G C A T T A C G G C A T

Article The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in

Klaudyna Krause 1, Monika Maci ˛ag-Dorszy´nska 2 , Anna Wosinski 3, Lidia Gaffke 3 , Joanna Morcinek-Orłowska 1,3, Estera Rintz 3, Patrycja Biela´nska 3, Agnieszka Szalewska-Pałasz 1, Georgi Muskhelishvili 4 and Grzegorz W˛egrzyn 3,*

1 Department of Bacterial Molecular Genetics, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (K.K.); [email protected] (J.M.-O.); [email protected] (A.S.-P.) 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 80-822 Gdansk, Poland; [email protected] 3 Department of Molecular Biology, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (A.W.); lidia.gaff[email protected] (L.G.); [email protected] (E.R.); [email protected] (P.B.) 4 School of Natural Sciences, Agricultural University of Georgia, 0131 Tbilisi, Georgia; [email protected] * Correspondence: [email protected]; Tel.: +48-58-5236024

 Received: 31 March 2020; Accepted: 16 April 2020; Published: 19 April 2020 

Abstract: A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

Keywords: DNA replication; central carbon metabolism; metabolites; replication control; suppression of replication mutants

1. Introduction The replication of genetic material is one of the fundamental metabolic processes in the living cell. The genetic stability as well as precise functioning of the organism is dependent on the proper regulation of this process. The copying of the DNA during replication, which precedes cell division, is performed by a complex of various enzymes. These proteins can be divided into different groups: proteins altering the DNA structure, enzymes synthesizing nucleic acids, proteins responsible for the

Genes 2020, 11, 447; doi:10.3390/genes11040447 www.mdpi.com/journal/genes Genes 2020, 11, 447 2 of 13 accuracy of copying process, and correction of occurring errors. The complexity of the replication and its dependency of the available energy sources require precise regulation. DNA replication is regulated at many sequential steps, beginning with initiation, through elongation, and to its end—the termination [1]. During the last decades, studies of the replication process helped to characterize the replication factory elements and to understand the role of its components. Nevertheless, until now, the entire picture of the molecular mechanism regulating this fundamental process has not been fully understood. In order to function correctly, the cell needs nutrients providing both the building blocks and energy, and consequently, a tight cooperation between all cellular processes. However, the core of this relationship remains unclear. The key element of this cooperation is central carbon metabolism (CCM). Similar to DNA replication, CCM is indispensable in every organism [1,2]. The reactions of CCM allow the maintenance of cellular homeostasis by keeping the optimal balance between energy production and usage in the cell under various growth conditions. Until recently, it was assumed that DNA replication and CCM are connected only indirectly through CCM-depended synthesis of the nucleic acids’ precursors and provision of metabolic energy. However, it has been demonstrated that effects of certain mutations in genes coding for replication proteins could specifically be suppressed by particular mutations in genes encoding CCM enzymes in Bacillus subtilis [3]. Since indirect suppression due to variations in bacterial growth rate was excluded, it appeared that there is a direct link between CCM and DNA replication [3]. Indeed, recent studies indicated that there are multiple links connecting CCM to DNA replication initiation and elongation in B. subtilis [4]. Other investigations indicated that similar relationships also occur in Escherichia coli [5], although differences in detailed effects of CCM on DNA replication between these two bacteria were evident (summarized in [6]). Interestingly, there are also reports suggesting that enzymes of CCM might contribute to regulation of DNA replication in eukaryotic cells, including human cells (for recent reports, see [7–9]; for reviews and discussions, see [6,10,11]). Despite the evidence that DNA replication regulation is directly linked to CCM, the molecular mechanism of this relationship remains unknown. Previous studies indicated that effects of various mutants of E. coli could be alleviated by dysfunction of specific genes. The suppression pattern was as follows: the effects of dnaA46(ts) mutation could be suppressed by dysfunction of pta or ackA, effects of dnaB(ts) by dysfunction of pgi or pta, effects of dnaE486(ts) by dysfunction of tktB, effects of dnaG(ts) by dysfunction of gpmA, pta, or ackA, and effects of dnaN159(ts) by dysfunction of pta or ackA [5]. The observed suppression suggested that some metabolic enzymes could modulate properties in response to the physiological state of the cell, moreover, the deficiency of CCM enzymes may lead to accumulation of certain metabolites (replication and CCM genes and enzymes, together with their functions, are listed in Table1). These metabolites, in the wild-type cells, could be substrates for particular metabolic enzymes, while in mutants lacking specific enzymes, accumulation of the metabolites would result in metabolic disturbance and possible inhibition of growth. The metabolites could also serve as small molecule signals transmitting information about the metabolic condition and causing the necessary adjustments in DNA replication and cell division. To test this hypothesis, we aimed to investigate the effects of carbon pathways metabolites (acetate, pyruvate, succinate, fumarate, malate, lactate, and α-ketoglutarate) on bacterial growth and DNA replication. Metabolites from glycolysis/gluconeogenesis, overflow pathway, and tricarboxylic acid (TCA) cycle were chosen to mimic effects of mutations in particular genes, which were previously reported to suppress the defects observed in replication mutants. Genes 2020, 11, 447 3 of 13

