribose and is responsible for the commited step in deoxyribonucleotide salvage (Figure 9).Closer analysis revealed that several of the mutations to deoB were identical.
It is possible that these mutations have occurred independently, but it may also be possible that these were the result of cross-contamination between lines during the evolution experiment.To assess this, we examined whether the affected lines carried a large number of shared mutations.Lines L1.1 and L1.8 had 47 and 157 unique mutations, with only the deoB mutation shared.For this pattern to be explained by cross-contamination, this mutation would have had to have been the very first mutation to appear, and cross-contamination would have had to have occurred before any other mutation occurred.With no evidence of deoB mutation in the progenitor line at transfer 30 (∆RNR250_T30_L1), we consider it highly unlikely that cross-contamination between these lines has occurred.In the second case, L1.3 (53 SNPs), L1.5 (40 SNPs)    and L1.10 (123 SNPs) share a common deoB mutation along with 13 other mutations.
On these data, it is harder to rule out cross-contamination. Conservatively, it thus appears that deoB has independently mutated five or six times under the conditions of our experiment.
To next sought to assess the impact of the mutations in deoB, on phosphopentomutase function.We first generated an alignment of 100 homologous phosphopentomutases, including from E. coli (REL606) and Bacillus cereus (Supplementary File 1).The latter was included as protein structure and active site residues have been characterised 28 .Using the alignment as a guide, we next assessed functional impact of observed deoB mutations by mapping these onto the structure of B. cereus phosphopentomutase (PDB ID: C3M8).Of the five unique mutations observed in our experiment (Table 1), three directly impact conserved active site residues, either through truncation or mutation of active site residues (Table 2).The remaining two mutations are not directly associated with the active site but result in amino acid substitutions not observed in any sequences in our alignment.We therefore conclude that these mutations to the deoB gene are all likely to impact phosphopentomutase function, with some expected to completely inactivate the enzyme.

Discussion
While all life is dependent on ribonucleotide reduction for the synthesis of deoxyribonucleotides, a handful of obligate intracellular species are known to lack ribonucleotide reductase genes 2 and presumably depend on their host for a source of dNTPs.In this work, we report the successful deletion of all ribonucleotide reductase operons from E. coli (Figure 1).The resulting line (∆RNR) was sequenced to confirm genomic deletion of ribonucleotide reductase genes.Our ∆RNR knockout line grows on MOPS minimal media +1% glucose supplemented with deoxyribonucleosides (dNS) (Figure 2), but does not grow when provided with their constituent molecules (deoxyribose and the four bases A, G, C, T).Growth experiments reveal that, of the four dNSs, only deoxycytidine (dC) is essential for growth (Figure 3).Under limiting levels of dNS, our lines exhibit a filamentous phenotype that can be reversed by increasing dNS supplementation (Figure 4).
In order to understand how obligate intracellular bacteria adapted to the loss of ribonucleotide reduction, we established an evolution experiment where we serially passaged replicate lines in either 1 mg/mL or 0.25 mg/ml dNS.The resulting lines were sequenced after 30 transfers and one line evolved in 0.25 mg/ml dNS (DRNR_250_T30_1) was selected for a second round of evolution at 0.01 mg/ml dNS through a further ten transfers (Figure 5).
Genome sequencing revealed independent mutations to two genes across our replicate lines lines.One gene, cdd, carried mutations in 8/10 lines by transfer 30.The cdd locus codes for cytidine deaminase, which converts deoxycytidine to deoxyuridine, which in turn will be available for dTTP production.Our data reveal two independent mutations (a segmental deletion in 7 lines, and an inactivating SNP in the eighth).The segmental deletion deleted part of the cdd ORF and part of ORF of the upstream gene, yohK, which, together with yohJ, has been shown to code a 3-hydroxypropionate transporter 34 , and was postitionally identical across all seven lines.This deletion likely occurred prior to the establishment of our replicate lines, but appeared not to have gone to fixation.An independent ∆RNR line (∆RNR34) lacked this deletion but rapidly lost cdd when passaged in the experimental growth media (Supplementary Figure 6).This, together with the independent truncation mutation in ∆RNR_T30_250_L5, which impacts cdd but not yohK, suggests there is selection against cdd but not necessarily yohK.Given the fact that our ∆RNR lines can grow with deoxycytidine (dC) as sole deoxyribonucleoside supplement but do not grow if dC is omitted from the deoxyribonucleoside mix, it may be that eliminating cytidine deamination prevents loss of this key deoxyribonucleoside through deamination.We have not assessed growth on dU, but one possibility is that deoxyuridine is less amenable as a substrate for production of all four dNTPs.One intriguing observation is that, despite the rapid loss of cdd, this loss of function appears not to have occurred across all replicates by transfer 30.This may be a consequence of the length of the evolution experiment.Comparing the genomes of species known to lack genes for ribonucleotide reduction 2 reveals absence of cdd frequently coincides with this state.For instance, all confirmed members of the genus Ureaplasma lack both nrd genes and cdd (Supplementary Text 2), suggesting loss of cdd function is selectively advantageous.Establishing whether loss of one gene drives the loss of the other is not straightforward however.An analysis of strains of Buchnera aphidicola revealed that 48/74 strains retain ribonucleotide reduction, but 0/74 possess cdd (Supplementary Text 2; Supplementary Table 14), indicating that the latter can be lost in lines capable of synthesising their own deoxyribonucleotides.The picture is also not clear in Borrelia, with cdd found in species variously possess or lack ribonucleotide reduction (Supplementary Text 2).
