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J Bacteriol. Jan 2007; 189(2): 591–602.
Published online Nov 17, 2006. doi:  10.1128/JB.01381-06
PMCID: PMC1797375

Essential Bacterial Functions Encoded by Gene Pairs[down-pointing small open triangle]||


To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.

Gene disruption is a useful tool to explore the function of genes and to identify essential genes. Several publications have described functional analysis of genomes by systematic disruption of genes, particularly in prokaryotes. Genes can be disrupted by a variety of methods, such as transposition or the use of plasmids with conditional replication and homologous recombination. In particular, in Bacillus subtilis, genes that encode essential functions were identified, while other nonessential genes were assigned to functional categories (56). This was based on the examination of key cellular processes, such as metabolism, cell division, DNA segregation, competence, or secretion, in generated mutant strains (56). Similarly, in Haemophilus influenzae (2), Staphylococcus aureus (35), Salmonella enterica (55), or Helicobacter pylori (98), a large number of genes that are required for growth under nutrient-rich conditions were identified.

So far, systematic gene disruption has been applied only to single genes. Yet, studies of genome structure have revealed the prevalence of sequence duplication (58, 92). Whole genes or parts of gene sequences are frequently duplicated. As a consequence, a number of proteins within the same organism have similar sequences. Experimental studies indicate that even if the level of sequence identity is high two proteins can have significant functional differences. For example, the type II topoisomerases gyrase and topoisomerase IV are 40 to 60% identical, although each has a distinct function essential for cell viability (17, 49). Nevertheless, the extent of sequence similarity between these essential proteins means that the same antibacterial inhibitors often inhibit both type II topoisomerases (49, 94).

Equally, pairs of proteins with little sequence similarity can nonetheless have significant functional similarity. In B. subtilis, the peptide deformylases Def and YkrB show similarity only across a short length (motif) but either is sufficient for carrying out a deformylase reaction essential for cell viability (40). That the function they carry out is essential was revealed when both genes were knocked out in combination, because the presence of one protein could compensate for the absence of the other (40). Indeed, both of these enzymes are inhibited by the same antibacterial inhibitor, despite the limited similarity in sequence and the redundancy in function (40). Our hypothesis is that many such pairs of genes, which we term isologous pairs, may have been missed by current functional genomics approaches. So far, this term has been applied to proteins in different organisms (72), but we now adopt it to describe proteins, and their encoding genes, within the same organism. Isologous proteins are similar to each other just as they are similar to their homologues in other species.

In this study, we have examined the prevalence of essential isologous proteins within B. subtilis by systematic disruption of gene pairs. Prioritization of the pairs of genes selected was based on the level of sequence identity between their encoded protein sequences and bioinformatic comparison with proteins from five other bacterial species. In addition, we characterized the growth of the mutants under several conditions. Our analysis confirms the existence of essential functions encoded by isologous gene pairs. Such pairs had not been recognized as targets for antibacterial discovery programs until recently (during the course of this study) (40, 42). Antibacterial targets tend to be single proteins that are conserved in a number of bacterial pathogens (broad spectrum), but not in humans. In an age where there is an escalating clinical need for new antibacterials, our findings could provide a new focus for future antibacterial discovery programs.


Analysis of protein sequence similarity.

Protein sequences of the whole genomes of B. subtilis 168, Escherichia coli K-12, S. aureus Mu50, Streptococcus pneumoniae R6, H. influenzae Rd, and Pseudomonas aeruginosa PAO1 were obtained in FASTA format from the NCBI (www.ncbi.nlm.nih.gov/genomes/lproks.cgi). They were converted into BLAST databases using the FORMATDB utility. Searches were carried out within the total protein sequences of one organism at a time, using the utility BLASTALL and preserving at most the top four matches. Initially, the query sequences and the sequences queried were the total proteins of a particular organism. In later searches the query sequences were more specific, while the sequences queried were the total protein sequences of a different organism. Expectation value (E value) thresholds were set at less than 10−10 for searches in which the query and the result sequences were obtained from one organism or at less than 10−3 for searches across different organisms. The E value is a descriptor of the statistical probability that the particular sequences of two proteins will be identical. It is much more quantitative than the percent identity descriptor often quoted and is therefore suitable for larger-scale analyses such as these. Identical proteins have E values of 10−180, while BLAST does not recognize alignments with E values greater than 10−3.