Table 1. E. coli replication and CCM genes and proteins involved in the replication-metabolism link.

Gene Protein ( Product) Protein Activity dnaA DnaA; replication initiator Binding to the replication origin dnaB DnaB; DNA DNA unwinding in replication forks dnaC DnaC Delivery of DnaB to the origin dnaE DnaE; a subunit of DNA III DNA synthesis during replication dnaG DnaG; Synthesis of primers during replication dnaN DnaN; b subunit of DNA polymerase III Formation of the clamp conferring during replication ackA Acetate kinase Formation of acetyl phosphate from acetate and ATP 2,3-bisphosphoglycerate-dependent gpmA Interconversion of 2-phosphoglycerate and 3-phosphoglycerate phosphoglycerate mutase pgi Glucose-6-phosphate isomerase Isomerization of aldehydo-D-glucose 6-phosphate to keto-D-fructose 6-phosphate pta Phosphate acetyltransferase Interconversion of acetyl-CoA and acetyl phosphate Production of xylulose-5-phosphate and ribose-5-phosphate from tktB Transketolase 2 sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate

2. Materials and Methods

2.1. Bacterial Strains E. coli MG1655 strain [12] was used in this work as a wild-type control, and other isogenic dna(ts) mutants are listed in Table2.

Table 2. E. coli strains employed in this work.

Strain Genotype Reference MG1655 F- l- ilvG rfb-50 rph-1 [12] dnaA46 F- l- ilvG rfb-50 rph-1 dnaA46 tna::Tn10 [13] dnaC(ts) leu thy rpsL dnaC(ts) [14] dnaB8 F- l- ilvG rfb-50 rph-1 dnaB8 cmR [5,14] dnaE486 F- l- ilvG rfb-50 rph-1 dnaE486 zae502::Tn10 [5] dnaG(ts) leu thy rpsL dnaG(ts) [15] dnaN159 F- l- ilvG rfb-50 rph-1 dnaN159 zid501::Tn10 [16]

2.2. Metabolites The tested compounds were purchased from Sigma Aldrich (Saint Louis, MO, USA). Stock solutions were prepared in distillated water and used to achieve indicated concentrations.

2.3. Effects of Metabolites on the Growth of Bacterial Strains

Bacteria were grown in liquid lysogeny broth (LB) medium at 30 ◦C in flasks. Following dilution (1:100) of overnight cultures with fresh LB, the cells were grown under the same conditions until A600 = 0.2. Next, 100 ml of each culture or its dilution were plated on solid LB medium (LB supplemented with 1.5% agar) containing indicated concentration of tested metabolite. The plates were then incubated at either 30 ◦C (controls) or following semi-restrictive temperatures (determined according to [5]): 39 ◦C for dnaA46(ts), 41 ◦C for dnaB8(ts), 35 ◦C for dnaC(ts), 36.5 ◦C for dnaE486(ts), 34 ◦C for dnaG(ts), and 37.5 ◦C for dnaN159(ts) for 16 h. Colony forming units (CFU) were calculated from plates where the colony number was between 100 and 1000.

2.4. Microscopic Analysis The optical microscopy analysis was performed as described previously [3]. Briefly, bacteria were grown exponentially at 30 ◦C until A600 = 0.2. Then, the cultures were shifted to semi-restrictive temperatures (as listed in the preceding subsection) for the time equal to five generations (as estimated at 30 ◦C). After the incubation, cells were stained for 10 min with Genes 2020, 11, x FOR PEER REVIEW 4 of 14