Deletion of yohK and cdd results in a small, but intact ORF (Supplementary Figure 5).While this seems unlikely to be functional, the creation of an ORF, particularly where the upstream gene codes for a transmembrane protein, may be consistent with the 'Car Trunk' hypothesis for the avoidance of mutations that would create a toxic protein 35 .The fact that deletion of these genes is reproducible under defined experimental conditions (Supplementary Figure 6) suggests that this region might be suitable for testing this hypothesis.
All lines at grown in 0.25 mg/ml dNS showed a marked mutational skew (A:TàG:C) that differs the expected G:CàA:T mutational skew expected for E. coli 36 , and more generally for bacteria 37 .Knockout of cdd does not appear linked to this skew; 4/5 lines grown at 1mg/ml dNS do not show an obvious A:TàG:C skew (Figure 8).For lines grown at 0.25 mg/ml dNS, two (L2, L3) carry intact cdd genes and both genomes show evidence of A:TàG:C mutational skew.Such skew has been previously shown to be associated with defective mismatch repair 36 .By transfer 30, we observe only two lines with SNPs in mismatch repair genes, with only one mutation being nonsynonymous (Supplementary Table 5).Thus, the skew we observe cannot be explained by widespread mutation to the mismatch repair pathway.The skew may instead be driven in part by the growth dependency; our data show that dC is the sole deoxyribonucleoside required for growth (Figure 3D), with the other dNSs unable to compensate for lack of dC (Figure 3E) but appearing to provide minor improvements to growth (Figure 2B).If these other dNSs are less readily taken up, the majority of intracellular dNTP production would derive from dC supplied in the media.If so, the dCTP pool would be higher than other dNTPs.
We also observed loss of function of deoB in our experiments (Table 1).This observation is interesting for several reasons.First, it indicates that loss of ribonucleotide reduction does not lead to compensatory deoxyribonucleotide synthesis via the DERA pathway, which has been speculated to be an ancestral route for dNTP synthesis, predating the advent of ribonucleotide reduction 20,21 .While the enzymes from this pathway can operate biosynthetically 16,17,38 , loss of phosphopentomutase function via mutation of deoB suggests that this pathway is not readily accessible for synthesis of deoxyribonucleotides in vivo, even though there would have been a strong selective advantage to such an alternative synthetic route during our experiment.We note however that both acetaldehyde toxicity (to both deoxyriboaldolase and to cells more generally) and low intracellular availability may preclude a simple switch to using the DERA pathway for dNTP production 17,21 .Indeed, under the conditions of our experiment, where dNS supplementation is reduced by 25-fold, we observe strong selection to dispense with deoB.This suggests that not only is the reverse DERA pathway is inaccessible for biosynthesis, but salvage is selected against when dNTPs are scarce.In the second phase of our experiment, dNS supplementation is dropped from 0.25mg/ml to 0.01mg/ml, and it is under these conditions of severe dNS restriction that we observe parallel loss-of function mutations at the deoB locus (Table 1).We conclude that, when deoxyribonucleotides are in short supply, deoB loss-of-function ensures dNTPs are not lost to glycolysis via salvage (Figure 9).The biochemistry of the DERA pathway supports mutation of deoB as the most plausible target, since this step is responsible for interconversion of deoxyribose-5-phosphate and deoxyribose-1-phosphate.The latter is the substrate for phosphorylase-mediated addition of nucleobases, generating deoxyribonucleosides, whereas the former is the substrate for deoxyriboaldolase-mediated salvage.Thus, deoB loss-of-function mutations serve to prevent deoxyribose loss to central metabolism while still permitting phosphorylase-mediated deoxyribonucleoside resynthesis (Figure 9).Thus, while conversion of DERA to a synthetic pathway might be a better long-term solution to loss of ribonucleotide reduction, loss of function of this pathway is advantageous in the short term, precluding its cooption to dNTP synthesis.This interpretation is consistent with a recent experiment where mutation of dihydrofolate reductase led to deoB loss of function to prevent loss of deoxyribose-5-phosphate to glycolysis 39 .
Finally, analysis of species that lack nrd genes reveals frequent loss of deoB, suggesting that loss of ribonucleotide reduction might drive loss of deoB, as observed in our experiments.For members of Ureaplasma, the pattern is clear (Supplementary Text 2): all members of this genus completely lack genes for ribonucleotide reduction and show no evidence of coding for either deoB or cdd, the two most mutated genes in our experiments.For Buchnera aphidicola cdd appears to have been completely lost from all sequenced strains.Close to half of sequenced Buchnera strains show co-occurrence of ribonucleotide reduction and phosphopentomutase genes (48/74 strains), with 30% (22/74 strains) lacking genes for both functions (Supplementary Table 14), suggesting that loss of one function results in loss of the other.However, two strains appear to carry deoB while lacking nrd genes, and two further strains show the opposite pattern.
