The Reacquisition of Biotin Prototrophy in Saccharomyces ......in Escherichia coli and Bacillus sp....

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.074963

The Reacquisition of Biotin Prototrophy in Saccharomyces cerevisiaeInvolved Horizontal Gene Transfer, Gene Duplication

and Gene Clustering

Charles Hall* and Fred S. Dietrich*,†,1

*Department of Molecular Genetics and Microbiology, Duke University Medical Center and †Institute for Genome Sciencesand Policy, Duke University, Durham, North Carolina 27710

Manuscript received April 25, 2007Accepted for publication October 16, 2007

ABSTRACT

The synthesis of biotin, a vitamin required for many carboxylation reactions, is a variable trait inSaccharomyces cerevisiae. Many S. cerevisiae strains, including common laboratory strains, contain only apartial biotin synthesis pathway. We here report the identification of the first step necessary for the biotinsynthesis pathway in S. cerevisiae. The biotin auxotroph strain S288c was able to grow on media lackingbiotin when BIO1 and the known biotin synthesis gene BIO6 were introduced together on a plasmidvector. BIO1 is a paralog of YJR154W, a gene of unknown function and adjacent to BIO6. The nature ofBIO1 illuminates the remarkable evolutionary history of the biotin biosynthesis pathway in S. cerevisiae.This pathway appears to have been lost in an ancestor of S. cerevisiae and subsequently rebuilt by acombination of horizontal gene transfer and gene duplication followed by neofunctionalization.Unusually, for S. cerevisiae, most of the genes required for biotin synthesis in S. cerevisiae are grouped in twosubtelomeric gene clusters. The BIO1–BIO6 functional cluster is an example of a cluster of genes of‘‘dispensable function,’’ one of the few categories of genes in S. cerevisiae that are positionally clustered.

BIOTIN (vitamin H) is an essential vitamin. InSaccharomyces cerevisiae it is required for lipid metab-

olism (Roggenkamp et al. 1980), leucine metabolism(Ohsugi and Imanishi 1985), and is a substrate of thebiotin protein ligase (BPL1) (Hoja et al. 1998). Biotin istaken up by VHT1, a plasma membrane biotin transporter(Stolz et al. 1999). Many prokaryotes are capable of syn-thesizing biotin, as are plants and some fungi. No animalis capable of synthesizing biotin. Some fungal species in-cluding Aspergillus sp. (Shchelokova and Vorob’eva

1982) are capable of synthesizing biotin, and some suchas Ashbya gossypii (Dietrich et al. 2004) lack the genesnecessary to synthesize biotin. Some fungal species thatare incapable of synthesizing biotin still have portionsof the biotin biosynthetic pathway and are capable of uti-lizing intermediates to synthesize biotin (Phalip et al.1999; Wu et al. 2005). In S. cerevisiae, many wild isolatescannot synthesize biotin but nevertheless retain the BIO2,BIO3, and BIO4 genes from this biosynthetic pathway.Other isolates of this species are biotin prototrophs.The steps in biotin biosynthesis have been determinedin Escherichia coli and Bacillus sp. (Streit and Entcheva

2003) (Figure 1). The first step of biotin biosynthesis,the formation of pimeloyl-CoA, is not conserved. Somespecies including E. coli, form pimeloyl-CoA from thecondensation of three molecules of malonyl-coenzymeA (CoA) by the action of the bioC–bioH complex (Ifuku

et al. 1994). Other species such as some species of Bacillusare capable of forming pimeloyl-CoA from pimelic acidwith a single gene (bioW) (Bower et al. 1995). The lastfour steps from pimeloyl-CoA to biotin are generallyconserved (Streit and Entcheva 2003).

In eukaryotes, less is known about the genes involvedin biotin formation than in bacteria. Most of what isknown about biotin biosynthesis in eukaryotes is derivedfrom work in Arabidopsis thaliana and S. cerevisiae (Zhang

et al. 1994; Phalip et al. 1999; Streit and Entcheva

2003; Pinon et al. 2005; Wu et al. 2005). In S. cerevisiae,three of the genes involved in biotin synthesis and in-termediate transport are found within a subtelomericcluster located on chromosome XIV (BIO3, BIO4, andBIO5) (Phalip et al. 1999). The final gene in the path-way, encoding biotin synthetase (BIO2), is found on chro-mosome VII (Zhang et al. 1994) (Figure 1). A screen forgenes of recent bacterial origin in S. cerevisiae and A.gossypii indicated, and subsequent phylogenetic analysisstrongly supports, that two of these three known genesfor biotin biosynthesis, BIO3 and BIO4, were acquired byhorizontal gene transfer (HGT) from bacteria (Hall

et al. 2005). Wu et al. (2005) identified and characterizedthe gene immediately upstream of the known S. cerevisiae

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. EF567080.

1Corresponding author: Department of Molecular Genetics and Microbi-ology and Institute of Genome Science and Policy, Duke UniversityMedical Center, 287 CARL Bldg., Box 3568 DUMC, Durham, NC 27710.E-mail: [email protected]

Genetics 177: 2293–2307 (December 2007)

biotin biosynthesis pathway. This gene, BIO6, was foundas a result of genomic sequencing of the sake-brewing,biotin prototrophic strain K7 of S. cerevisiae. This gene isa paralog of the horizontally transferred BIO3, but wasshown to have acquired a new function. On the basis ofanalysis of gene order, this duplication appears to havebeen independent of the genome duplication (Wolfe

and Shields 1997). BIO6 is necessary but not sufficientfor biotin production indicating the presence of one ormore additional genes. We have identified the open read-ing frame adjacent to BIO6 as the upstream step of biotinsynthesis in S. cerevisiae. This gene, which is now namedBIO1, is a paralog of the gene YJR154W and appears tohave acquired the function of pimeloyl-CoA synthetase(PCAS) and as such is a functional homolog of the bioWgene in Bacillus sp. and the bioC–bioH complex of E. coli.In S288c, there are two pseudogene copies each of BIO1and BIO6 located on chromosomes I and VIII given thesystematic names YAR069W-A (BIO6) and YHR214W-F(BIO8) and YAR070W-A (BIO1) and YHR214W-G (BIO7).

MATERIALS AND METHODS

Phylogenetic methods: Accession numbers for all sequen-ces used in this analysis can be found in supplemental Table 6at http://www.genetics.org/supplemental/. Gene sequencesand ribosomal small subunit ribosomal (SSU) DNA sequencesused in this analysis were acquired from GenBank (Benson

et al. 1999). Ribosomal SSU sequences were aligned by primarystructure using ClustalX (Thompson et al. 1997). Amino acidsequences for all biotin biosynthesis genes were aligned byprimary structure using ClustalX. Ambiguously aligned regions

were excluded from analysis. Estimates of phylogenetic re-latedness among species were determined using neighbor-joining (NJ) (Saitou and Nei 1987) analysis of SSU sequences.NJ trees were constructed in ClustalX using the IUB matrix. NJtrees were bootstrapped in ClustalX using 1000 replicates.Estimates of phylogenetic relatedness among biotin biosyn-thesis genes were determined using NJ analyses of proteinsequences. NJ trees were constructed in ClustalX using themethod of Saitou and Nei (1987) and the Gonnet matrix(Gonnet et al. 1992).