temperatures (as listed in the preceding subsection) for the time equal to five generations (as Genes 2020estimated, 11, 447 at 30 °C). After the incubation, cells were stained for 10 min with DAPI (4’,6’‐diamidino4 of 13 ‐2‐ phenylindole) (Sigma Aldrich, Saint Louis, MO, USA) and SynaptoRed™ (N‐(3‐ DAPItriethylammoniopropyl) (4’,6’-diamidino-2-phenylindole)‐4‐(6‐(4‐(diethylamino)phenyl) (Sigma Aldrich, Saint Louis,hexatrienyl) MO, USA) pyridinium and SynaptoRed dibromide,™ (N-(3-triethylammoniopropyl)-4-(6-(4-(diethylamino)phenyl)pyridinium, 4‐[6‐[4‐(diethylamino) phenyl]‐ 1,3,5‐hexatrienyl] hexatrienyl)‐1‐[3‐(triethylammonio) pyridinium dibromide, propyl]‐, pyridinium,dibromide) 4-[6-[4-(diethylamino) ( Sigma Aldrich, Saint phenyl]- Louis, 1,3,5-hexatrienyl]-1-[3-(triethylammonio)MO, USA), as described previously [17]. Bacteria propyl]-, were dibromide)observed ( Sigma under Aldrich, the Leica Saint DMI4000B Louis, MO,microscope USA), asat describedthe magnification previously of 100 [17 ].X Bacteriaand photographed were observedwith under Leica DFC365FX. the Leica DMI4000B The length microscope of 100 randomly at the magnification chosen cells was of 100 measured X and photographed by ImageJ Ink with program Leicaand DFC365FX. analyzed The with length GraphPad of 100 Prism randomly 5. chosen cells was measured by ImageJ Ink program and analyzed with GraphPad Prism 5. 2.5. Kinetics of DNA Replication In Vivo 2.5. KineticsOvernight of DNA Replication bacterial cultures In Vivo grown at 30 °C in LB were diluted (1:100), and the incubation was

600 3 Overnightcontinued bacterialuntil A cultures = 0.1. Then, grown [ atH]thymidine 30 ◦C in LB was were added diluted to (1:100),indicated and concentration the incubation and to radioactivity of 5 μCi/mL, and the3 culture was transferred to semi‐restrictive temperature. At was continued until A600 = 0.1. Then, [ H]thymidine was added to indicated concentration and to radioactivityindicated of 5times,µCi/mL, samples and the were culture placed was transferredonto Whatman to semi-restrictive 3MM paper temperature.filters and then At indicated immediately times,transferred samples were to 10% placed trichloroacetic onto Whatman acid (TCA) 3MM paper for 10 filters min. Following and then immediately this incubation, transferred the filters to were 10% trichloroaceticwashed in 5% TCA acid (TCA)and 96% for ethanol. 10 min. The Following filters were this incubation,dried and their the filtersradioactivity were washed was measured in 5% in TCAthe and scintillation 96% ethanol. counter The filters (Microbeta2 were dried®, Perkin and their Elmer, radioactivity Waltham, was MA, measured USA). in the scintillation counter (Microbeta2®, Perkin Elmer, Waltham, MA, USA). 3. Results 3. Results 3.1. Growth Defect Suppression of Replication Mutant Strains by Certain CCM Metabolites 3.1. Growth Defect Suppression of Replication Mutant Strains by Certain CCM Metabolites Since suppression of growth defects of the temperature‐sensitive replication mutations was Sinceobserved suppression in pta, ackA of, pgi growth, tktB, defectsand gpmA of theCCM temperature-sensitive mutants ([5]; see also replicationFigure 1), we mutations asked whether was the observedexcess in ofpta chosen, ackA, metabolitespgi, tktB, and (thatgpmA mightCCM potentially mutants accumulate ([5]; see also in Figure metabolically1), we asked defective whether cells) can the excessimprove of the chosen growth metabolites of replication (that mutants. might potentially The semi‐restrictive accumulate temperatures in metabolically were chosen defective based on cells)previous can improve report the [5] to growth achieve of decreased replication efficiency mutants. of The plating semi-restrictive by 2–3 orders temperatures of magnitude. were Various chosenconcentrations based on previous of metabolites report [5 ](pyruvate, to achieve acetate, decreased fumarate, efficiency succinate, of plating malate, by 2–3 lactate, orders and of α‐ magnitude.ketoglutarate) Various were concentrations added to the of solid metabolites medium, (pyruvate, which was acetate, followed fumarate, by plating succinate, of relevant malate, strains. lactate,The and metabolites’α-ketoglutarate) concentrations were added were tochosen the solid by monitoring medium, whichof bacterial was followedgrowth in by liquid plating cultures of at relevantpermissive strains. temperatures The metabolites’ and concentrations selecting concentrations were chosen which by monitoring did not impair of bacterial bacterial growth viability. in No liquidsignificant cultures at differences permissive in temperatures viability and and culture selecting growth concentrations rate were observed which did at 30 not °C impair between bacterial wild‐type viability.and Nomutant significant bacteria, diff erencesas well inas viability between and wild culture‐type growthcells growing rate were at observeddifferent attemperatures, 30 ◦C between at any wild-typetested and concentration mutant bacteria, of metabolites. as well as between As expected, wild-type efficiency cells growing of plating at of di fftheerent tested temperatures, mutants at at semi‐ any testedrestrictive concentration temperatures of metabolites. were about 2–3 As expected,orders of magnitude efficiency of lower plating than of that the testedof the wild mutants‐type at strain semi-restrictivein the absence temperatures of respective were metabolites about 2–3 (Figure orders 1). of magnitude lower than that of the wild-type strain in the absence of respective metabolites (Figure1).