Computer scripts (written in Perl) were used to extract the protein identifiers for each query sequence that had found at least one matching sequence (hit), the E value of the match, and the protein identifiers of the best hits resulting from the search. For the searches within one organism, only information on searches in which the query sequences had found one matching sequence (other than themselves) was extracted. This is essentially a modified version of the bidirectional best-hit strategy (31). For the searches across organisms, the best two hit sequences were extracted from the search. Hit sequences resulting from such searches were used as query sequences in searches of the organism from which the original query sequences had been derived (reciprocal searches). Data extracted from the results of both searches were analyzed, using Microsoft Access and Excel, to identify similar proteins. For example, if four proteins (two pairs, each from a separate organism) found each other as top hits in the four corresponding BLAST searches (one within each organism and two across organisms), then the proteins were considered similar.

Creation of pHT35.

Standard methods for cloning and PCR were used (99). The gus neo fragment was extracted from pMLK83 (53) digested with NotI. This fragment was ligated to pACYC184 (93) that had been cut with SalI and BamHI and blunted, thus disrupting the tetracycline gene and resulting in vector pHT32. Additional cloning sites were created upstream of the Pxyl promoter of pRD96 (21) by the annealing of complementary oligonucleotide sequences topL (5′-AGCTATCGATGGATCCACTAGTAAGCTTACTGCA-3′) and botL (5′-TAGCTACCTA GGTGATCATTCGAATG-3′) and the ligation of the resulting linker molecule to pRD96 cut with PstI. The cat Pxyl polylinker fragment from the resulting recombinant vector was extracted following ClaI and BglII digestion and blunting at both ends. This fragment was ligated to pHT32 that had been cut with HindIII and ClaI and then blunted at both ends to create pHT34. Primers 34-F (5′-ACGGGATCCTGGCGACCACACCCGTCCTGTGGG-3′) and 34-R (5′-CGATGGATCCACTAGTAAGCTTACTGCAGG-3′) were used in a PCR to introduce a second BamHI site into pHT34 upstream of the gusA ribosome binding site. This vector was digested with BamHI and reannealed, selecting for loss of a 323-bp fragment and resulting in pHT35 (see Table S1 in the supplemental material).

Plasmid constructs for disruption and conditional mutants.

PCR was used to amplify an ~300-bp fragment from each gene of the putative pair, starting from approximately 200 bp within the gene for the generation of knockout constructs or from immediately upstream of the predicted ribosome binding site for the generation of conditional constructs. Amplification primers were designed to contain a restriction site for subsequent cloning into a relevant vector (pMUTIN4 [119] for one member of the potential pair and pHT35 for the other). Following digestion of both the insert and the vector with the relevant restriction enzymes, insert and vector were ligated. The ligation mixture was used to transform competent E. coli MC1061 (12) cells. Transformants were selected on 25 μg/ml chloramphenicol (pHT35) or 100 μg/ml ampicillin (pMUTIN4). Insert-positive constructs were identified by PCR using vector-specific primers. Plasmids isolated from insert-positive E. coli colonies (see Table S1 in the supplemental material) were sequenced and sequence data checked using Vector NTI software.

Creation of fabHA conditional mutant.

Primers 99BI-F (5′-TCGAAGCTTAAGATATAAAGGAGTC-3′) and 99BI-R (5′-TTAGGGATCCTGTGTGCACCTCACCTT-3′) were used to amplify the entire fabHA gene and to introduce HindIII and BamHI primers, respectively. The amplified and digested product was then ligated to similarly digested pJPR1 (J. Rawlins, unpublished). The resulting plasmid was pHT48.

Creation of polA conditional mutant.

Primers polAmis-F (5′-GAAGGAAATTGATTGACGGAACGAAAAAAATTAGTGCTTGTAG-3′) and polAmis-R (5′-CAATTTCCTTCTAAGAAGCCTCAAGCTTAATTG-3′) were used to introduce a mutation of the start codon of polA, from ATG to TTG, into plasmid pLI135B (see Table S1 in the supplemental material) by amplifying around the plasmid by PCR. This mutation was necessary because the original efficient translation signal seemed to give PolA product sufficient to allow growth even under repressive conditions.

Creation of polA and ypcP deletion mutants.

