Metabolic and genetic basis for auxotrophies in Gram-negative species

Yara Seifa,1 , Kumari Sonal Choudharya,1 , Ying Hefnera, Amitesh Ananda , Laurence Yanga,b , and Bernhard O. Palssona,c,2

aSystems Biology Research Group, Department of Bioengineering, University of California San Diego, CA 92122; bDepartment of Chemical Engineering, Queen’s University, Kingston, ON K7L 3N6, Canada; and cNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Lyngby, Denmark

Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved February 5, 2020 (received for review June 18, 2019)

Auxotrophies constrain the interactions of bacteria with their exist in most free-living microorganisms, indicating that they rely environment, but are often difficult to identify. Here, we develop on cross-feeding (25). However, it has been demonstrated that an algorithm (AuxoFind) using genome-scale metabolic recon- amino acid auxotrophies are predicted incorrectly as a result struction to predict auxotrophies and apply it to a series of the insufficient number of known gene paralogs (26). Addi- of available genome sequences of over 1,300 Gram-negative tionally, these methods rely on the identification of pathway strains. We identify 54 auxotrophs, along with the corre- completeness, with a 50% cutoff used to determine auxotrophy sponding metabolic and genetic basis, using a pangenome (25). A mechanistic approach is expected to be more appropriate approach, and highlight auxotrophies conferring a fitness advan- and can be achieved using genome-scale models of metabolism tage in vivo. We show that the metabolic basis of auxotro- (GEMs). For example, requirements can arise by means of a sin- phy is species-dependent and varies with 1) pathway structure, gle deleterious mutation in a conditionally essential gene (CEG), 2) enzyme promiscuity, and 3) network redundancy. Various levels or as a result of a combination of deletions, in which case they of complexity constitute the genetic basis, including 1) delete- would escape detection via comparative genomics. Given the rious single-nucleotide polymorphisms (SNPs), in-frame indels, high interconnectedness of metabolic networks, finding such sets and deletions; 2) single/multigene deletion; and 3) movement of manually is not a trivial task but can be efficiently approached mobile genetic elements (including prophages) combined with computationally using GEMs. genomic rearrangements. Fourteen out of 19 predictions agree GEMs are assembled based on genome annotation and cura- with experimental evidence, with the remaining cases high- tion of published literature (27, 28). They contain the most up-to- lighting shortcomings of sequencing, assembly, annotation, and date metabolic networks linking reactions with genes according reconstruction that prevent predictions of auxotrophies. We thus to experimentally validated mechanisms (28–30). Once they are develop a framework to identify the metabolic and genetic basis converted into a mathematical format (27, 31), flux balance anal- for auxotrophies in Gram-negatives. ysis (32) can be used to identify essential genes and auxotrophies that result from gaps in the network (15, 31, 33, 34). However, systems biology | mathematical modeling | auxotrophy | pangenome | a workflow for this purpose has yet to be formalized into a comparative genomics Significance ost–pathogen interactions and pathogen–microbiota inter- Hactions are dictated by the availability of nutrients as well Nutrient requirements play an important role in host–microbe as the metabolic capability of each participant to transform interactions, and can heavily affect the composition of micro- the nutrients into metabolic energy and biomass components. bial communities by setting hard constraints on microbe– Many bacterial strains (both commensal and pathogenic) lose microbe interactions. The auxotrophic capabilities of strains the capability to synthesize essential biomass precursors and and their underlying genetic and metabolic basis have rarely become dependent on extracellular resources for survival despite been studied on a large scale. This paper presents a compu- having prototrophic ancestors. Some auxotrophies arise as a tational approach using genome-scale models of metabolism result of the strain’s adaptation to a specific host or environ- and genomic sequences to predict auxotrophies in over 1,300 ment through the formation of small-scale deleterious mutations Gram-negative strains. Our network-based approach identi- leading to gene loss (1). For example, a methionine require- fies auxotrophs, their nutrient requirements, and the corre- ment is common among Pseudomonas aeruginosa strains isolated sponding causal genetic and metabolic basis, making it one of from cystic fibrosis patients (2–4), a requirement likely satisfied the most comprehensive efforts in large-scale strain-specific by the high concentration of amino acids in the patient’s spu- auxotrophy prediction. tum (5). Nutrient obligate pathogens are often host-associated (6), have a reduced genome (7), and retain a fitness advan- Author contributions: Y.S. and B.O.P. designed research; Y.S., K.S.C., Y.H., and A.A. per- tage over the free-living bacteria in their specific niche (8). formed research; Y.S., Y.H., A.A., L.Y., and B.O.P. contributed new reagents/analytic tools; Additionally, auxotrophs dictate the carbon and energy flow as Y.S. and K.S.C. analyzed data; and Y.S. and K.S.C. wrote the paper.y well as the stability of endosymbiotic communities (9). Aux- The authors declare no competing interest.y otrophies have been exploited 1) as markers for strain detec- This article is a PNAS Direct Submission.y tion and identification (10–12), 2) for the elucidation of their This open access article is distributed under Creative Commons Attribution-NonCommercial- lifestyle and microenvironment (13–17), 3) for the design of NoDerivatives License 4.0 (CC BY-NC-ND).y microbial ecosystems (18, 19), 4) for the design of attenuated Data deposition: AuxoFind is available on GitHub, with an example notebook. live vaccines (20, 21), and 5) for molecular therapy and tumor https://github.com/yseif/AuxoFind. All genomic sequences analyzed in this study are targeting (22–24). publicly available on The Pathosystems Resource Integration Center (PATRIC).y The identification of an auxotroph’s nutrient requirements 1 Y.S. and K.S.C. contributed equally to this work.y experimentally is difficult, occurs on a strain-by-strain basis, and 2 To whom correspondence may be addressed. Email: [email protected] is rarely accompanied with an identified causative genetic or This article contains supporting information online at https://www.pnas.org/lookup/suppl/ genomic lesion. Comparative genomic analyses suggest that dele- doi:10.1073/pnas.1910499117/-/DCSupplemental.y terious disruption of biomass precursor biosynthetic pathways First published March 4, 2020.