(a)

Figure 1. Cont.

Genes 2020, 11, 447 5 of 13 Genes 2020, 11, x FOR PEER REVIEW 5 of 14

(b) Acetate 10 9 dnaA46 dnaB8 dnaC(Ts) dnaG(Ts) dnaE486 dnaN159 10 8

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Figure 1. Cont.

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Figure 1. The growth of replication-impaired temperature sensitive mutants in the presence of followingFigure CCM 1. The metabolites: growth of (a )replication pyruvate,‐ (impairedb) acetate, temperature (c) succinate, sensitive (d) fumarate, mutants (e) a-ketoglutarate, in the presence of (f) malate,following (g) lactate. CCM metabolites: Bacteria (wild (a type—empty) pyruvate, (b columns,) acetate, mutants—filled (c) succinate, (d columns)) fumarate, were (e) cultivated a‐ketoglutarate, in semi-restrictive(f) malate, ( temperatures,g) lactate. Bacteria established (wild type—empty for each mutant, columns, and the mutants—filled efficiency of plating columns) was were measured cultivated as CFUin /mL.semi The‐restrictivedna(ts) mutationstemperatures, and metabolitesestablished are for indicated each mutant, above and graphs. the Theefficiency experiments of plating were was repeatedmeasured at least as in CFU/mL. triplicates The (SD indicated).dna(ts) mutations and metabolites are indicated above graphs. The experiments were repeated at least in triplicates (SD indicated). However, we found that particular metabolites were able to suppress effects of replication mutations,However, whereby, we the found efficiency that ofparticular suppression metabolites depended were on metaboliteable to suppress concentration effects (Figureof replication1). To achievemutations, complete whereby, suppression the efficiency (i.e., when of suppression the efficiency depended of plating on of themetabolite mutant strainconcentration was at the (Figure level 1). of wild-typeTo achieve strain) complete of the dnaA46suppressionmutation, (i.e., when 10 mM the pyruvate, efficiency 10 mMof plating acetate, of 2.5 the mM mutant fumarate, strain 15 was mM at the malate,level 15 of mM wild lactate,‐type strain) or 5 mM of theα-ketoglutarate dnaA46 mutation, was necessary.10 mM pyruvate, Complete 10 mM suppression acetate, 2.5 of mM the dnaB8 fumarate, mutation15 mM required malate, 5 15 mM mMα -ketoglutarate,lactate, or 5 mM or α‐ 250ketoglutarate mM succinate, was whilenecessary. partial Complete suppression suppression occurred of the in thednaB8 presence mutation of 10 mMrequired lactate 5 mM or 20 α‐ mMketoglutarate, malate. For or the 250dnaC mMmutation, succinate, the while presence partial of 12.5 suppression mM acetate,occurred 5 mM inα -ketoglutarate,the presence of 15 10 mM mM lactate, lactate 0.5 or mM20 mM fumarate, malate. or For 250 the mM dnaC succinate mutation, could the suppress presence of the growth12.5 mM defect. acetate, The 5 mM effect α‐ketoglutarate, of the dnaG mutation 15 mM lactate, was suppressed 0.5 mM fumarate, only in or the 250 presence mM succinate of 5 mM could α-ketoglutarate,suppress the though growth partial defect. suppression The effect of by the pyruvate dnaG mutation was also was observed. suppressed Complete only in suppression the presence of of the5 dnaE486mM α‐ketoglutarate,mutation occurred though at 5partial mM acetate suppression or 5 mM byα-ketoglutarate, pyruvate was while also pyruvate,observed. lactate, Complete malate,suppression and succinate of the caused dnaE486 partial mutation suppression. occurred Finally, at 5 mM only acetate partial or suppression 5 mM α‐ketoglutarate, of the dnaN159 while mutationpyruvate, could lactate, be observed malate, in and the succinate presence caused of succinate. partial These suppression. results indicateFinally, only that partial the addition suppression of variousof the metabolites dnaN159 maymutation efficiently could suppress be observed temperature in the presence sensitivity of ofsuccinate. different These mutants results in replication indicate that genes,the estimated addition as of the various ability tometabolites form colonies may at efficiently temperatures suppress which normallytemperature (without sensitivity any treatment) of different causedmutants a decrease in replication in viability genes, of mutants estimated by 2–3 as orders the ability of magnitude. to form Therefore,colonies at we temperatures asked whether which normally (without any treatment) caused a decrease in viability of mutants by 2–3 orders of magnitude. Therefore, we asked whether other mutant phenotypic properties of the tested strains could also be suppressed in the presence of an excess of different metabolites.