Yeast strains and plasmids: The S. cerevisiae strains used inthis study are listed in supplemental Table 7 at http://www.genetics.org/supplemental/. Strains SCCH015 (bio1THygB),SCCH016 (bio3THygB), and SCCH017 (bio6THygB) were cre-ated by the PCR-mediated gene disruption technique, usingthe hygromycin B resistance cassette from plasmid pGA32 andthe primer pairs BIO1KO1–BIO1KO2, BIO3KO1–BIO3KO2,and BIO6KO1–BIO6KO2, respectively (Wach et al. 1994; Lorenz

et al. 1995). All gene disruptions were confirmed by PCR andsequencing by standard methods using primer pairs BIO6PFX–HYGB and HYGC–PHO11R for BIO1, BIO3A–HYGB andHYGC–BIO3D for BIO3, and BIO6A–HYGB and HYGC–BIO6D for BIO6 (Mullis et al. 1986). Plasmids used in this studyare listed in supplemental Table 8 at http://www.genetics.org/supplemental/. Primer sequences used in this study arelisted in supplemental Table 9 at http://www.genetics.org/supplemental/.

To test whether BIO1 is necessary for biotin biosynthesisin S. cerevisiae, BIO1, BIO6, and both genes together were ex-pressed in S288c on derivatives of the 2 m plasmid YEplac195containing the BIO1, BIO6, and BIO6–BIO1 (with intergenicsequence) genes, respectively. BIO1, BIO6, and BIO6–BIO1 werePCR amplified from A364a genomic DNA using primers BIO1PF–BIO1PR, BIO6PF–BIO6PR, and BIO6PF–BIO1PR, respectively.PCR products were inserted by homologous plasmid gap repairin vivo (Lorenz et al. 1995). Plasmid PPH001 was digested withEcoRI. Linearized plasmid and PCR products were used to

Figure 1.—Diagram of the biotin biosynthesis pathway in S. cerevisiae. (A) Genomic position of the six known genes involved inbiotin biosynthesis and intermediate transport. The chromosomal positions of BIO1/BIO6 genes are based on strain S288C andrepresent duplicated regions on chromosomes I and VIII. (B) Position of the genes in the biotin biosynthesis pathway. The bio-chemical intermediates in this pathway are based on work in E. coli, Bacillus sp., and S. cerevisiae. The precursor of pimeloyl-CoA ismalonyl-CoA in E. coli and pimelic acid in Bacillus sp.

2294 C. Hall and F. S. Dietrich

transform S. cerevisiae strain BY4743. Cells were selected onminimal media lacking uracil (Burke et al. 2000). Plasmid in-serts were confirmed by PCR and sequencing using primersPTHQOUT1, PTHQOUT2, PTHQOUT3, PTHQIN1, PTHQIN2,and PTHQIN3.

To determine if YJR154W can complement a BIO1 deletion,BIO1 and YJR154W were PCR amplified and cloned into the2 m plasmid vector (p427-TEF) under the control of a TEF1promoter and introduced into the bio1D (SCCH015) deletionstrain. BIO1 and YJR154W were PCR amplified from A364agenomic DNA using primer pairs BIO1PF427–BIO1PR427and YJR154WPF427–YJR154WPR427, respectively. PCR prod-ucts were inserted by homologous plasmid gap repair in vivo(Lorenz et al. 1995). Plasmid p427-TEF was digested withEcoRI. Linearized plasmid and PCR products were used totransform S. cerevisiae strain SCCH015. Cells were selected onyeast peptone dextrose (YPD) with G418. PCR and sequencingusing primers TEFfw, CYCtREV, and CYCtREV2 confirmedplasmid inserts.

To determine if BIO3, BIO6, and the BIO3 homolog from K.lactis have overlapping function, BIO3, BIO6, and the BIO3homolog from K. lactis were PCR amplified and cloned intothe 2 m plasmid vector (p427-TEF) under the control of a TEF1promoter and introduced into the bio3D (SCCH016) andbio6D (SCCH017) deletion strains. BIO3, BIO6, and the BIO3homolog from K. lactis were PCR amplified from S. cerevisiaestrain A364a and K. lactis strain CBS 683 genomic DNA usingprimer pairs BIO3PF427–BIO3PR427, BIO6PF427–BIO6PR427,

and KLACBIOPF427–KLACBIOPR427, respectively. PCR pro-ducts were inserted by homologous plasmid gap repair in vivo(Lorenz et al. 1995). Plasmid p427-TEF was digested with EcoRI.Linearized plasmid and PCR products were used to transformS. cerevisiae strains SCCH016 and SCCH017. Cells were selectedon YPD with G418. PCR and sequencing using primers TEFfw,CYCtREV, and CYCtREV2 confirmed plasmid inserts.

Media and growth conditions: For general cultivation pur-poses all strains were grown on YPD broth and agar (Burke

et al. 2000). Selection for plasmids PPH001, PCH001, PCH002,and PCH003 was performed on synthetic complete minusuracil media (SC �Ura) (Burke et al. 2000). Selection forplasmids p427-TEF, PCH004, PCH005, PCH006, PCH007, andPCH008 was performed on YPD media with G418 (Burke et al.2000). Tests for biotin prototrophy were performed on biotin-free media composed of ammonium sulfate (15 mm), mono-potassium phosphate (6.6 mm), dipotassium phosphate (0.5 mm),sodium chloride (1.7 mm), calcium chloride (0.7 mm), mag-nesium chloride (2 mm), boric acid (0.5 mg/ml), copper chlo-ride (0.04 mg/ml), potassium iodide (0.1 mg/ml), zinc chloride(0.19 mg/ml), calcium pantothenate (2 mg/ml), thiamine(2 mg/ml), pyridoxine (2 mg/ml), inositol (20 mg/ml), and glu-cose (2%). It was found that agar contained too much contam-inating biotin to prevent background growth of auxotrophicstrains of S. cerevisiae. When plates were required, agarose wasused as a gelling agent to a final concentration of 2%. Forgrowth of strain A364a this media was supplemented withadenine sulfate (20 mg/liter), histidine (20 mg/liter), lysine

Figure 2.—Phylogeny of BIO1 from S. cerevisiae and related genes and pimeloyl-CoA synthetase genes from prokaryotes (left)and species phylogeny based on SSU rDNA (right). BIO1 (clade a) is a duplicate copy of YJR154W. The pimeloyl-CoA synthetaseactivity of the genes in species in boldface type has been experimentally verified (Streit and Entcheva 2003). Pimeloyl-CoAsynthetase activity appears to be highly divergent among the prokaryotes with some bacteria requiring the two-gene bioC–bioHcomplex (clades b and e) and others requiring a single gene such as bioW or bioZ (clades c and d). Trees were constructed byNJ in ClustalX. Numbers indicate bootstrap support for each node of 1000 replicates. Taxa marked with asterisks indicate putativephytanoyl-CoA dioxygenases.

BIO1 in Saccharomyces cerevisiae 2295

(30 mg/liter), and tyrosine (30 mg/liter). We found it nec-essary to titrate the level of biotin by transferring from two ormore plates of media lacking biotin to eliminate backgroundgrowth. For media containing biotin, biotin was added to afinal concentration of 2 mg/liter.