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AuxoFind() (CEGs) Comparative genomics presence/absence ihsm tan aigmlil pcfi n/rnonspecific and/or specific specific, ( were multiple auxotrophies requirements having predicted predicted strains of some 149) of with out (107 in acceptor 72% electron tetrathionate respiratory and a (43), vide system immune the (n pathogen by the detection and its (n host and niacin the which (42), over compete resource a Similarly, critical pathogenic- constitutes 41). from 2) (40, a both activation isolated affecting play immune and were interactions, BCAAs ity which host–pathogen of of in levels 5 role Intracellular strains, samples. 11 human across branched shared for were auxotrophies (BCAAs: acids specific amino chain example, For host–pathogen these interactions. during be advantage that to selective suggesting give known may interactions, auxotrophies nutrients host–pathogen involved in par- isolates which important of across auxotrophies promiscuity predicted multiple of the were Interestingly, lat- few from enzymes. pathways, the resulting ticipating redundant In irreversible, multiple interconversion. are are and which there salvage subsystem, nucleotide ter via as biosynthesis), achieved pyrimidine well and be as purine can (including biosynthesis of intermediate routes structure nucleotide of multiple the contrast, irreversibility to the In due and steps. 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SYSTEMS BIOLOGY A B

Fig. 2. In silico predictions of metabolic nutrient requirements across multiple Gram-negative species. (A) Nutrients for which a specific auxotrophy was predicted in at least one strain. (B) Top 15 metabolites for which a nonspecific auxotrophy was predicted (see Dataset S2 for the full results). Nutrient requirements were predicted for a total of 54 strains across Escherichia coli, Salmonella, Klebsiella, and Yersinia, with amino acids auxotrophies appearing with the highest frequency.

specific L-lysine requirement due to the absence of argD across fore nutrient dependence) resulting in reduced fitness indicates 11 Yersinia pestis strains, a specific biotin requirement (due to an unfavorable environment and/or insufficient access to impor- the absence of either bioAB, fabH, or fabI) across 5 Escherichia tant metabolites. Nutrient dependence was predicted in both coli strains, multiple amino acid auxotrophies in Yersinia ruck- aerobic and anaerobic conditions for each transposon mutant eri strains, and an L-leucine requirement in 3 E. coli K-12 by AuxoFind, by knocking out the disrupted gene in silico and strains. To determine which strains were closely related, we simulating for auxotrophy (Datasets S3 and S4). TRADIS yields constructed phylogenetic trees from the nucleotide polymor- a fitness measure (log2 fold change) for each mutant, which phism (single-nucleotide polymorphism [SNP]) count from the we filtered for adjusted P value smaller than 0.05. A total of concatenation of all core genes using rapid core genome multi- 960 measured log2 fold changes passed these thresholds. We alignment (ParSNP) (45). While the Y. pestis L-lysine auxo- considered log2 fold changes in fitness smaller than −1 to be trophs were not clustered together in a single subclade, the L- detrimental and those larger than 1 to be beneficial. Only 25 leucine E. coli auxotrophs, Y. ruckeri amino acid auxotrophs, CEGs yielded increased fitness upon disruption in at least one and E. coli K-12 strains were (SI Appendix, Figs. S1 and S2). condition, while 70 were detrimental (in ≥1 conditions) (Fig. 3). Otherwise, predicted auxotrophs were generally spread across At the gene level, 5 out of 15 in E. coli and 6 out of 12 in S. the phylogenetic tree, with many subclades containing only one enterica of the CEGs whose disruption increased fitness were lost auxotrophic strain. in one or more natural isolates (highlighted in bold in Fig. 3; Datasets S3 and S4). Amino Acid and Vitamin Auxotrophies Confer a Fitness Advantage Of note, the disruption of two out of five and five out of six In Vivo. We next sought to identify the effect of metabolic CEGs in S. enterica and E. coli, respectively, yielded increased requirements on the fitness of auxotrophic strains in their native fitness in one condition but decreased fitness in another, sug- environment. We evaluated published fitness profiles for E. coli gesting that the conferred fitness advantage is niche-specific. strains UTI89 and EC958 and S. enterica subsp. enterica ser. For example, argH and frdD disruption in S. enterica were ben- Typhimurium str. SL1344 mutants across various in vivo and eficial in chicken intestine but detrimental in cattle intestine. in vitro environments (including cattle, pig and chicken intes- Additionally, fitness change upon CEG disruption varied across tine, mouse spleen, human serum, and bladder cell infection phases of infection. For example, the disruption of bioH in model) (46–51). Briefly, fitness profiles are derived through E. coli was advantageous in the intracellular bacterial commu- transposon-directed insertion-site sequencing (TRADIS), and nities (IBC) phase but detrimental in later phases (dispersal a fitness measure is calculated by comparing the number of and postdispersal phase). In contrast, leuA disruption yielded reads across mutants between the inocula and output samples. decreased fitness in the dispersal phase but increased fitness We posit that a strain with a disrupted CEG (as defined in in the reversal and postreversal phases. Some beneficial nutri- AuxoFind Predicts Auxotrophy from Genomic Sequences Using ent requirements carried over from one stage of infection to GEMs) has an increased fitness, that is, when the gene’s func- the next. For example, L-arginine and L-cysteine auxotrophic tion is dispensable. In this case, it is evident that the mutant’s mutants exhibited elevated fitness in three consecutive phases auxotrophy is at least partially compensated for by a favorable including IBC, dispersal, and postdispersal phases. In addi- nutritional background. Conversely, loss of function (and there- tion, a thiamin requirement was beneficial in four out of the