Genes 2020, 11, 447 7 of 13 other mutant phenotypic properties of the tested strains could also be suppressed in the presence of an excess of different metabolites.

3.2. Effects of Metabolites on Filamentation of the Replication Mutants at Elevated Temperature Changes in environmental conditions lead to growth rate alterations. This effect could be connected to changes in the cell cycle, cell mass doubling, chromosome replication, and chromosome segregation in the bacterial cell. The replication deregulation may lead to severe defects in cell morphology (if cells do not divide correctly and make filaments) or in chromosomes’ segregation during cell division. Growth defect of replication mutants at the restrictive temperatures is reflected also in the impairment of cell division, leading to appearance of filamentous cells. Suppression of E. coli replication mutant phenotype in the presence of metabolic mutants included not only improvement of bacterial growth at restriction temperature [5], but also rescue of otherwise drastically elongated cell shape [17]. Therefore, we tested the effects of CCM metabolites on this parameter in the dna(ts) strains. Examples of the microscopic pictures of bacterial cells obtained in experiments with and without CCM metabolite supplementation of wild-type and dna(ts) cultures at 30 ◦C and the semi-restrictive temperature are presented in Figure2. The results were quantitated by length measurements of 100 randomly chosen cells. At semi-restrictive temperatures, the length of the dnaA46, dnaB8, dnaC(ts), dnaG(ts), dnaE486, and dnaN159 mutant cells varied from 10 to 30 µm, while it was 2–3 µm in the case of the wild-type strain (Figure3). No significant di fferences between wild-type bacteria and mutants could be observed at 30 ◦C (in all cases, the length of cells was 2–3 µm). However, at semi-restrictive temperatures, effects of all the tested metabolites on mutant cell filamentation could be observed, although only some metabolites used at certain concentrations caused complete suppression of the filamentation phenotype. These conditions included: 12.5 mM pyruvate, 250 mM succinate, 10 mM fumarate, and 20 mM lactate for dnaA46; 250 mM succinate, 10 mM fumarate, and 20 mM lactate for dnaB8; 10 mM fumarate and 20 mM lactate for dnaC; 10 mM pyruvate and 20 mM lactate for dnaG; 250 mM succinate and 20 mM lactate for dnaE486; and 20 mM lactate for dnaN159. Other concentrations of metabolites caused partial suppression effects of various mutations in replication genes on cell shape (Figure3). Moreover, changes in nucleoidGenes 2020 localization,, 11, x FOR PEER evident REVIEW in mutants at semi-restrictive temperatures, were abolished8 of 14 by certain metabolites (Figure2).

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Figure 2.FigureThe 2. microscopic The microscopic analysis analysis of of the the e ffeffectect of of CCM metabolites metabolites on on bacterial bacterial cell morphology cell morphology and and nucleoidnucleoid position. position. Wild Wild type type and anddnaA46 dnaA46 strainsstrains were incubated at at 39 39 °C◦C in in the the presence presence of of the the metabolitemetabolite as indicated, as indicated, prior prior to staining to staining with with DAPI DAPI and and SynaptoRed. SynaptoRed. Bar Bar indicatesindicates 10 10 mm. mm. Panels Panels represent:represent: (a) MG1655 (a) MG1655 (wild-type), (wild‐type), no metabolite no metabolite added added (b )(bdnaA46) dnaA46, no, no metabolite metabolite added added (c ()c )MG1655 MG1655 (wild-type),(wild lactate‐type), lactate 20 mM 20 ( dmM) dnaA46 (d) dnaA46, lactate, lactate 20 mM. 20 mM.