RESULTS

To identify the remaining gene(s) necessary for biotinbiosynthesis, crosses were carried out between the biotinproducing strains YJM627 and A364a and the biotinauxotroph S288c. Thirty tetrads from the YJM627–S288ccross yielded a complex segregation pattern of 4 4:0(1, �), 17 3:1 (1, �), and 9 2:2 (1, �) for biotin pro-duction. This pattern is consistent with two redundantand unlinked loci. Results from 25 tetrads of the A364a–S288c cross showed 2:2 segregation in all cases. S288clacks at least two biotin biosynthesis genes, one of whichis BIO6. The observed segregation pattern suggested a

single-gene trait, and indicated that the missing gene(s)was closely linked to BIO6. Analysis of the genome se-quences of the Saccharomyces sensu stricto species as wellas compiled data from the S. cerevisiae genome projectsongoing at the Sanger Centre (http://www.sanger.ac.uk/Teams/Team71/durbin/sgrp/) allowed constructionof a gene map of the region surrounding BIO6 (Figure1). Several genes neighbor BIO6, including members ofthe multigene families FLO1 (flocculation), PHO11 (acidphosphatase), and IMD1 (inosine monophosphate de-hydrogenase), as well as genes of unknown function.Examination of the map suggested that the most likelycandidate for ‘‘BIO1’’ was the gene immediately flankingBIO6. This gene is a paralog copy of YJR154W, a gene ofunknown function but with sequence similarity to genesthat bind phytanoyl-CoA, a molecule structurally similarto pimeloyl-CoA (Figure 2 and supplemental Figure 1 athttp://www.genetics.org/supplemental/), the likely prod-uct of the missing biotin biosynthesis gene. PCR and

Figure 4.—Growth of S. cerevisiae strains on media contain-ing 2 mg/liter biotin (A) and lacking biotin (B). Cells weregrown overnight in YPD, transferred to liquid minimal medialacking biotin, grown overnight, transferred a second time toliquid minimal media lacking biotin, and then spotted ontoplates. S288c is a biotin auxotroph. A364a is a biotin proto-troph. All plasmids were transformed into strain SCCH015(A364a derived, bio1THygB). 1vector indicates vector alone(plasmid p427-TEF), 1BIO1 includes BIO1 gene (plasmidPCH004), and 1YJR154W includes the YJR154W gene (plas-mid PCH005).

Figure 3.—Growth of S. cerevisiae strains on media contain-ing 2 mg/liter biotin (A) and lacking biotin (B). Cells weregrown overnight in YPD, transferred to liquid minimal medialacking biotin, grown overnight, transferred a second time toliquid minimal media lacking biotin, and then spotted ontoplates. S288c is a biotin auxotroph. YJM627 is a biotin proto-troph. 1vector indicates vector alone (plasmid PPH001),1BIO1 includes BIO1 gene (plasmid PCH001), 1BIO6 in-cludes BIO6 gene (plasmid PCH002), and 1BIO1BIO6 in-cludes both genes on a single plasmid (plasmid PCH003).


sequencing confirmed that BIO6 is flanked by this pu-tative BIO1 in S. cerevisiae strains A364a and YJM627.

To test whether BIO1 is necessary for biotin biosyn-thesis in S. cerevisiae, BIO1 and BIO6 were PCR amplifiedand cloned into a plasmid vector (YEplac195 derived),both individually under the control of an ADH1 pro-moter and together on the same plasmid with BIO6 underthe control of an ADH1 promoter and BIO1 under thecontrol of its native promoter. S288c strains with bothBIO1 and BIO6, but not with either individually or withthe vector control, became biotin prototrophs (Figure3). Growth on 5-FOA to select for loss of the plasmidresulted in loss of biotin prototrophy (data not shown).These results indicate that BIO1 and BIO6 are togethernecessary and sufficient for the conversion of S288c into

a biotin prototroph and suggests that BIO1 is the likelypimeloyl-CoA synthetase of S. cerevisiae.

To determine if BIO3 and BIO6 have overlapping func-tion, and if YJR154W can complement a BIO1 deletion,we constructed deletions of BIO1, BIO3, and BIO6 in thebiotin prototrophic strain A364a. BIO1, BIO3, and BIO6were disrupted by the replacement of the open readingframe with the hphMX4 (Goldstein and McCusker 1999)cassette by homologous recombination. All three dele-tions were confirmed by PCR and sequencing. All threedeletions resulted in a strain auxotrophic for biotin.

BIO1 and YJR154W were PCR amplified and clonedinto a plasmid vector (p427-TEF) under the control of aTEF1 promoter and introduced into the bio1D deletionstrain. The plasmid expressing BIO1 was able to com-

Figure 5.—Growth of S. cerevisiae strains on media contain-ing 2 mg/liter biotin (A) and lacking biotin (B). Cells weregrown overnight in YPD, transferred to liquid minimal medialacking biotin, grown overnight, transferred a second time toliquid minimal media lacking biotin, and then spotted ontoplates. S288c is a biotin auxotroph. A364a is a biotin proto-troph. All plasmids were transformed into strain SCCH016(A364a derived, bio3THygB). 1vector indicates vector alone(p427-TEF ), 1BIO3 and 1BIO3 K. lactis includes the BIO3gene from S. cerevisiae and K. lactis, respectively (plasmidsPCH006 and PCH007), and 1BIO6 includes the BIO6 gene(plasmid PCH008).

Figure 6.—Growth of S. cerevisiae strains on media contain-ing 2 mg/liter biotin (A) and lacking biotin (B). Cells weregrown overnight in YPD, transferred to liquid minimal medialacking biotin, grown overnight, transferred a second time toliquid minimal media lacking biotin, and then spotted ontoplates. S288c is a biotin auxotroph. A364a is a biotin proto-troph. All plasmids were transformed into strain SCCH017(A364a derived, bio6THygB). 1vector indicates vector alone(p427-TEF ), 1BIO3 and 1BIO3 K. lactis includes the BIO3gene from S. cerevisiae and K. lactis, respectively (plasmidsPCH006 and PCH007), and 1BIO6 includes the BIO6 gene(plasmid PCH008).


plement the bio1D strain, while the plasmid expressingYJR154W was not able to complement the bio1D strain(Figure 4). Similarly, BIO3, BIO6, and the BIO3/BIO6homolog from K. lactis were PCR amplified and clonedinto a plasmid vector (p427-TEF ) under the control of aTEF1 promoter and introduced into the bio3D and bio6D

deletion strains. The plasmids expressing BIO3 from S.cerevisiae and K. lactis were able to complement the bio3D

strain, while the plasmid expressing BIO6 did not com-plement (Figure 5). In the bio6D strain, the plasmids ex-pressing BIO3 from S. cerevisiae and K. lactis were unableto complement the bio6D strain, while the plasmid ex-pressing BIO6 was able to complement (Figure 6), con-firming the results reported by Wu et al. (2005).

DISCUSSION

A controversy arose following the seminal work ofPasteur (1860) as to the nutrient requirements of yeast.While some supported the observation of Pasteur thatfermentation could be obtained with a media of mineralsalts in the form of yeast ash, sucrose, ammonium tar-trate, and a pinhead-sized inoculum of levure (Duclaux

1864), others reported that this mixture was insufficientto allow growth (von Leibig 1869). Extensive work byWildiers (1901) suggested the existence of an essentialmicronutrient that is water soluble, insoluble in alcohol,absent from yeast ash, dialyzable, and while present inpeptone and beer wort, absent from urea, asparagine,and several other nitrogenous compounds. He namedthis compound ‘‘bios.’’ Efforts to purify this compoundfailed, and controversy continued over the nature of thiscompound and even its existence (Tanner 1924). Workby Farries and Bell (1930) showed that A. gossypii andtwo related species are unable to grow on asparagine as asole nitrogen source, while some other fungi includingFusarium fructigenum were capable of growth. Theyfound that a growth factor was necessary for growth ofA. gossypii, which they furthermore fractionated andextensively analyzed. Work by Buston and Pramanik

(1931a,b) showed that this factor necessary for growthof A. gossypii contained two components, one beinginositol, and the other being identified several yearslater by purification, crystallization, and compositionaldetermination as biotin (Kogl and Tonnis 1936).