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L-tryptophan (frdD tetrathionate (leuB), (nadA), nicotinate for (hisBF auxotrophic histidine were fitness elevated with with cattle glutamate, of infection 4-aminobenzoyl intestinal biotin, and with mutants included L-isoleucine auxotrophic fitness Other infection. increased of phases full tested the five for S4 detrimental and iso- S3 a natural Datasets yields See across urinary knock-out conditions. CEG for lost other that proxy in dataset. are fitness indicates a bold on (*) as effect in asterisk designed highlighted An was lates. Genes model applied. infection. infection was were auxo- workflow tract profiles cell be TRADIS fitness to the bladder was The which predicted disrupted. in The fitness is been sources it mutant various has which from the which for obtained gene which nutrient the change, the in and both fold trophic condition out each list log2 we For one tested, screens. than mutant (more in fitness increased 3. Fig. biotin B hs Dispersal IBC phase Pyridoxine L-histidine eosre ae nwihtesmlaeu supplementa- simultaneous the which in cases observed We usto ee ofre ohseicadnonspecific and specific both conferred genes of subset A as species across diverges auxotrophies for basis metabolic The Salmonella L-leucine (pdxA) niacin tytpa isnhsscnb civdvatredifferent three via achieved be can biosynthesis L-tryptophan t.19wr isn h full the missing were 159 str. (bioH*) Cattle intestine Escherichia opttoal rdce Esta eefudt eutin result to found were that CEGs predicted Computationally ( nadA w in two Escherichia, L-arginine (argA orargG*) L-cysteine (cysIorcysN) (hisBF) ( leuB Salmonella glutamate (pabA*) 4-aminobenzoyl- ) ) tytpa rmcoimt (via chorismate from L-tryptophan and tytpa i both via L-tryptophan ), tetrathionate csen (cysC), L-cysteine L-valine, L-arginine (argBH*) Yersinia L-tryptophan (trpA) and L-cysteine Farm animalinfectionmodel Bladder cellinfectionmodel S. entericastrainSL1344 trpCDE and Salmonella E. colistrainUTI89 Post-dispersal biotin nyudraarbccniin) and conditions), anaerobic under only ( trpAB ( frdD* lsn,and L-lysine, u sntcniinlyesnilin essential conditionally not is but n nyoein one only and Salmonella, cysC Escherichia thiamin ( , O2-) bioA ofr oseicauxotro- nonspecific a confers ) uorpi mutants auxotrophic enterica, S. .coli E. ofr pcfi auxotrophy specific a confers ( ) agnn (argBH L-arginine thiG orthiF* u pcfi uorpyin auxotrophy specific a but trp tnaA L-isoleucine andL-valine glutamate (pabB) 4-aminobenzoyl- t.D and D6 str. tytpa auxotroph, L-tryptophan prn oee,strain However, operon. Reversal tan r aal of capable are strains luie(9.I an In (49). L-leucine L-tryptophan-specific and L-leucine L- Histidine(hisG) ) Escherichia L-lysine (lysA) BALB/c liver Pig intestine niacin trpAB P esnaalek- Yersinia ( value ( trpABCDE), Post-reversal leuA* nadC ), pathways. L-leucine ) ) Yersinia < strains ( ilvC 0.05) ) L- odfeetseis o xml,C2YadA1Vmuta- A111V to and to C128Y restricted to were restricted example, A). 5 restricted were For (Fig. were tions species. ( nadB). mutations D218N different deleterious and to C128Y, of A28V A111V, subsets and included Interestingly, (nadA) found SNPs P219L the and and rohdei, Yersinia and dysenteriae, variations species affected structural The acids 57). protein (56, amino were deleterious) in 10 be to result than assume validated we more to the which of of likely deletion one are and or least (which mutations at indel carrying these an strains for and/or 71 SNPs dataset of our total searched a found We niacin 55). a 43, in (14, of result strains A111V) natural and in requirement C297A, in C200A, and C113A, C128Y, G74E) and D218N, con- of proof case for dataset. in well-known our