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FigureFigure 3. The 3. The e ffeffectect of CCM CCM metabolites metabolites on the on cell the length cell of lengthreplication of mutants. replication Bacteria mutants. (wild type— Bacteria (wildempty type—empty columns, columns,dna(ts) mutants—filleddna(ts) mutants—filled columns) were grown columns) at semi were‐restrictive grown temperatures at semi-restrictive in the temperaturespresence inof thefollowing presence metabolites: of following (a) pyruvate, metabolites: (b) acetate, (a) pyruvate, (c) succinate, (b) ( acetate,d) fumarate, (c) succinate,(e) a‐ (d) fumarate,ketoglutarate, (e) a-ketoglutarate, (f) malate, (g) lactate. (f) malate, The cell (g length) lactate. was The measured cell length for 100 was bacteria measured per each for strain. 100 bacteria The per eachdna strain.(ts) mutations The dna and(ts) metabolites mutations are and indicated metabolites above graphs. are indicated The experiments above graphs. were repeated The experiments at least were repeatedin triplicates at least (SD indicated). in triplicates (SD indicated).

3.3. Kinetics3.3. Kinetics of DNA of DNA Replication Replication in the in the Presence Presence of of CCM CCM Metabolites Metabolites To testTo whether test whether the the effects effects of CCMof CCM metabolites metabolites onon E.E. coli replicationreplication mutants’ mutants’ growth growth and and filamentationfilamentation at semi-restrictive at semi‐restrictive temperatures temperatures are are duedue toto their direct direct and and rapid rapid action action on onDNA DNA replication process, we aimed to estimate the efficiency of bacterial DNA synthesis in vivo. replication process, we aimed to estimate the efficiency of bacterial DNA synthesis in vivo.

We measured the efficiency of incorporation of the labelled DNA synthesis precursor, [3H]thymidine, into the DNA. However, we found that the addition of vast majority of tested compounds did not improve the efficiency of DNA synthesis in mutants at semi-restrictive temperatures. Genes 2020, 11, x FOR PEER REVIEW 11 of 14

We measured the efficiency of incorporation of the labelled DNA synthesis precursor, 3 [GenesH]thymidine,2020, 11, 447 into the DNA. However, we found that the addition of vast majority of tested10 of 13 compounds did not improve the efficiency of DNA synthesis in mutants at semi‐restrictive temperatures. Rather, inhibition of DNA replication by CCM metabolites was observed in both wild‐ typeRather, and inhibition mutant cells of DNA (Figure replication S1; note by that CCM for metabolites technical reasons, was observed experiments in both wild-typewere repeated and mutant three timescells (Figure separately S1; notefor each that metabolite. for technical Therefore, reasons, experiments efficiencies of were 3H repeatedincorporation, three timesexpressed separately in CPM, for 3 mayeach differ metabolite. between Therefore, experiments efficiencies with different of H incorporation, metabolites, and expressed even values in CPM, measured may di atff timeer between 0 may vary,experiments which, withhowever, different did metabolites,not affect the and general even values picture measured and conclusions. at time 0 mayMoreover, vary, which, in some however, cases, particulardid not aff ectmetabolites the general caused picture strong and conclusions. inhibition of Moreover, 3H incorporation, in some cases, thus, particular effects of metabolites selected 3 metabolitescaused strong are inhibition demonstrated). of H incorporation, The only exception thus, effects was of 250 selected mM metabolites succinate‐mediated are demonstrated). efficient improvementThe only exception of DNA was replication 250 mM succinate-mediated in the dnaB8 mutant e (Figurefficient improvement4). Therefore, ofwe DNA conclude replication that in inmost the cases,dnaB8 themutant observed (Figure effects4). Therefore, of metabolites we conclude on mutants’ that in phenotypes most cases, were the observed indirect. e ffects of metabolites on mutants’ phenotypes were indirect.