In retrospect, much of the confusion over theexistence and nature of bios may have stemmed fromvariability of the organisms used—we now know thatsome S. cerevisiae strains are biotin auxotrophs and thata few S. cerevisiae strains and many other fungal speciesare prototrophic (Shchelokova and Vorob’eva 1982)(Figure 7 and Table 1). Furthermore, many S. cerevisiaeautotrophic strains can utilize not only biotin, but alsocertain intermediates in the biotin biosynthetic pathway(Phalip et al. 1999), and even a dilution of 4 3 10�11 ofcrystallized biotin is sufficient to allow yeast growth(Kogl et al. 1937). It is possible that either the strain

studied by Pasteur was a biotin prototroph or that hispinhead-sized inoculum transferred sufficient biotin toenable growth. It is also clear that A. gossypii was a for-tuitous subject for the analysis of biotin by Farries andBell. The species is not only vigorously growing and abiotin auxotroph, but, as genome sequencing has re-vealed (Dietrich et al. 2004), is lacking the biotin syn-thetic genes BIO2, BIO3, BIO4, and BIO5 found in biotinauxotrophic strains of S. cerevisiae and is thus unable toconvert biotin precursors to biotin.

The identification of biotin, its role as a cofactor, andthe resolution of its structure had a protracted history.The biotin synthesis pathway in S. cerevisiae has provendifficult to study largely because the evolution of the bio-tin biosynthesis pathway in S. cerevisiae has been complex.As we discuss in more detail below, the biotin biosyn-thesis pathway in S. cerevisiae has evolved by mechanismsof gene loss and gene gain by both HGT and gene du-plication, making the biotin synthesis pathway an interest-ing model of gene pathway evolution. Also, unusuallyfor S. cerevisiae, five of the six genes required for biotinsynthesis, including all of the horizontally derived andduplicated genes, are localized to two subtelomericclusters.

Gene clustering: The BIO1 and BIO6 genes areadjacent, as are the BIO3, BIO4, and BIO5 genes in S.

Figure 7.—Variability of biotin biosynthesis in hemiasco-mycete fungi. Strains of Candida albicans (2), K. lactis (2), S.cerevisiae (37), S. paradoxus (35), and S. kluyveri (2) were rep-lica plated from minimal media onto media lacking biotin.Some strains show no growth, some weak growth, and othersrobust growth. This experiment was repeated three times.Growth indicated is an average for all three experiments. Itis likely that growth differences are due to copy number differ-ences in BIO6 and BIO1 (Wu et al. 2005). Strains used andtheir relative growth on minimal media lacking biotin aresummarized in Table 1.


cerevisiae. We have examined the extent of clustering ofgenes of related function and found that clusters ofgenes of related function are unusual, and generally,there is very little clustering of genes of related function

in S. cerevisiae. For most pathways, genes are scatteredacross the genome. With the exception of five specificcategories of genes, discussed below, we identified only14 pairs of adjacent, functionally related genes. This

TABLE 2

Dispensable pathway genes that are clustered in S. cerevisiae

Cluster function Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6

Galactose utilization YBR018C (GAL7) YBR019C (GAL10) YBR020W (GAL1)Maltose utilization YBR297W (MAL33) YBR298C (MAL31) YBR299W (MAL32)Maltose utilization YGR288W (MAL13) YGR289C (MAL11) YGR292W (MAL12)Fructose uptake? YJR158W (HXT16) YJR159W (SOR1) FSY1, not in S288c

(fructose_symporter)Biotin synthesis YNR056C (BIO5) YNR057C (BIO4) YNR058W (BIO3)Biotin synthesis YAR069W (BIO6) YAR070W (BIO1)Aryl-sulfate

utilizationYOL164W (BDS1) YOL165W (AAD15)

Resistanceto arsenic

YPR199C (ARR1) YPR200C (ARR2) YPR201W (ARR3)

SAM utilization YPL273W (SAM4) YPL274W (SAM3)Vitamin B1/B6

metabolismYFL058W (THI5) YFL059W (SNZ3) YFL060C (SNO3)

Vitamin B6metabolism

YMR095C (SNO1) YMR096W (SNZ1)

Vitamin B1/B6metabolism

YNL332W (THI12) YNL333W (SNZ2) YNL334C (SNO2)

Use allantoinas nitrogen

YIR027C (DAL1) YIR028W (DAL4) YIR029W (DAL2) YIR030C(DCG1)

YIR031C(DAL7)

YIR032C(DAL3)

Iron uptake YOR381W (FRE3) YOR382W (FIT2) YOR383C (FIT3) YOR384W(FRE5)

The fructose symporter (GenBank no. AJ250992) is from Saccharomyces pastorianus, now considered a strain of S. cerevisiae, and islocated adjacent to SOR3. FRE3 and FRE5 are duplicated genes, as are FIT2 and FIT3.

TABLE 1

Strains used in Figure 8 and their relative growth on minimal media lacking biotin

Vigorous growth S. cerevisiae YJM627 (6), S. cerevisiae Y55 (8), S. cerevisiae SK1 (9), S. cerevisiae Y9 (31),S. cerevisiae Y12 (33), S. paradoxus T21.4 (46), S. paradoxus Y7 (47), S. paradoxus Y6.5 (48),S. paradoxus Q59.1 (50), S. paradoxus S36.7 (54), S. paradoxus Z1.1 (55), S. paradoxus Y9.6 (56),S. paradoxus Y8.1 (63), S. paradoxus YPS138 (66), S. paradoxus A4 (68), S. paradoxus A12 (69),S. paradoxus UFRJ50791 (70), S. paradoxus UFRJ50816 (71), S. paradoxus UWOPS91-917.1 (76),S. kluyveri PMY043 (77), S. kluyveri CBS3082 (78)

Moderate growth S. cerevisiae 378604X (15), S. cerevisiae UWOPS03-461.4 (27), S. cerevisiae UWOPS05-217.3 (28),S. cerevisiae UWOPS05-227.2 (29), S. cerevisiae K11 (30), S. cerevisiae DBVPG6044 (35),S. paradoxus N-17 (43), S. paradoxus Q62.5 (51), S. paradoxus Q89.8 (52), S. paradoxus Q95.3 (53),S. paradoxus Q79.4 (57), S. paradoxus Q96.8 (58), S. paradoxus Y8.5 (61), S. paradoxus Z1 (62)

Weak growth S. cerevisiae A364a (5), S. cerevisiae DBVPG1953 (32), S. paradoxus CBS432 (42),S. paradoxus CBS5829 (44), S. paradoxus DBVPG4650 (45), S. paradoxus KPN3828 (64),S. paradoxus KPN3829 (65)

No growth C. albicans SC5314 (1), C. albicans MMRL 2010 (2), K. lactis CBS 141 (3), K. lactis CBS 683 (4),S. cerevisiae S288c (7), S. cerevisiae W303 (10), S. cerevisiae YJM978 (11), S. cerevisiae YJM981 (12),S. cerevisiae YJM975 (13), S. cerevisiae 322124S (14), S. cerevisiae 273614X (16),S. cerevisiae DBVPG1788 (17), S. cerevisiae DBVPG1373 (18), S. cerevisiae YIIc17-E5 (19),S. cerevisiae DBVPGG040 (20), S. cerevisiae NCYC 361 (21), S. cerevisiae YPS606 (22),S. cerevisiae YPS128 (23), S. cerevisiae YS2 (24), S. cerevisiae YS4 (25), S. cerevisiae YS9 (26),S. cerevisiae NCYC 110 (34), S. cerevisiae DBVPG6765 (36), S. cerevisiae L-1374 (37),S. cerevisiae L-1528 (38), S. cerevisiae UWOPS07-2421 (39), S. cerevisiae DBVPG1106 (40),S. cerevisiae UWOPS83-787.3 (41), S. paradoxus Q32.3 (49), S. paradoxus L07 (59), S. paradoxus

Q31.4 (60), S. paradoxus DBVPG6304 (67), S. paradoxus N-43 (72), S. paradoxus N-44 (73),S. paradoxus N-45 (74), S. paradoxus IFO1804 (75)


TABLE 3

Functionally related pairs of adjacent genes in S. cerevisae

Adjacent inA. gossypii?