However, the auxotrophy for own. niacin analysis such of would its one to this demonstrate of we as level) cept, effort do events, gene we an pseudogenization the predict Here, constitute at to mutations. attempt lesions deleterious not limited genetic smaller-scale currently for of prediction is account identification the (which the for requirements workflow to nutrient our bacterial expand of strains, should host-adapted efforts in development future including auxotrophy strains study to in order observed been aeruginosa has loss-of- small-scale mutations auxotrophy through function adaptation niacin Niche predominant conserved. highly a and in patients fibrosis for reports cystic extensive across despite auxotrophy acid auxotrophic, amino be to predicted Auxotro- were the for of Basis none Genetic the in phies Constitute Mutations Small-Scale requirement shikimate 4D). the (Fig. pseudospecific making predicted, L-tyrosine, is for phenylalanine) requirement (including a nutrients supplementations, acceptable multiple excluded of is set the shikimate from If (aroD). dehydratase 3-dehydroquinate fergusonii Escherichia pre- for with was auxotrophy alterna- supplement dicted shikimate to the a example, was which For nutrient nutrients. in one multiple with instances supplementing observed to we tive Finally, 4C). isozyme (Fig. carbamoyltransferase acetylor- ornithine both and of deacetylase absence nithine the to due supplementation or L-arginine confer two to of predicted enterica absence example, was For isozymes) simultaneous auxotrophy. encoding the an (e.g., only genes which more in cases also ing dniyi eei tan of strains in here it in identify described initially was and and of absence biosyn- both the essential of In biosynthesis multiple the in for essential CEG (ilvC) is reductoisomerase a ketol-acid example, of For pathways. participation thetic pathways the different by across distributed or CEGs multiple of absence tan) hs eut eosrt htcnegn evolution convergent that demonstrate results These strains). either in deletions 16 observe aslls ffnto uain in mutations function of loss Causal Shigella .coli E. and enterica, S. pneumoniae, Klebsiella .Interestingly, 4B). (Fig. required is L-isoleucine ilvE hglaflexneri, Shigella .aeruginosa P. e.Nwotsr 3723wr rdce orequire to predicted were 0307-213 str. Newport ser. eels cossrisi utpeseisinclud- species multiple in strains across lost were Klebsiella and tan 3 4.Ised efudta Eswere CEGs that found we Instead, 14). (3, strains .WieteA1Vmutation A111V the While S5). (Dataset strains .enterica S. hglaboydii, Shigella .aeruginosa P. Shigella .dysenteriae S. ilvC PNAS nadA G5, .enterica, S. and t.AC349det h bec of absence the to due ATCC35469 str. eoa neiii tan ocrylarge carry to strains Enteritidis serovar ln,splmnaino both of supplementation alone, 5–4.Ti eutepaie ht in that, emphasizes result This (52–54). | lbilamichiganensis Klebsiella .flexneri S. xedn tt l tan in strains all to it extending Shigella, ac 7 2020 17, March Shigella or .enterica S. esnaenterocolitica, Yersinia or nadA .pneumoniae K. .aeruginosa P. nadB .flexneri S. tan,adA8 a restricted was A28V and strains, Shigella hglasonnei, Shigella .coli, E. . mn h pce studied, species the Among icuigW9X P219L, W299X, (including tan,D1NadP219L and D218N strains, o ohi h aeo 5 of case the in both (or eoa uln eonly we Dublin, serovar vln and L-valine | nadB pce nordataset our in species and Shigella, 5) diinly we Additionally, (55). o.117 vol. tytpa,and L-tryptophan, tan sltdfrom isolated strains hr were There coli. E. tanL0 and L201 strain icuigA28V, (including .coli, E. t.R1,and RC10, str. | and pestis, Y. L-isoleucine. o 11 no. .enterica S. lC ilvD, ilvC, L-valine Shigella | 6267 P. S. L-