Acetate Succinate 40000 40000 t=60' 30000 30000 t=60' t=45'

20000 20000 t=45' [CPM] [CPM] H]thymidine intake H]thymidine t=30' H]thymidine intake 3

3 t=30' 10000 t=15' 10000 t=15' t=0' t=0' 0 0 0 0 0 0

Relative [ 0 10 10 10 10 10 0 0 0 0 0 Relative [ 50 50 50 50 50 12,5 12,5 12,5 12,5 12,5 125 250 125 250 125 250 125 250 concentration (mM) 125 250 concentration (mM)

(a) (b)

Fumarate -Ketoglutarate

40000 20000

30000 t=60' 15000 t=60' t=45' 20000 t=45' 10000 t=30'

[CPM] t=15' [CPM]

H]thymidine intake t=0' H]thymidine intake H]thymidine 3 t=30' 3 10000 t=0' t=15' 5000

0 0 Relative [ Relative [ Relative 0 0 0 0 0 0 5 0 5 0 5 0 5 0 5 10 10 10 10 10 10 10 10 10 10 0,5 2.5 0,5 2,5 0,5 2,5 0,5 2,5 0,5 2,5 7,5 7,5 7,5 7,5 7,5 concentration (mM) concentration (mM) (c) (d)

Malate Lactate 20000 20000 t=60' t=60' 15000 15000 t=45' t=45' 10000

10000 [CPM]

H]thymidine intake H]thymidine t=30' 3 [CPM]

H]thymidine intake H]thymidine t=30' 5000 t=0' t=15' 3 5000 t=0' t=15' 0 Relative [ Relative 0 0 0 0 0 0 15 20 15 20 15 20 15 20 15 20 17,5 17,5 17,5 17,5 17,5 Relative [ Relative 0 0 0 0 0 concentration (mM) 15 20 15 20 15 20 15 20 15 20

17,5 17,5 17,5 17,5 17,5 concentration (mM) (e) (f)

FigureFigure 4. 4. TheThe kinetics kinetics of of DNA DNA replication replication in invivo vivo in inthe the presence presence of following of following CCM CCM metabolites: metabolites: (a) acetate,(a) acetate, (b) (succinate,b) succinate, (c) (fumarate,c) fumarate, (d) ( da)‐ketoglutarate, a-ketoglutarate, (e) ( emalate,) malate, (f ()f lactate.) lactate. The The wild wild type type (empty (empty

columns)columns) and and dnaB8 (filled(filled columns) bacteriabacteria were were cultured cultured at at 41 41◦ C.°C. The The DNA DNA synthesis synthesis was was assessed assessed at attime-points time‐points as indicated.as indicated. The The results results are from are atfrom least at three least repetitions three repetitions of experiments of experiments with SD indicated.with SD indicated. 4. Discussion 4. DiscussionDirect links between the regulation of DNA replication and CCM have been reported in various organisms,Direct links from between bacteria [ the3,5, 17regulation] to humans of DNA [7–9]. replication This implies and that CCM the modulationhave been reported of DNA in replication various organisms,control by centralfrom bacteria carbon [3,5,17] metabolism to humans occurs commonly[7–9]. This inimplies both prokaryotes that the modulation and eukaryotes of DNA [6]. replicationMediated by control CCM, itby appears central that carbon regulation metabolism of DNA occurs replication commonly might bein ofboth particular prokaryotes importance and for all organisms, and defects in the CCM-replication regulation link may lead to serious problems in cell physiology, including eukaryotic cell transformation and formation of cancer cells [11]. On the Genes 2020, 11, 447 11 of 13 other hand, molecular mechanisms of this link are largely unknown. Whether DNA replication regulation by CCM is based on protein-protein interactions or signals mediated by metabolites remains an unsolved question. Only recently, some insights into understanding detailed mechanisms of the CCM-replication link in bacterial cells have been published. In B. subtilis, multiple links appear to connect CCM to DNA replication initiation and elongation [4]. It was suggested that changes in CCM may affect functions and/or recruitment of the DNA helicase, primase, and DNA polymerase at the replication origin, thus modulating the initiation stage, while the elongation process could be influenced due to changes in DNA polymerase operating at the replication fork. Although several CCM reactions have been suggested to influence DNA replication machinery, providing signals involved in the control of DNA synthesis at different stages [4], detailed signal transduction mechanisms remain unknown. In E. coli, it was demonstrated that temperature sensitivity of dnaA46 mutant could be suppressed by defects in the acetate overflow pathway [18]. Such suppression is correlated with accumulation of pyruvate due to dysfunction of pyruvate dehydrogenase activity. In fact, double mutants, revealing thermosensitivity of DnaA initiator protein and lacking activity of pyruvate dehydrogenase, could grow at elevated temperature as efficiently, as wild-type strains [18]. Taking into consideration the above mentioned results, in this work, we tested the effects of various CCM metabolites (pyruvate, acetate, α-ketoglutarate, lactate, malate, fumarate, succinate) on temperature-sensitive phenotype of several E. coli mutants in replication genes including dnaA, dnaB, dnaC, dnaG, dnaE, and dnaN, coding for the replication initiator protein, DNA helicase, DnaA-DnaB linker, DNA primase, subunit of DNA polymerase III, and sliding clamp of DNA polymerase III, respectively. We found that various metabolites can suppress the growth defects of temperature-sensitive mutants at semi-restrictive temperatures. This suppression was observed as both an improvement in the efficiency of plating (Figure1) and reduction of cell filamentation (Figures2 and3). In this work, we not only confirmed previous observation that excess of pyruvate results in suppression of the dnaA46 mutation [18], but also extended this phenomenon to other metabolites and other mutations. Regarding dnaA46, thermosensitivity of bacteria bearing this mutation could be suppressed by mutations in either pta or ackA genes, coding for phosphate acetyltransferase and acetate kinase, respectively (see Table1). Dysfunctions of these genes may cause accumulation of acetate in cell, either indirectly or directly, respectively. Hence, suppression of effects of the dnaA46 mutation by excess of not only pyruvate (as reported previously, [18]) but also acetate (Figures1 and3) is compatible with previous observations [ 5], as well as with the hypothesis that the suppression in mediated by metabolites. Dysfunctions of genes coding for 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, glucose-6-phosphate isomerase, and transketolase 2, which were reported previously to suppress effects of temperature sensitivity of certain dnaB, dnaE, dnaG, and dnaN mutations in different combinations [5], lead to accumulation of various metabolites as these enzymes operate at key stages of CCM. Therefore, the data indicating suppression of growth and cellular morphology defects by different metabolites (Figures1 and3), observed in corresponding E. coli replication mutants, are also compatible with previous results [5] and they can support the above presented hypothesis. As discussed above, our data suggests that in E. coli, the CCM-replication link, is mediated by small metabolites rather than by direct protein-protein interactions. On the other hand, the excess of metabolites could not suppress defects in the direct DNA synthesis of the mutants, at least in short-term experiments (up to 60 min) (Figure4). Therefore, the e ffects of metabolites on DNA replication appear to be indirect. The pattern of suppression effects also corroborates this conclusion. The mutants that were most responsive to metabolites included dnaA46 and dnaE486, which harbor defects in the main proteins involved in DNA replication initiation and elongation, respectively, and are subject to extensive regulatory impacts. The least responsive mutant was dnaN159, bearing a change in the clamp of DNA polymerase III, which is an essential protein for DNA replication, but appears rather uninvolved in extensive control mechanisms. Genes 2020, 11, 447 12 of 13