Adjacent inK. lactis? Gene SGD description line

Yes Yes YBR111W-A (SUS1) Protein involved in mRNA export coupled transcription activation; componentof the SAGA histone acetylase complex

YBR112C (CYC8) General transcriptional corepressor, acts together with Tup1p; also acts as partof a transcriptional coactivator complex that recruits the SWI/SNF andSAGA complexes to promoters

Yes Yes YDR402C (DIT2) N-formyltyrosine oxidase, sporulation-specific microsomal enzyme required forspore wall maturation, involved in the production of a soluble LL-dityrosine-containing precursor of the spore wall, homologous to cytochrome P-450s

YDR403W (DIT1) Sporulation-specific enzyme required for spore wall maturation, involved inthe production of a soluble LL-dityrosine-containing precursor of the sporewall; transcripts accumulate at the time of prospore enclosure

Yes Yes YDR522C (SPS2) Protein expressed during sporulation, redundant with Sps22p for organizationof the b-glucan layer of the spore wall; S. pombe ortholog is a spore wallcomponent

YDR523C (SPS1) Putative protein serine/threonine kinase expressed at the end of meiosis andlocalized to the prospore membrane, required for correct localization ofenzymes involved in spore wall synthesis

Yes Yes YHR102W (KIC1) Protein kinase of the PAK/Ste20 kinase family, required for cell integritypossibly through regulating 1,6-b-glucan levels in the wall; physicallyinteracts with Cdc31p (centrin), which is a component of the spindlepole body

YHR103W (SBE22) Protein involved in the transport of cell wall components from the Golgi tothe cell surface; similar in structure and functionally redundant with Sbe2p;involved in bud growth

Yes Yes YNL261W (ORC5) Subunit of the origin recognition complex, which directs DNA replication bybinding to replication origins and is also involved in transcriptional silencing

YNL262W (POL2) Catalytic subunit of DNA polymerase epsilon, one of the major chromosomalDNA replication polymerases characterized by processivity and proofreadingexonuclease activity; also involved in DNA synthesis during DNA repair

Yes No YCL039W (GID7) Protein of unknown function, involved in proteasome-dependent cataboliteinactivation of fructose-1,6-bisphosphatase; contains six WD40 repeats;computational analysis suggests that Gid7p and Moh1p have similarfunctions

YCL040W (GLK1) Glucokinase, catalyzes the phosphorylation of glucose at C6 in the firstirreversible step of glucose metabolism; one of three glucosephosphorylating enzymes; expression regulated by nonfermentablecarbon sources

Yes No YLR115W (CFT2) Subunit of the mRNA cleavage and polyadenlylation factor (CPF); requiredfor pre-mRNA cleavage, polyadenylation, and poly(A) site recognition, 43%similarity with the mammalian CPSF-100 protein.

YLR116W (MSL5) Component of the commitment complex, which defines the first step in thesplicing pathway; essential protein that interacts with Mud2p and Prp40p,forming a bridge between the intron ends; also involved in nuclear retentionof pre-mRNA

No No YMR306W (FKS3) Protein involved in spore wall assembly, has similarity to 1,3-b-d-glucan synthasecatalytic subunits Fks1p and Gsc2p; the authentic, nontagged protein isdetected in highly purified mitochondria in high-throughput studies

YMR307W (GAS1) b-1.3-glucanosyltransferase, required for cell wall assembly; localizes tothe cell surface via a glycosylphosphatidylinositol (GPI) anchor

No No YBR087W (RFC5) Subunit of heteropentameric replication factor C (RF-C), which is a DNAbinding protein and ATPase that acts as a clamp loader of the proliferatingcell nuclear antigen (PCNA) processivity factor for DNA polymerases deltaand epsilon

(continued )


finding contradicts several published claims. Cohen et al.(2000, p. 184) reported that ‘‘of the 2081 adjacent pairsexamined, 387 fell into the same functional category,significantly more than would be expected by chance(P ¼ 10�8).’’ Lee and Sonnhammer (2003, p. 875)reported on the basis of the KEGG pathways (Kanehisa

et al. 2006) that ‘‘virtually all pathways in S. cerevisiaeshowed significant clustering.’’ Teichmann and Veitia

(2004, p. 2124) reported that ‘‘a significant fraction ofgenes encoding subunits of stable complexes are closeto each other on yeast chromosomes.’’

Analysis of adjacent genes in S. cerevisiae using theKEGG pathways (Kanehisa et al. 2006), GO annotation(Ashburner et al. 2000), gene names, and Saccharomy-ces Genome Database (SGD) descriptions (Cherry et al.1997) identifies five categories of genes that are clus-tered by function. The genes involved in biotin synthesis

are an example of a ‘‘dispensable pathway,’’ a pathwaythat is found in some strains and species but not inothers within a phylogenetic clade. In S. cerevisiae thiscategory includes specific carbon source utilization clus-ters, aryl-sulfate utilization, siderophore-bound iron uti-lization, utilization of S-adenosylmethionine, and theutilization of allantoin as a nitrogen source (Wong andWolfe 2005). This category furthermore includes arsenicresistance and vitamin B1/B6 metabolism (Table 2).The limited number of dispensable pathway clusters inS. cerevisiae is likely a reflection of the absence of sec-ondary metabolism. Dispensable pathway clusters havebeen widely reported in other fungi including indole-diterpene biosynthesis in Neotyphodium lolii (Young et al.2006) and Aspergillus flavus (Zhang et al. 2004), thepenicillin biosynthetic gene cluster in Penicillium chryso-genum (Smith et al.1990; Abe et al. 2002), P. nalgiovense

TABLE 3

(Continued)

Adjacent inA. gossypii?

Adjacent inK. lactis? Gene SGD description line

YBR088C (POL30) Proliferating cell nuclear antigen (PCNA), functions as the sliding clampfor DNA polymerase delta; may function as a docking site for otherproteins required for mitotic and meiotic chromosomal DNA replicationand for DNA repair

No No YKL113C (RAD27) 59 to 39 exonuclease, 59 flap endonuclease, required for Okazaki fragmentprocessing and maturation as well as for long-patch base-excision repair;member of the S. pombe RAD2/FEN1 family

YKL114C (APN1) Major apurinic/apyrimidinic endonuclease, 39-repair diesterase involved inrepair of DNA damage by oxidation and alkylating agents; also functionsas a 39-59 exonuclease to repair 7,8-dihydro-8-oxodeoxyguanosine

No No YMR275 (BUL1) Ubiquitin-binding component of the Rsp5p E3-ubiquitin ligase complex,functional homolog of Bul2p; disruption causes temperature-sensitivegrowth, overexpression causes missorting of amino acid permeases

YMR276W (DSK2) Nuclear-enriched ubiquitin-like polyubiquitin-binding protein, required forspindle pole body (SPB) duplication and for transit through the G2/Mphase of the cell cycle, involved in proteolysis, interacts with theproteasome