To construct a complete deletion of PolA, primers prepolA-F2 (5′-CGAGGGCCCAAAAGGCGAATTGTGGGGGATGCTG-3′) and prepolNK-R (5′-TTTAAGCTTCCGTCATTCAATTTCCTCC-3′) were used to amplify a fragment upstream of polA by PCR and to introduce a HindIII site at the downstream end for cloning. Primers postpolNK-F (5′-GTAGGATCCGAAATAAACAGAGATAGG-3′) and postpolA-R (5′-CAAGCATGCGATTGAGCATTTCCTGCCTGAC-3′) were used in a PCR to amplify a fragment downstream of polA and introduce a BamHI site at the upstream end for cloning. The kanamycin resistance cassette from pBEST501 (4) was extracted following digestion with HindIII and BamHI. The cassette was ligated to the two PCR fragments, which had been digested with HindIII and BamHI. Primers postpolA-R and prepolA-F2 were used to amplify the ligation product by PCR.

To obtain a C-terminal deletion of PolA, primers prepoltrn-F (5′-GACGGATCCAAAGGCGAATTGTGGGGGATGCTG-3′) and prepoltrn-R (5′-ATCGGTACCTCTACTCTTCATGATAATTATCGCCAATCTGTTCAACG-3′) were used to amplify a fragment within polA and introduce BamHI and KpnI sites for cloning. The PCR product, digested with BamHI and KpnI, was ligated to pBEST501 that had been cut similarly. The resulting plasmid was then digested with SphI and SalI and ligated to a fragment downstream of polA that had been amplified by PCR, using primers postpolAtrn-F (5′-GTAGCATGCGAAATAAACAGAGATAG-3′) and postpoltrn-R (5′-GGGGTCGACAATTGCATTTAAAGCGAGACG-3′), and digested with SphI and SalI. The resulting plasmid was pED5-2 (see Table S1 in the supplemental material).

To construct a complete deletion of ypcP, primers preYpcPN-F (5′-CAGAAGCTTAAACAGTGACCGATTATGTCAGCG-3′) and preYpcPN-R (5′-GCAAAGATCTATTATTATTCATCAAATAACTCC-3′) were used to amplify a fragment upstream of ypcP and introduce cloning sites by PCR. The fragment was digested with HindIII and BglII and ligated to similarly cut pRD96. The resulting vector was digested with BamHI and ligated to a fragment downstream of ypcP, which had been amplified by PCR using postYpcPN-F (5′-GCTGGATCCTAGAGAGATCGTTTAGATCC-3′) and postYpcPN-R (5′-GACGGATCCCGTTCCTTTTGCTTCTATTTTCAAATGCG-3′) primers and cut with BamHI. The resulting vector was partially cut with BamHI and EcoRI. Its staggered ends were blunted and ligated. The resulting vector was pED4-2 (see Table S1 in the supplemental material).

Cloning in B. subtilis.

For single gene disruptions, deletions, and creation of conditional mutants (see Table S1 in the supplemental material), B. subtilis 168CA was made competent (59, 70) and transformed with recombinant plasmids or linear DNA fragments, created as described above. Selection was for resistance to kanamycin (5 μg/ml) in the presence of 0.5% xylose (pHT35 and pBEST501) or to erythromycin (0.5 μg/ml) in the presence of 1 mM isopropyl-β-d-thiogalactoside (IPTG) (pMUTIN4) or resistance to chloramphenicol (5 μg/ml) (pRD96 and pJPR1). To create the C-terminal PolA deletion, loss of the original selective marker, the kanamycin cassette, was achieved by growth for several generations in the absence of antibiotic. To create double gene knockouts, a single knockout strain was made competent and transformed with chromosomal DNA from the strain with a knockout of its gene partner, selecting only for the incoming mutation. Resulting colonies were patched onto separate antibiotic plates to check for retention of the resident mutation in the presence and absence of inducer. Colonies were also diagnosed based on two PCRs; one reaction used two plasmid-specific primers and one insert amplification primer to diagnose integration of the plasmid, and the other used two primers in regions outside the insert to diagnose disruption of the target sequence.

Analysis of medium dependence.

Single knockouts, double knockouts, and conditional strains were all streaked on nutrient agar or minimal agar (as described in reference 39, but without trace elements) media in the presence or absence of appropriate antibiotics and inducers. Selective conditions and colonies were examined by eye after at least 16 h at 37°C.