SYSTEMS BIOLOGY A Yersinia Escherichia Salmonella Chorismate Chorismate Chorismate

159 trpDE D6 trpDE trpDE

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159 trpAB D6 trpAB trpAB

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BIOMASS BIOMASS BIOMASS BCMulti-species Klebsiella Cytoplasm Predicted L-threonine L-Glutamate argA Periplasm auxotroph Extracellular strain name ilvA pyruvate argB L201 Nonspecific 3-dehydroquinate ilvB and ilvN ilvB and ilvN argC L201 D or or RC10, G5 aroD ATCC35469 shikimate ilvH and ilvI ilvH and ilvI argD or astC MEM, L021 RC10, G5 aroE (or ydiB) ilvC ilvC SA20084644 argE L201 BL60006 aroK or aroL Ornithine Ornithine argI or argF MEM, L021 KP11 ilvD aroA and aroC ilvE BL60006 yzsuk-4 chorismate

argH L-phe L-valine ilvE MEM, L021 L-trp BL60006 L-arginine L-arginine BIOMASS L-tyr L-isoleucine BIOMASS BIOMASS BIOMASS Multi-species

Fig. 4. (A) Specificity of L-tryptophan requirement as a function of species-specific systems-level pathway structure. (B) Multiple simultaneous specific auxotrophies as a result of single gene deletion. (C) Auxotrophies as a result of deletion of multiple isozymes. In K. pneumoniae str. KP11, argI and argF are both absent. (D) Pseudospecific auxotrophies in which the alternative to one nutrient requirement (e.g., shikimate) is the simultaneous requirement for multiple nutrients (e.g., L-phenylalanine, L-tryptophan, and L-tyrosine).

may have led to loss of the nicotinate biosynthesis capabil- deletion events (Fig. 5C). For example, the deleted region in E. ity across species. That the deleterious mutations were main- coli strains DHB4, C3026, and DH10B consisted of 21 genes, tained across descendants indicates that a niacin requirement and the genes upstream and downstream of the deletion end- confers a selective advantage, an observation which is also sup- points in E. coli MC4100 (rapA and fruR) are adjacent in the ported in Amino Acid and Vitamin Auxotrophies Confer a Fitness three auxotrophs. Conversely, K. pneumoniae strain L201 is miss- Advantage In Vivo. ing a total of 246 contiguous ORFs with respect to strain L491. One deletion edge was located at position 4,943,680, marking Genomic Basis of Auxotrophy. Next, we proceeded to examine the the end of the chromosomal GenBank file, while the other was genomic basis of the auxotrophies predicted from our workflow. located at position 371, marking the start of the sequence for To assess the genetic changes at the strain level, we compared plasmid p1-L201. We suspect that an assembly error may explain the genomic region of each predicted auxotroph with a closely this observation. related strain (SI Appendix, SI Materials and Methods). We In the remaining instances, the genes which were located at observed multiple cases in which a missing CEG constituted a the edges of the missing fragment were separated by multiple part of a larger deleted genomic fragment, in which multiple ORFs in the auxotroph. Interestingly, these ORFs constituted syntenic operons were lost simultaneously. We found that the a prophage in four auxotrophs, suggesting that insertion of number of missing syntenic genes surrounding an absent CEG viral DNA may have mediated the deletion event (Fig. 5D). varied from n = 1 to n = 251 open reading frames (ORFs) aver- In particular, E. coli strain C321.∆A, which is a genomically aging 49 genes. S. enterica str. U288 was the only strain for which recoded organism lacking bioAB and ybhB, carried enterobac- the missing chromosomal region contained only one CEG (hisD) teria phage Λ [containing 15 genes, predicted by PHAge Search (Fig. 5B). Conversely, S. enterica str. 0112-791 was missing two Tool (PHAST) (58)] in the deletion locus. Similarly, E. coli strain genomic regions (n = 251 ORFs and n = 28 ORFs) and did not S50 (isolated from forest soil) carried a prophage sharing three carry a total of four CEGs. ORFs with phage Stx2 at the locus for 152 missing contiguous When the conserved genes flanking the missing genomic frag- genes. ment were adjacent in the auxotroph, we classified the observed A slightly more complex sequence of events affected S. enterica loss as a simple deletion. There were 15 cases of simple multigene ser. Newport strain 0211-109 which was isolated from a cow with

6268 | www.pnas.org/cgi/doi/10.1073/pnas.1910499117 Seif et al. Downloaded by guest on September 30, 2021 Downloaded by guest on September 30, 2021 asdb h netdcutro ee.Smlry 4gnsare genes 14 was Similarly, proA genes. rearrangement of in strain cluster genomic deleted inserted in the that elsewhere rel- by hypothesize relocated close caused a was We in but ORFs) 0211-109. 0112-791), 56 (with (strain 2 ative gifsy the prophage of (span- of region downstream upstream between deletion directly genes the Notably, ning DNA) cluster. transforming sequence insertion of binding internalization- in uptake DNA (involved and ComEC/Rec2 a element protein carried mobile competence also of related copies auxotroph three The mul- small and proteins). of transporters, copies three efflux beta-lactamase, tidrug C Class clus- of containing sequence copies ORFs, insertion two (18 resistance an beta-lactam found conferring likely we ter genes), 252 of (consisting S6 (Dataset gastroenteritis ( E deletion. in multigene hisD of phage-mediated deletion and (F gene insertion sequences. Single Phage insertion (B) and (D) acids). prophages edges. by amino deletion mediated (>30 recombination of deletions/insertions homologous rejoining in-frame with large as deletion well multigene as nadB and nadA 5. Fig. toh orsoddt pce o hc hr a nyone only was example, there For dataset. which our in for genome species representative to corresponded otrophs of bacterial downstream of rearrangement located (59). the genomes ORFs promote sequence can insertion 16 elements an Insertion of with apart, consisting ORFs (pepD cluster 2,251 region located deletion the are flanking ykfl) repeat genes multiple the In the carries HST04, occurred. have strain RR1 across may deletion, transposition that redistributed of denoting regions locus are the transposases) At genome. 3 (including ment efe al. et Seif B C AD Y. enterocolitica ial,w bevdfu ntne nwihtepeitdaux- predicted the which in instances four observed we Finally, MC4100