No No YOL124C (TRM11) Catalytic subunit of an adoMet-dependent tRNA methyltransferase complex(Trm11p-Trm112p), required for the methylation of the guanosinenucleotide at position 10 (m2G10) in tRNAs; contains a THUMP domainand a methyltransferase domain

YOL125W (TRM13) 29-O-methyltransferase responsible for modification of tRNA at position 4;exhibits no obvious similarity to other known methyltransferases

No No YOR340C (RPA43) RNA polymerase I subunit A43YOR341W (RPA190) RNA polymerase I subunit; largest subunit of RNA polymerase I

— — YML058W (SML1) Ribonucleotide reductase inhibitor involved in regulating dNTP production;regulated by Mec1p and Rad53p during DNA damage and S phaseHUG1 found only in sensu stricto species

YML058W-A (HUG1) Protein involved in the Mec1p-mediated checkpoint pathway that respondsto DNA damage or replication arrest; transcription is induced by DNAdamage

Pairs of genes are on consecutive lines. First two columns indicate if the homologs of each gene pair are also adjacent in K. lactisand A. gossypii. Descriptions are from SGD (Cherry et al. 1997).


(Laich et al. 1999), P. griseofulvum, and P. verrucosum(Laich et al. 2002), the aflatoxin pathway in A. nidulans(Keller and Adams 1995) and A. parasiticus (Trailet al.1995), ergot synthesis in Claviceps purpurea (Tudzynski

et al. 1999), and genes involved in plant pathogenesis inUstilago maydis (Kamper et al. 2006).

The additional four categories of clustered genesas well as two categories of artifacts that have possiblybeen confused with functionally related gene clustersin previous analyses, are summarized in Tables 1–3, andin supplemental Table 4 at http://www.genetics.org/supplemental/. Beyond these five categories of clus-tered genes, only 14 pairs of adjacent genes of relatedfunction were identified (Table 3). Of these 14 pairsonly 8 pairs are also adjacent genes in A. gossypii.

The clustering of the biotin biosynthesis genes is con-sistent with the observation that these genes are notubiquitous in the hemiascomycetes nor are they consis-tently found in the species S. cerevisiae. It is unclearwhether the clustering of genes in dispensable pathwaysis a result of selection for more efficient transmission ofthe trait to meiotic progeny, or of selection against toxicintermediates produced by incomplete pathways inmeiotic progeny, or some other explanation.

Horizontal gene transfer: A comparison of S. cerevisiaeand A. gossypii (Hall et al. 2005) identified 10 geneshorizontally acquired from prokaryotes by S. cerevisiaesince the divergence of these species, including BIO3and BIO4. We here report an updated analysis of hori-zontally acquired genes in S. cerevisiae including an

Figure 8.—Phylogeny of KAPA synthetase (bioF and BIO6) and DAPA synthetase (bioA and BIO3) indicates horizontal genetransfer of BIO3 into fungi from proteo bacteria (A) and gain of KAPA synthetase function by duplication of BIO3 to formBIO6 (B). Gene phylogeny KAPA synthetase is shown in a and DAPA synthetase in b. Species phylogeny was determined by ribo-somal small subunit (SSU). Both trees were determined by distance methods. The numbers indicate bootstrap support for eachnode of 1000 replicates.


additional 3 genes (supplemental Table 5 and supple-mental Figures 3, 4, and 5 at http://www.genetics.org/supplemental/).

In our initial analysis of HGT in S. cerevisiae and A.gossypii, the majority of the genes of recent bacterialorigin in S. cerevisiae appeared to be enzymes involved inthe scavenging of nutrients such as the horizontally ac-quired alkyl-aryl sulfatase BDS1. This observation fitswell with our understanding of HGT in bacteria in whichHGT appears to be a mechanism for the acquisition ofnovel metabolic characteristics. These genes appear tobe single-gain events that provide an immediate selec-tive advantage. Uniquely, the two genes of the biotinbiosynthesis pathway identified in our screen as likely

recent horizontal acquisitions, BIO3 and BIO4, encodetwo adjacent steps of the same biosynthetic pathway. Phy-logenetic analysis strongly supported the view that thesetwo genes were of recent bacterial origin (Figures 8 and9). Phylogenetic analysis also indicated that these twogenes were not acquired simultaneously as part of an‘‘operon transfer’’ but individually from different pro-karyotic donors. BIO3 appears to come from gamma-proteo bacteria and BIO4 from alpha-proteo bacteria.Further evidence for HGT of BIO3 and BIO4 is that inthe eukaryotic biotin biosynthesis pathway, the activitiesof dethiobiotin synthetase (DTBS) and DAPA synthe-tase (DS) appear to be performed by a single proteinencoded by a single gene based on sequence homology.

Figure 8.—Continued.


Species with eukaryotic type DTBS and DS show thisdual function enzyme structure. In prokaryotes and insome hemiascomycetes, these two activities are encodedon separate genes (supplemental Figure 6 at http://www.genetics.org/supplemental/).

Reconstruction of the biotin synthesis pathway:Identification of S. cerevisiae genes in amino acid andnucleotide synthesis pathways dates to the 1950s, yet thefirst gene identified in biotin biosynthesis was BIO2 in1995 (Zhang et al. 1994). With the identification of BIO3,BIO4, BIO5 (Phalip et al. 1999), BIO6 (Wu et al. 2005),and BIO1 (this report), it now appears that the completeset of genes for biotin biosynthesis in S. cerevisiae hasbeen identified.

The identification of BIO6 and now BIO1 allows us toreconstruct the evolutionary history of the entire path-way. This history is detailed in Figure 10. All of theknown genes for biotin biosynthesis form distinct eu-karyotic and prokaryotic clades (Figures 2, 8, 9, 10and supplemental Figure 2 at http://www.genetics.org/supplemental/). Construction of gene phylogenies foreach step of biotin biosynthesis reveals that eukaryotic-type biotin biosynthesis genes are found among plantsand most fungi, but disappear after the divergence of

Yarrowia lipolytica from the rest of the hemiascomycetesexcept for the final step biotin synthetase (BS). Otherthan Y. lipolytica, most hemiascomycete fungi possessprokaryotic-type DTBS and DS genes. The most likelyexplanation for this observation is that the eukaryoticbiotin biosynthetic pathway was lost in the ancestor ofCandida/Debaryomyces/Kluyveromyces/Saccharomy-ces after the divergence with the ancestor of Y. lipolyticaexcept for the final step, BS (Figure 10 and supplemen-tal Figure 2 at http://www.genetics.org/supplemental/).The next two steps, DTBS and DS, were then reacquiredby HGT from prokaryotes. It is difficult to preciselydetermine at which point in the evolution of thehemiascomycetes the loss of the pathway occurred orwhen prokaryotic genes were acquired. This uncertaintyis due to limited genomic sampling of more basalhemiascomycetes.

The partial construction of the biotin biosynthesispathway accomplished by the acquisition of bacterialgenes left the pathway still incomplete, yet this partialpathway appears to have been maintained (Figure 10).With the appearance of the Saccharomyces sensu strictocomplex, reconstruction of the biotin biosynthesis path-ways was completed with the appearance of BIO6, a

Figure 9.—Phylogeny of dethiobiotin synthetase (bioD and BIO4) indicates horizontal gene transfer of BIO4 into fungi frombacteria. Gene phylogeny of dethiobiotin synthetase (left) and species phylogeny (right) were determined by ribosomal smallsubunit (SSU). Both trees were determined by distance methods. The numbers indicate bootstrap support for each node of1000 replicates.