Some strains were also examined in liquid media, either Oxoid Penassay broth or minimal medium (as described in reference 39, but without trace elements), by diluting cultures 1/1,500 and incubating them at 37°C in the presence (1% xylose or 1 mM IPTG, as appropriate) or absence of inducer. For measurement of reporter activity, samples were incubated with assay buffer containing lysozyme, 4-methyl-umbelliferyl β-d-galactoside, resorufin β-d-glucuronide, and Triton X-100 for 1 h at room temperature before measurement of fluorescence on a BMG Fluostar Galaxy instrument. To rescue growth on minimal medium, 200 μg/ml of different amino acids was added separately and in combination.


Protein duplication in bacteria.

The B. subtilis genome contains a high level of sequence duplication that does not appear to result from duplication of large segments of DNA (60). To quantify the extent of protein duplication, BLASTP was used to compare the gene products of the whole B. subtilis genome against itself. Seven hundred two proteins that could be reciprocally paired (351 pairs) were identified, based on an average threshold E value for the reciprocal similarities of ≤10−20. This cutoff is reasonably, but not greatly, different from the minimum level of similarity recognized in BLASTP searches of 10−3 (Fig. (Fig.1).1). Triplets and larger groupings of similar proteins were excluded from this analysis.

FIG. 1.
Distribution of protein pairs based on the sequence similarity (E value) between the proteins of each pair in B. subtilis, E. coli, H. influenzae, P. aeruginosa, S. aureus, and S. pneumoniae. Proteins were paired if their overall level of sequence similarity ...

Analysis of five other bacterial genomes also revealed large numbers of duplicated proteins in each of them (Fig. (Fig.1;1; Table Table1).1). Of the organisms analyzed, H. influenzae appeared to contain the least redundancy. However, of the 194 proteins (97 pairs) in H. influenzae, 130 had previously been reported to have essential functions (2). Such analyses confirmed that pairs of similar proteins exist in a number of bacterial species and as such were worth experimental investigation to find uncharacterized essential functions.

Frequency of protein pairs in various bacteria

Systematic disruption of 135 gene pairs.

Several groups have completed functional analysis programs based on high-throughput disruption of single genes and aimed at finding essential genes (2, 35, 55-56, 98). However, essential functions protected by redundancy, so that the loss of one protein can be compensated for by the activity of another, would not have been detected. We therefore undertook a systematic analysis of gene pairs in B. subtilis.

Bioinformatic analysis suggested that extending the threshold E value to 10−10 would include protein pairs whose sequence similarity is restricted to short motifs. We therefore identified a total of 523 pairs in B. subtilis, and to these a number of criteria were applied to select gene pairs for experimental evaluation. These pairs loosely fell into three categories. In the first category were pairs encoding proteins with high similarity. Seventy-one pairs were chosen based on an arbitrary cutoff value (E value of ≤10−70, equivalent to an observed >26% sequence identity). The second category consisted of 61 pairs selected from the remaining 452 gene pairs encoding proteins connected with lower similarity scores (E values of 10−10 to 10−69). This category had been filtered for the proteins that were duplicated in at least one more bacterial species, on the simple basis that they were more likely to contain common motifs. Within these we did discern some proteins with characterized motifs. In addition, these 61 pairs lacked protein homologues in humans and therefore represented higher-priority antibacterial targets. Finally, we included protein pairs that were also significantly similar to other proteins in B. subtilis and so were parts of larger similarity groupings but were putative homologues of protein pairs identified in H. influenzae. The three pairs in this third category were included to ensure coverage of duplication in pathogenic bacteria. In total, 135 pairs of genes were selected for systematic disruption individually and in combination (Table (Table22).

Single and double gene disruptions in B. subtilis

Examination of the 270 genes making up these pairs indicated that 123 had been functionally characterized to some extent elsewhere (Table (Table2)2) (1, 5-11, 13-15, 18-19, 22, 24, 28-30, 32-34, 37-38, 41-48, 51-52, 54, 57, 61-69, 71, 73-81, 83-89, 91, 95-97, 101-118, 120-128). For the majority of the pairs, only one gene had been characterized. Where both genes had been characterized, the paired genes appeared to have a function in common (28 pairs) (1, 5, 7-9, 11, 15, 29, 32-33, 37, 42, 44-45, 54, 57, 65, 74, 83-84, 86, 88, 97, 105, 108, 110, 114-117, 123-124, 126). Primarily, the characterization for these pairs was either incomplete or based solely on biochemical data. This made the need for an in vivo study more relevant. Therefore, all of the genes selected bioinformatically were included in our experimental analysis.