K. pneumoniae E. coli S. enterica S. dysenteriae Small scalemutations Multi-gene deletion Single genedeletion L201 L491 DH10B C3026 Y. aleksiciae DHB4 S. enterica S. enterica T000240 and Y. arohdei S. sonnei icnaxtoh u okonls-ffnto uain in mutations loss-of-function known to due auxotrophy Niacin (A) complexity. of levels various spans auxotrophy nutrient for basis genetic The S. boydii chromosome Y. pestis panK dgoT S. enterica U288 E. coli n ee ptemo h eee frag- deleted the of upstream genes 9 and proB, .coli E. rluA h h h dgoD iAdo insH dgoD birA rapA rapA rapA rapA tanR1( eiaieo -2,including K-12), of derivative (a RR1 strain No. strains ilvC pepN murB fruR fruR fruR leuD plasmid hisG hisG lysR mraZ birA .A h ou fdeletion of locus the At 5E). Fig. and and h h 221 bp SNP -A111V( SNP -P219L( SNP -D218N( SNP -C128Y( SNP -A28V( Insertion ( Insertion Insertion ( Insertion Deletion ( Deletion ( 1404 bp leuC hisD ilvA panK mraZ mraZ a oae immediately located was potA) hisC n =21 leuB n hisC ilvD =246 nadB nadA nadB nadA nadB nadA nadA nadA nadB leuA ) ) ilvE ) ) ) ) ) ) )

argB E. coli E. coli S. enterica E. coli 0112-791 0211-109 C321.deltA E ER3440 NEB 5α BW25113 esnafred- Yersinia HST04 fruR argC RR1 IAI1 Combined deletionandhomologousrecombination S50 Prophage inductionmediateddeletion .coli E. pepD. h mmuP mmuP rpsA rpsA rHpp p frsA gpt pepD prfH prfH and ybhC ybhC ftsH ftsH eDpf rtcB prfH pepD ihfB ihfB h h ybhB eriksenii aae,adfudta oa f4 tan eludraprob- (includ- our a auxotrophs in under predicted fell value were strains six each 41 those, to ing of Of a equal total 5%. of or of a ability that than probability found the less the and be using dataset, calculated to col- distribution We length we value distri- genome approach. genus, observed extreme the maxima each generalized fit For block a and genus. to length same of sequence bution the strains’ result the of a lected strains respect predicted as with other the length sequence timescale to whether genomic reduced evolutionary asked a had We losses small auxotrophs (60). gene a bottlenecks massive on population and symbiosis, occur toward can evolution and tion these of history evolutionary the regions. retrace chromosomal to necessary speciation be after would occurring events 5 evolutionary (Fig. of are cases result these a in CEGs likely of protein absence The hypothetical biosynthesis). isoleucine a carries families) kristensenii Yersinia oee,aFse’ xc etrvasta hr sn signifi- no strains is of (P adaptation. population there genomes niche the reduced that among with of reveals auxotrophs of test result enrichment exact strains cant a Fisher’s these as a that However, auxotrophy hypothesis the developed supporting have further 9-65), G5, str. K. yzusk-4, and iegn n nin vltoaytaetr cosspecies. across trajectory evolutionary ancient and Divergent ) insH insH dgoD comEC eoesralnn sotnascae ihnceadapta- niche with associated often is streamlining Genome Phage genome(n=15) Phage genome(n=25) rec2 pyrC .michiganensis K. bioA dgoT Phage gifsy 2(n=56) F K. pneumoniae K. Y. frederiksen pepD F ,adalre ubro etnn eoi sequences genomic pertinent of number larger a and ), n =152 carries proA bioB n =22 Y. kristensenii crl blaC prfH proB MGE bioF n =16ORFs pdxT n =18ORFs rtcB FDAARGOS_417 PNAS phoE KP11 wihsae h ags ubro gene of number largest the shares (which tanRC10, strain .enterica S. fabH prfH ATCC33639 and pepN crl | yafP K. michiganensis K. n =2,251genes setA n =334 ac 7 2020 17, March bioF au .) niaigta genome that indicating 0.6), = value potB MGE Divergent evolutionarytrajectory gadCB pepD pepT pyrD yafO phoE lysR .enterica S. potB rtcB t.01-9,and 0112-791, str. utgn eeinculdwith coupled deletion Multigene ) h yafN between yjjG yjjG .pneumoniae K. rBproA proB aroD yafP pyrC RC10 | pdxT ivlN tanT020 (C T000240. strain ydiM ilvNB o.117 vol. yafO yjjG K. G5 K. gadC ivlB pabC yafN aroE Deleted CEG Deleted gene Repeat region Insertion element setA h ivle in (involved and | gadB tan KP11 strains o 11 no. h setA fadH ykfl ykfl pepT .enterica S. h while lpp, lpp lpp Simple ) leuD leuD yafW yafW leuD | h 6269 L-