In summary, we demonstrated that various CCM metabolites suppress the effects of mutations in different replication genes of E. coli, providing evidence that the CCM-replication regulation link is mediated by small metabolites rather than direct protein-protein interactions. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation [4,19]. Therefore, one may suggest that molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important common feature of the control of cell physiology in bacteria. Since CCM-DNA-replication link has been also reported in human cells [7–9], we speculate that this regulatory mechanism might reflect a general biological adaptation, evolutionarily conserved from bacteria to humans.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/4/447/s1, Figure S1: The kinetics of DNA replication in vivo in the presence of CCM metabolites. The wild type (empty columns) and dna(ts) (as indicated, filled columns) bacteria were cultured at the semi-restrictive temperatures in the presence of indicated metabolites at various concentrations. DNA synthesis was assessed by measurement of [3H]thymidine incorporation at time-points as indicated. The results are from at least three repetitions of experiments with SD indicated. Author Contributions: Conceptualization, A.S.-P., G.M. and G.W.; methodology, K.K., M.M.-D., A.W., L.G., A.S.-P. and G.W.; validation, K.K., and M.M.-D.; formal analysis, A.S.-P., G.M. and G.W.; investigation, K.K., M.M.-D., A.W., L.G, J.M.-O.; resources, K.K., M.M.-D., A.W., L.G., A.S.-P. and G.W.; writing—original draft preparation, G.W..; writing—review and editing, K.K., M.M.-D., A.W., L.G., E.R., P.B., A.S.-P., G.M. and G.W.; visualization, K.K., M.M.-D., A.W., E.R., P.B.; supervision, A.S.-P., G.M. and G.W.; project administration, G.W.; funding acquisition, M.M.-D., G.M. and G.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Science Center (Poland), project grants 2012/04/M/NZ2/00122 (to G.W., in collaboration with G.M.) and 2016/23/D/NZ1/02601 (to M.M-D.). Acknowledgments: The authors thank Joanna Bart, Agnieszka Kołodziejska and Barbara Wojnowska for support in project administration. Conflicts of Interest: The authors declare no conflicts of interest.

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