If confirmed, this would indicate loss of one function may not always precipitate loss of the other.
We observe a very clear pattern in members of the Borreliaceae.The NCBI Taxonomy Browser 40 lists two genera with genome data, Borrelia and Borreliella following a recent proposal [41][42][43] to split the genus Borrelia in two.(though this has been contested 44- 46 ).Our analyses indicate that Borrelia (sensu Adeolu & Gupta 41 ) code class Ib ribonucleotide reductases but lack deoB (Supplementary Text 2), while Borrelliela lack both deoB and RNR genes.This pattern could be taken to indicate that members of the Borrelia lost deoB but retained ribonucleotide reduction.However, phylogenetic analyses 47 and location on linear plasmids 48 indicate that the class Ib RNR genes (nrdEF) in Borrelia has been acquired via horizontal gene transfer, presumably following initial loss of ribonucleotide reduction in the Borreliaceae.
It is noteworthy that class Ib RNR, carried by members of the Borrelia (Supplementary Text 2), is manganese-dependent 49 , whereas class Ia is an irondependent enzyme 50 .Interestingly, the most well-studied member of the Borreliaceae, Borreliella burgdorferi (sensu Adeolu & Gupta) does not require iron 51 .It is tempting to speculate that initial loss of ribonucleotide reduction was driven by selection for iron independence, with subsequent reacquisition of ribonucleotide reduction favouring the manganese-dependent enzyme.
More generally, the switch to an intracellular existence may result in relaxed selection on ribonucleotide reduction if host-derived deoxyribonucleotides are sufficient to replace endogenous production.This appears to be an infrequent outcome as most intracellular bacteria retain ribonucleotide reduction.This rare event might be driven by additional pressures, as evolution of iron independence in B. burgdorferi suggests.
Our experiments indicate that, in the absence of endogenous ribonucleotide reduction, disruption of the DERA pathway via mutations to deoB may serve to minimise loss of these building blocks when dNTP availability is limited.Given the necessity of genome replication, we conclude that life without ribonucleotide reduction drives the loss of dNTP catabolism via DERA, enabling building block reuse.It remains to be seen whether there are conditions wherein the reverse DERA pathway 21 can operate synthetically in vivo as an alternative to ribonucleotide reduction.type progenitor (REL606) were established at one of two conditions (1 mg/mL or 0.25 mg/mL dNS in MOPS+1% glucose), and serially passaged for 30 transfers.Genomic material from each line was then extracted and sent for sequencing.To further investigate adaptation to low concentrations of dNS, the "fittest" DRNR line grown at 0.25 mg/mL dNS (DRNR_250_T30_1) was used to seed a subsequent experiment.Ten replicate lines of DRNR_250_T30 _1 were serially passaged for an additional 10 transfers in MOPS+1% glucose and 0.01 mg/ml dNS.DNA from each of these 10 lines (DRNR_10_T40_1-10) was then extracted and sent for whole genome sequencing.

Figure 1 .
Figure 1.Deletion of ribonucleotide reductase genes in E. coli. A. Schematic of each of the three RNR operons in E. coli and the genomic regions following scarless deletion .Arrows indicate locations of PCR primers (SupplementaryTable 2) used to confirm RNR operon presence/absence.B. Results of

Figure 4 .Figure 5 .
Figure 4. DRNR exhibit a reversible elongated, filamentous cell morphology with multiple nucleoids when grown in limiting dNS concentrations.A. 100x Brightfield photographs of REL606 and ∆RNR at 1 and 0.01 mg/mL B. FITC and DAPI staining at 100x of REL606 and ∆RNR at 0.01 mg/mL C. ∆RNR phenotype is reversible if dNS concentration is increased.Loss of clumpy phenotype occurs when ∆RNR cells are transferred from low (0.01 mg/mL) to high (1 mg/mL) dNS.Brightfield microscopy images reveal that this change is accompanied by a reduction in elongated cell morphology upon transfer to high [dNS].(Magnification 1000X; Scale bar 20 µM)

Figure 6 .Figure 7 .
Figure 6.The ∆RNR elongate cell phenotype diminishes over the course of an evolution experiment.A. Mean cell length during the course of a 30-transfer evolution experiment.Cell length was determined for 20 cells for replicate line 1 of ∆RNR and REL606 evolved in either 1 mg/mL or 0.25 mg/mL dNS.Cell measurements were taken every 5 transfers.Over the course of the evolution experiment ∆RNR cell length gradually reduces at both dNS concentrations.B. Morphology of wild-type (top row) and ∆RNR (bottom row) cells at Transfer 1 and Transfer 30 at each dNS concentration (1 mg/mL or 0.25 mg/mL).(Magnification 1000X; Scale bar 2µM)