KAPA synthetase (KS), and BIO1, a putative PCAS. Phy-logenetic construction indicates that these genes are theresult of duplication of BIO3 and YJR154W, respectively.BIO3 does not have the function of BIO6 (Wu et al. 2005;this study), nor does YJR154W have the function of BIO1(this study). These two genes represent cases of geneduplication with subsequent neofunctionalization.

One of the most important largely unresolved issuesin evolutionary biology concerns the genetic origins ofmorphological and biochemical novelties. The mostcommon source of new genetic material in eukaryotesappears to be gene duplication. Gene duplication refersto the production of two duplicate loci, through un-equal crossing over, tandem duplication, or other illegit-imate chromosomal rearrangements. Gene duplicationwas originally discussed in the work of Muller (1936),but its full importance in the evolutionary process was

not generally realized until Ohno’s seminal book .30years later (Ohno 1970). Previously, models havepredicted that it is rarer for a duplicated gene to reacha stable frequency in a population than to be silencedthrough mutation (made into a nonfunctional ‘‘pseu-dogene’’) (Haldane 1933; Ohno 1970). Some insightinto the extent of gene loss after duplication can begained by examining the whole-genome duplication inthe lineage of S. cerevisiae. In that case it has been shownthat 92% of the duplicated genes were lost within 100million years after genome duplication (Seoighe andWolfe 1998). It seems more likely for a gene to be lostafter duplication than to acquire a new function. Thereconstructed biotin biosynthesis pathway observed inS. cerevisiae acquired two genes by this process. It is alsointeresting to note that BIO5, although not part of thecore biosynthetic pathway, also appears to have formed

Figure 10.—Loss of the eukaryotic biotin biosynthesis pathway in the hemiascomycete lineage and reconstruction by horizontalgene transfer and gene duplication. Phylogram represents the evolutionary relationship between fungi. Rows indicate enzymetype. E indicates eukaryotic-type enzyme and P indicates prokaryotic-type enzyme. D indicates genes formed by duplicationand neofunctionalization. Question marks indicate that the gene responsible for this step is unknown. Columns indicate enzymefunction. PCAS indicates pimeloyl-CoA synthetase, KAPAS indicates KAPA synthetase, DAPAS indicates DAPA synthetase, DTBSindicates dethiobiotin synthetase, and BS indicates biotin synthetase. While S. kluyveri is a biotin prototroph (Figure 8), the ge-nome of this organism does not appear to encode either a eukaryotic or a prokaryotic type PCAS or KAPAS.


as the result of gene duplication and possible neo-functionalization (supplemental Figure 7 at http://www.genetics.org/supplemental/).

In this work we report the identification of BIO1 as thefirst step of the known biotin biosynthetic pathway in S.cerevisiae. We also examine the remarkable evolutionaryhistory of biotin biosynthesis in S. cerevisiae and relatedfungi. We conclude that the biotin biosynthetic pathwayrepresents an example of gene pathway evolution. Inthis lineage, biotin biosynthesis has been nearly lost andthen rebuilt by two rare genetic phenomena: horizontalgene transfer and gene neofunctionalization after du-plication. More work is needed to determine whetherpimelic acid, malonyl-CoA, or another molecule func-tions as the first intermediate in biotin synthesis inS. cerevisiae.

LITERATURE CITED

Abe, Y., T. Suzuki, C. Ono, K. Iwamoto, M. Hosobuchi et al.,2002 Molecular cloning and characterization of an ML-236B(compactin) biosynthetic gene cluster in Penicillium citrinum.Mol. Genet Genomics 267: 636–646.

Ashburner, M., C. Ball, J. A. Blake, D. Botstein, H. Butler et al.,2000 Gene ontology: tool for the unification of biology. Nat.Genet. 25: 25–29.

Benson, D. A., M. S. Boguski, D. J. Lipman, J. Ostell, B. F.Ouellette et al., 1999 GenBank. Nucleic Acids Res. 27: 12–17.

Bower, S., J. Perkins, R. R. Yocum, P. Serror, A. Sorokin et al.,1995 Cloning and characterization of the Bacillus subtilis birAgene encoding a repressor of the biotin operon. J. Bacteriol.177: 2572–2575.

Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li et al.,1998 Designer deletion strains derived from Saccharomyces cerevisiaeS288c: a useful set of strains and plasmids for PCR-mediated genedisruption and other applications. Yeast 14: 115–132.

Burke, D. T., D. Dawson and T. Stearns, 2000 Methods in Yeast Ge-netics: A Cold Spring Harbor Laboratory Course Manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY.

Buston, H., and B. N. Pramanik, 1931a The accessory factor neces-sary for the growth of Nematospora gossypii: part I. Biochem. J. 25:1656–1670.

Buston, H., and B. N. Pramanik, 1931b The accessory factor nec-essary for the growth of Nematospora gossypii: part II. Biochem.J. 25: 1671–1673.

Cherry, J. M., C. Ball, S. Weng, G. Juvik, R. Schmidt et al.,1997 Genetic and physical maps of Saccharomyces cerevisiae. Na-ture 387: 67–73.

Cohen, B. A., R. D. Mitra, J. D. Hughes and G. M. Church, 2000 Acomputational analysis of whole-genome expression data revealschromosomal domains of gene expression. Nat. Genet. 26: 183–186.

Dietrich, F. S., S. Voegeli, S. Brachat, A. Lerch, K. Gates et al.,2004 The Ashbya gossypii genome as a tool for mapping the an-cient Saccharomyces cerevisiae genome. Science 304: 304–307.

Duclaux, M., 1864 Sur la fermentation alcoolique. Comp. Rend.Acad. Sci. 58: 1114–1116.

Farries, E. H. M., and A. F. Bell, 1930 On the metabolism of Nem-atospora gossypii and related fungi, with special reference to thesource of nitrogen. Ann. Bot. 44: 423–455.

Goldstein, A. L., and J. H. McCusker, 1999 Three new dominantdrug resistance cassettes for gene disruption in Saccharomyces cer-evisiae. Yeast 15: 1541–1553.

Gonnet, G. H., M. A. Cohen and S. A. Benner, 1992 Exhaustivematching of the entire protein sequence database. Science 256:1443–1445.

Haldane, J., 1933 The part played by recurrent mutation by evolu-tion. Am. Nat. 67: 5–9.

Hall, C., S. Brachat and F. S. Dietrich, 2005 Contribution of hor-izontal gene transfer to the evolution of Saccharomyces cerevisiae.Eukaryot. Cell 4: 1102–1115.

Hoja, U., C. Wellein, E. Greiner and E. Schweizer, 1998 Pleio-tropic phenotype of acetyl-CoA-carboxylase-defective yeast cells–viability of a BPL1-amber mutation depending on its readthroughby normal tRNA(Gln)(CAG). Eur. J. Biochem. 254: 520–526.

Ifuku, O., H. Miyaoka, N. Koga, J. Kishimoto, S. Haze et al.,1994 Origin of carbon atoms of biotin. 13C-NMR studies on bi-otin biosynthesis in Escherichia coli. Eur. J. Biochem. 220: 585–591.

Kamper, J., R. Kahmann, M. Bolker, L. J. Ma, T. Brefort et al.,2006 Insights from the genome of the biotrophic fungal plantpathogen Ustilago maydis. Nature 444: 97–101.

Kanehisa, M., S. Goto, M. Hattori, K. F. Aoki-Kinoshita, M. Itoh

et al., 2006 From genomics to chemical genomics: new develop-ments in KEGG. Nucleic Acids Res. 34: D354–D357.