SYSTEMS BIOLOGY streamlining is not a predominant phenomenon across the six isoleucine, and L-arginine requirement in Y. ruckeri strains YRB genera. and NHV 3758. In addition, we found literature evidence for a biotin requirement in E. coli strain C321.∆A and its two Experimental Validation of Auxotrophies Highlight Technological genomically recoded derivatives (CP006698.1, CP010455.1, and Shortcomings at Multiple Levels. We proceeded to validate our CP010456.1) (71). Three predicted auxotrophs could grow nei- predictions experimentally by evaluating growth requirements ther in minimal medium nor in minimal medium supplemented of the predicted auxotrophs. We were able to obtain six E. with the corresponding predicted nutrient. For example, E. coli (61–64), three Yersinia (65), three Salmonella (66, 67), coli strain RR1 is a predicted L-proline auxotroph, S. dysen- three Shigella (68, 69), and one Klebsiella (70) strain (Fig. 6). teriae strain BU53M1 is a predicted niacin auxotroph, and Y. Out of 16 strains that we experimentally tested, 8 (4 E. coli, aleksiciae strain 159 was predicted to have multiple auxotro- 2 Y. ruckeri, 1 S. flexneri, and 1 S. sonnei) grew only when phies. However, neither exhibited any growth upon supple- glucose + M9 media was supplemented with the predicted mentation, suggesting that they may have additional nutrient essential nutrient(s). The confirmed auxotrophies included 1) requirements. an L-proline requirement in E. coli strain HST04; 2) an L- Notably, we tested growth of two Y. ruckeri strains on a leucine requirement in E. coli strains DHB4 and DH10B; 3) reduced chemically defined medium. While there is no precedent a niacin requirement in E. coli strain SF-173, S. flexneri strain for such an effort for this species, a chemically defined medium 2457T, and S. sonnei strain 2015C-3794; and 4) an L-valine, L- was nonetheless derived for multiple clinical Y. enterocolitica

Missing Observatio Follow-up Genetic basis Strains Essential nutrients gene/SNPs n results E. coli strain SF-173 nadB (A28V) Niacin √ - S. flexneri 2a str. nadA Niacin √ - 2457T (A111V) nadA Pseudogenes S. sonnei 2015C- (Deletion (bp Niacin √ - 3794 = 31)) S. dysenteriae strain nadA Additional Niacin √* BU53M1 (P219L) auxotrophies Gene found K. pneumoniae Arginine AND argI,purA × through PCR strain KP11 Adenine Single gene primer loss Y. ruckeri strain argG Arginine √ - NHV_3758 Y. ruckeri YRB argG Arginine √ - Gene found K. pneumoniae pyrI,pyrB Orotate C5H3N2O4 × through PCR strain KP11 primer Y. ruckeri strain ilvB, L-Valine AND L- √ - NHV_3758 YPO4089 Isoleucine ilvB, L-Valine AND L- Y. ruckeri YRB √ - YPO4089 Isoleucine Cytosine AND Partial Cytidine AND operon loss Y. aleksiciae strain Deoxyinosine AND Additional pyrF, prsA √* 159 L-Histidine AND auxotrophies NMN AND L- Tryptophan S. enterica serovar Gene found Enteritidis str. thrB L-Threonine × through PCR EC20100101 primer S. enterica serovar Gene found Enteritidis str. nadA Niacin × through PCR SA20094177 primer E. coli K-12 strain K- leuACD L-Leucine × Unknown 12 C3026 E. coli K-12 strain K- leuACD L-Leucine √ - 12 DHB4 E. coli str. K-12 leuACD L-Leucine √ - substr. DH10B E. coli strain HST04 proAB L-proline √ - Additional E. coli strain RR1 proAB L-proline √* auxotrophies - Full operon Uridine AND L- loss Valine AND L- trpA,trpB,trp Isoleucine AND L- C, Y. aleksiciae strain Tryptophan AND Additional trpD,trpE,ilvB √* 159 NMN AND auxotrophies , Guanosine AND L- ilvN Histidine AND 2',3'- Cyclic CMP S. enterica serovar Hypoxanthine AND Gene found Bovismorbificans str. purDH Adenosine AND L- × through blastn 3114 Histidine

Fig. 6. Results of in-house experimental validations for nutrient requirements across 16 Gram-negative strains and outcome of follow-up experiments and analysis for failure cases. Growth curves, PCR primers, and details regarding the list of strains can be found in SI Appendix. An asterisk (*) denotes that these strains couldn’t grow upon predicted nutrient supplementation, suggesting that they have additional nutrient requirements. Additionally, we found literature evidence for a biotin requirement in E. coli strain C321.∆A and its two genomically recoded derivatives. Note that one strain may have multiple genetic basis of auxotrophy, for example, Yersinia strains. (71).