To make double disruption strains readily, two vectors were used in parallel. One was pMUTIN4 (119), previously used for the functional analysis of single genes in B. subtilis (56). The other vector, pHT35, was constructed to be pMUTIN4-like. The selective marker (neo), reporter gene (gus), and inducible promoter (Pxyl) of the new vector, pHT35, were all different from those of pMUTIN4. pHT35 was based on a nonhomologous E. coli replicon, P15A (93), to ensure that its DNA sequence was unrelated to that of pMUTIN4 (Fig. (Fig.2).2). This plasmid combination should be valuable in many situations in which expression of two genes needs to be manipulated simultaneously and/or measured in parallel.

FIG. 2.
Map of pHT35, showing key features.

Of the 270 genes analyzed, 244 were successfully disrupted (Table (Table2).2). Our results were in agreement with earlier publications, as only two (topA and yqiD) of the 244 genes we disrupted had previously been reported to be essential (56).

Identification of new essential genes.

The remaining 26 genes could not be disrupted, despite multiple attempts (Table (Table2).2). Eleven of these genes (ctaD, gapA, mntA, yfkN, ysgA, ycgG, yloU, yddT, yomL, ytbE, and yqhY) had previously been reported to be nonessential (14, 32, 56, 88, 120). Five of these genes (yloU, yqhY, gapA, ysgA, and ycgG) had, however, been defined as “persistent” based on the strong conservation of homologues in gram-positive bacteria (31). We propose that these genes should be added to the list of essential genes in B. subtilis. Of these 11, we have prioritized mntA, ctaD, ysgA, ycgG, yloU, and yfkN for future characterization as potential antibacterial targets because they are widely conserved and absent from humans.

Other single gene effects.

The mutants of 11 nonessential genes were auxotrophic (Table (Table3).3). Eight of these genes (yjbV, thiE, pyrC, purK, gltA, tenI, hisF, and hisA) had known metabolic roles (61, 64, 82, 83, 90, 104) or were significantly similar to characterized metabolic proteins in other species (25). An additional set of 13 mutants showed defective sporulation (Table (Table3).3). Seven of these genes (nasA, oppA, yvgN, ytxN, speA, opuE, and recG) had been characterized previously at least partly (3, 80, 85, 96, 109, 113, 122), but only oppA and ytxN had been implicated directly in sporulation (85, 113). These observations provided preliminary information on the possible general functions of ysaA, ypvA, yfjR, yodR, yqiD, yjbM, and ywaC, genes that were previously without functional assignment.

Single mutants affected in growth on minimal medium or during sporulation

Remarkably, the effects of one single knockout were suppressed by disruption of the partner gene (Table (Table3).3). Disruption of yodR blocked sporulation, but when a yodR mutation was combined with a scoB mutation the double knockout sporulated as well as the wild type. Whereas the double mutant had approximately 70% spores after a 2-day incubation on nutrient agar, no spores were detected with the yodR mutant. Similarly, the yodR mutant was unable to grow in minimal medium, while the double mutant could (data not shown). All three strains grew well in rich medium. Bioinformatic analysis suggests that both genes encode probable coenzyme A transferases but with different substrates. Indeed the two proteins are reasonably similar (E value of 10−53 or 50% identity across their length). We speculate that this phenotypic effect may be indirect and a consequence of variation in levels of different metabolites in these strains. Alternatively, ScoB may be partially isologous to YodR, causing the protein substitution to be detrimental under some growth conditions.

Auxotrophic isologous pairs.

Knockouts of the nonessential genes, comprising 114 pairs in total, were combined to produce double mutants. This resulted in 108 viable double knockouts (Table (Table2).2). One hundred one of these mutants had not been examined previously. Our results also confirmed the nonessentiality of six pairs characterized in vivo elsewhere (29, 33, 45, 65, 97). There is controversy over the essentiality of the ykuA pbpA pair (124). All of the double knockout mutants were examined for defects in growth and sporulation on both rich and minimal media.