Keller, N. P., and T. H. Adams, 1995 Analysis of a mycotoxin genecluster in Aspergillus nidulans. SAAS Bull. Biochem. Biotechnol. 8:14–21.

Kogl, F., P. Fildes, A. Lwoff, B. C. J. G. Knight and G. M. Richardson,1937 Discussion Meeting on Growth Factors. Proc. R. Soc. Lond.B Biol. Sci. 124: 1–13.

Kogl, F., and B. Tonnis, 1936 Uber das bios-problem. Darstellungvon krystallisiertem biotin aus eigelb. Mitteilung uber pflanzlichewachstumsstoffe. Zeitschrift fur Physiologie Chemie 241: 43–73.

Laich, F., F. Fierro, R. E. Cardoza and J. F. Martin, 1999 Or-ganization of the gene cluster for biosynthesis of penicillin inPenicillium nalgiovense and antibiotic production in cured drysausages. Appl. Environ. Microbiol. 65: 1236–1240.

Laich, F., F. Fierro and J. F. Martin, 2002 Production of penicillinby fungi growing on food products: identification of a completepenicillin gene cluster in Penicillium griseofulvum and a truncatedcluster in Penicillium verrucosum. Appl. Environ. Microbiol. 68:1211–1219.

Lee, J. M., and E. L. Sonnhammer, 2003 Genomic gene clusteringanalysis of pathways in eukaryotes. Genome Res. 13: 875–882.

Lorenz, M. C., R. S. Muir, E. Lim, J. McElver, S. C. Weber et al.,1995 Gene disruption with PCR products in Saccharomyces cere-visiae. Gene 158: 113–117.

Muller, H., 1936 Bar duplication. Science 83: 528–530.Mullis, K., F. Faloona, S. Scharf, R. Saiki, G. Horn et al.,

1986 Specific enzymatic amplification of DNA in vitro: the poly-merase chain reaction. Cold Spring Harb Symp. Quant. Biol.51(Pt 1): 263–273.

Ohno, S., 1970 Evolution By Gene Duplication. Springer, Berlin.Ohsugi, M., and Y. Imanishi, 1985 Microbiological activity of biotin-

vitamers. J. Nutr. Sci. Vitaminol. 31: 563–572.Pasteur, M. L., 1860 Memoires sur la fermentation alcoolique.

Ann. Chim. Phys. 3: 323–426.Phalip, V., I. Kuhn, Y. Lemoine and J. M. Jeltsch, 1999 Char-

acterization of the biotin biosynthesis pathway in Saccharomycescerevisiae and evidence for a cluster containing BIO5, a novel geneinvolved in vitamer uptake. Gene 232: 43–51.

Pinon, V., S. Ravanel, R. Douce and C. Alban, 2005 Biotin synthe-sis in plants. The first committed step of the pathway is catalyzedby a cytosolic 7-keto-8-aminopelargonic acid synthase. Plant Phys-iol. 139: 1666–1676.

Roggenkamp, R., S. Numa and E. Schweizer, 1980 Fatty acid-requiring mutant of Saccharomyces cerevisiae defective in acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. USA 77: 1814–1817.

Saitou, N., and M. Nei, 1987 The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.

Seoighe, C., and K. H. Wolfe, 1998 Extent of genomic rearrange-ment after genome duplication in yeast. Proc. Natl. Acad. Sci.USA 95: 4447–4452.

Shchelokova, E., and L. Vorob’eva, 1982 Biotin formation by thefungus Rhizopus delemar. Prikl. Biokhim. Mikrobiol. 18: 630–635.

Smith, D. J., M. K. Burnham, J. Edwards, A. J. Earl and G. Turner,1990 Cloning and heterologous expression of the penicillinbiosynthetic gene cluster from Penicillum chrysogenum. Biotech-nology 8: 39–41.

Stolz, J., U. Hoja, S. Meier, N. Sauer and E. Schweizer,1999 Identification of the plasma membrane H1-biotin sym-porter of Saccharomyces cerevisiae by rescue of a fatty acid-auxotro-phic mutant. J. Biol. Chem. 274: 18741–18746.

Streit, W. R., and P. Entcheva, 2003 Biotin in microbes, the genesinvolved in its biosynthesis, its biochemical role and perspectives


for biotechnological production. Appl. Microbiol. Biotechnol.61: 21–31.

Tanner, F. W., 1924 The ‘‘BIOS’’ question. Chem. Rev. 1: 397–472.Teichmann, S. A., and R. A. Veitia, 2004 Genes encoding subunits

of stable complexes are clustered on the yeast chromosomes: aninterpretation from a dosage balance perspective. Genetics 167:2121–2125.

Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin and D. G.Higgins, 1997 The CLUSTAL_X windows interface: flexiblestrategies for multiple sequence alignment aided by quality anal-ysis tools. Nucleic Acids Res. 25: 4876–4882.

Trail, F., N. Mahanti, M. Rarick, R. Mehigh, S. H. Liang et al.,1995 Physical and transcriptional map of an aflatoxin genecluster in Aspergillus parasiticus and functional disruption ofa gene involved early in the aflatoxin pathway. Appl. Environ.Microbiol. 61: 2665–2673.

Tudzynski, P., K. Holter, T. Correia, C. Arntz, N. Grammel et al.,1999 Evidence for an ergot alkaloid gene cluster in Clavicepspurpurea. Mol. Gen. Genet. 261: 133–141.

von Leibig, J., 1869 Ueber die Gahrung and die Quelle der Musk-elkraft. Annalen der Chemie und Pharmacie 153: 1–47.

Wach, A., A. Brachat, R. Pohlmann and P. Philippsen, 1994 Newheterologous modules for classical or PCR-based gene disrup-tions in Saccharomyces cerevisiae. Yeast 10: 1793–1808.

Wildiers, E., 1901 Nouvelle substance indespensable au developpe-ment de la levure. Le Cellule 18: 313–336.

Wolfe, K. H., and D. C. Shields, 1997 Molecular evidence for anancient duplication of the entire yeast genome. Nature 387:708–713.

Wong, S., and K. H. Wolfe, 2005 Birth of a metabolic gene clusterin yeast by adaptive gene relocation. Nat. Genet. 37: 777–782.

Wu, H., K. Ito and H. Shimoi, 2005 Identification and characteriza-tion of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.Appl. Environ. Microbiol. 71: 6845–6855.

Young, C. A., S. Felitti, K. Shields, G. Spangenberg, R. D. Johnson

et al., 2006 A complex gene cluster for indole-diterpene biosyn-thesis in the grass endophyte Neotyphodium lolii. Fungal Genet.Biol. 43: 679–693.

Zhang, S., B. J. Monahan, J. S. Tkacz and B. Scott, 2004 Indole-diterpene gene cluster from Aspergillus flavus. Appl. Environ. Mi-crobiol. 70: 6875–6883.

Zhang, S., I. Sanyal, G. H. Bulboaca, A. Rich and D. H. Flint,1994 The gene for biotin synthase from Saccharomyces cerevisiae:cloning, sequencing, and complementation of Escherichia colistrains lacking biotin synthase. Arch. Biochem. Biophys. 309:29–35.

Communicating editor: A. P. Mitchell


The Reacquisition of Biotin Prototrophy in Saccharomyces ......in Escherichia coli and Bacillus sp....

Documents

Transcript of The Reacquisition of Biotin Prototrophy in Saccharomyces ......in Escherichia coli and Bacillus sp....