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L- L- L- iessesbooyefr ie tpeitn n under- of and models predicting mechanistic at using aimed auxotrophies effort nutrient biology standing systems sive for hypotheses obser- (76). testable studies experimental generate follow-up and contradictions predictions These incon- silico mutations vations. of in function sources between of additional sistency constituted loss strains, in-frame technological three of unknown affecting biosynthesis of these acid positives. presence amino overcome false the related in and to gaps true closely between Knowledge used of distinguish that level) and be assem- shortcomings observed ORF can We genome the annotation. incorrect prototrophs (at genome 2) alignment erroneous genome, pangenome 3) the sequenc- and uneven is across bly, 1) genes by quality sequences, deletions/missing ing became complete of in sequence call- identification even genomic hampered, behind the a challenges and from apparent, technological The absent highlight levels. genes/functions of to ing multiple growth served strains at support latter could shortcomings The media were mutants. strains minimal 5 11 that that observed but and auxotrophs, strains 16 in social requirements ent bacterial highly full and and and permanent space deletion more colonization gene be network. strain’s (75). to full the likely SNPs their as constrain are as when such could removal such conditions They events operon variations right dataset. major small our the However, constitutes in under basis (3.8%) reversed genetic rare (one be are deletions perhaps 9-65. ORFs) large-scale strain more from A both or arising Paratyphi for serovar auxotrophies case niche and Overall, the 0112-791 are strain indeed they had Newport beta-lactam is that auxotrophs conferring this predicted suggesting genes 54 adapted; genomes, the of smaller of cluster 6 significantly due a particular, CEGs In of four move- losing resistance. insertion strain sequence the one accompa- insertion with to and/or genome, and/or mediated the anal- insertion across likely pangenome ment prophage was a CEGs by in of nied loss alleles the of which number of single largest ysis a the of have case affected to event only multigene deletion the large gene homologous Interestingly, to extensive events. mutation with recombination coupled function deletions of polymor- multioperon loss nucleotide and single a from causing ranging phism auxotrophy, for basis of function a as reactome. pathway full same strain’s the a in vary participating and strains CEGs loss across across differently to paralogs leading that affect may suggest development function therefore of path- auxotrophy We CEG. for alternative the pressures of of selective that position to the respect with on ways depending deletion, specificity upon simulated different the confer in pathways participating biosynthetic CEGs aux- same two nonspecific Additionally, a other. but the species in can one otrophy fashion. strains in different auxotrophy strain-specific two specific in a a function confer same in the out varied carrying pathway CEGs or therefore functional 3) and and activity, redundancy, enzymatic promiscu- protein’s the 2) a pathway, of metabolic ity the of structure entire on the depends 1) auxotrophies of multiplicity) and ficity/nonspecificity development. auxotrophy in nutritional role context-specific a plays the likely that background suggest availability and nutritional niches in between differences reflect variations hypothesize bladder these We stages. that of across varies biotin) stages and benefits (L-leucine multiple others fitness as over the (such carries infection while auxotrophies addition, some In of another. in detrimental loehr u eut osiuetems comprehen- most the constitute results our Altogether, nutri- for predictions our verified experimentally we Finally, genetic the of complexity the in continuity a observed We speci- (including basis metabolic the that found We .coli E. tan 3) hr eemlil ntne in instances multiple were There (36). strains PNAS L-arginine, | ac 7 2020 17, March eewihwsobserved was which gene a hisD, csen,adtimn,ta of that thiamin), and L-cysteine, | o.117 vol. .enterica S. and ruckeri , Y. | o 11 no. serovar | 6271

SYSTEMS BIOLOGY metabolism. The approach developed can be applied to quickly Data Availability. All data generated in this study are included in this pub- and systematically predict nutrient requirements from genomic lished article (and in SI Appendix and Datasets S1–S6). AuxoFind is available sequences. on GitHub, with an example notebook. All genomic sequences analyzed in this study are publicly available on PATRIC (38), and accession numbers are Materials and Methods available in Dataset S1. The specific procedure of data collection and quality control, prediction of CEGs,andhomologousgeneidentificationisdescribedin SI Appendix, ACKNOWLEDGMENTS. We thank Dr. Karsten Zengler and Marc Abrams for reviewing the manuscript and providing constructive suggestions. This work SI Materials and Methods. The detailed workflow for prediction of nutrient was supported by NIH Grants AI124316 and GM057089, and Novo Nordisk auxotrophy is described in SI Appendix, SI Materials and Methods. Deter- Foundation Grant NNF10CC1016517. We are grateful to Drs. Rebecca mination of gene neighborhood and synteny is described in SI Appendix, SI Lindsey, Nancy Strockbine, Shi Chen, Sang Jun Lee, Dana Boyd, Mehmet Materials and Methods. Sixteen strains were tested as a part of this study. Berkmen, Henning Sørum, David Rozak, Shannon Lyn Johnson, Craig The experimental validation methods and conditions are described in SI Winstanely, Roger Johnson, and Weihua Huang for generously providing Appendix, SI Materials and Methods. bacterial strains for this study.

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