One double knockout (ywaA ybgE) showed medium-dependent growth. For the ywaA ybgE pair, the double knockout strain showed good growth in rich medium, whereas in minimal medium the mutant showed little growth over 5 h (Fig. (Fig.3).3). In contrast, the single mutants grew well under both conditions, indicating that YwaA and YbgE are isologous, with a common function essential only in minimal medium. Bioinformatic analysis suggested that YwaA and YbgE were likely branched-chain amino acid aminotransferases. Indeed, when minimal medium was supplemented with both isoleucine and valine, growth of the double knockout strain was rescued, confirming an effect on branched-chain amino acid metabolism (Fig. (Fig.3).3). The presence of a leucine dehydrogenase (Bcd) in B. subtilis (23) may explain why the amino acid requirements we observed in vivo did not include leucine, the sole other branched-chain amino acid. During the period of this study, YwaA and YbgE were shown to catalyze an aminotransferase reaction with these amino acids as substrates (rather than products) for the synthesis of methionine (8). This is only partially consistent with our data as, in vivo, methionine did not rescue activity (data not shown).

FIG. 3.
Growth curves for strains B. subtilis 168CA, ΩywaA, ΩybgE, and ΩywaA ΩybgE in rich (A) and minimal (B) media. Minimal medium was supplemented with 200 μg/ml isoleucine and 200 μg/ml valine for rescue of ...

Essential isologous pairs.

Consistent with our original hypothesis, we also identified six pairs of genes that appeared to be essential when both genes were inactivated (Table (Table4).4). In these instances, the genes in the pair could be knocked out individually but not in combination. Within these pairs, the genes were far enough apart on the chromosome to preclude gene linkage as an explanation to these results. To validate that these pairs were essential, for each pair one gene was disrupted and its partner was made conditional. As shown in Fig. Fig.4,4, for all six pairs, repressing the expression of one gene led to a decrease in growth, thus confirming that the proteins were both essential and isologous. Their characteristics will now be described in turn.

FIG. 4.
Growth curves for strains B. subtilis 168CA, ΩfabHB Pxyl-fabHA, ΩtopB Pxyl-topA, ΩthrS Pxyl-thrZ, ΩyqjG Pxyl-spoIIIJ, ΩypcP Pspac-polA, and ΩfabI Pspac-fabL. Strains were streaked on antibiotic selective ...
Isologous pairs identified

ThrS and ThrZ.

ThrS and ThrZ have been described previously as isologous threonyl tRNA synthetases (36, 86). We also find that the isologous pair is essential for cell growth (Fig. (Fig.4)4) (86). They share the strongest level of similarity of all of the essential pairs newly identified. Most bacteria outside the Bacillus spp. possess a single gene encoding threonyl tRNA synthetase, rather than two genes. Similar threonyl tRNA synthetases exist in humans, making these proteins less attractive targets for antibacterial discovery.

TopA and TopB.

Most bacteria have either topoisomerase I (TopA) or topoisomerase III (TopB), but B. subtilis, E. coli, and S. aureus are relatively unusual in having both proteins. In B. subtilis, one of the proteins, TopA, was considered to be essential (56). Based on our results, the proteins in B. subtilis are isologous and essential as a pair. It is likely that so are those in E. coli and S. aureus. Owing to sequence similarity to human topoisomerases, TopA and TopB are lower-priority antibacterial targets.

FabI and FabL.

The genes coding for enoyl reductases FabI and FabL were shown elsewhere during the course of this study to have an essential function in fatty acid biosynthesis, so that the absence of one protein can be compensated for by the presence of the other (42). Our results corroborate that these proteins are isologous and essential as a pair (Fig. (Fig.4;4; Table Table4).4). Interestingly, depletion of both B. subtilis proteins was shown to increase expression of fabI, whereas depletion of either protein alone caused no effect on fabI expression (Fig. (Fig.5).5). This feedback effect confirmed that both enoyl reductases are active in vivo. They are conserved as a single enzyme in E. coli, H. influenzae, Enterococcus faecalis, and S. aureus. They are absent from humans, which makes them potential antibacterial targets, as validated by the antibacterial triclosan (42).

FIG. 5.
Expression of the PfabI-gus fusion following depletion of FabI (squares), FabL (circles), or both FabI and FabL (triangles). Pspac-fabL Pxyl-fabI (squares and circles) or Pspac-fabL ΩfabI (triangles) strains were grown in different inducer concentrations ...

FabHA and FabHB.

For FabHA and FabHB, inducer dependence was seen only with fabHA as the conditional allele (not shown). Presumably, the repressible construct for fabHB allows sufficient residual expression to allow growth. FabHA and FabHB are therefore isologous, and biochemical data (15, 20) suggest that they are 3-oxoacyl-[acyl carrier protein] synthases involved in fatty acid biosynthesis. They are duplicated in P. aeruginosa. They are conserved as single enzymes in E. coli, S. aureus, S. pneumoniae, E. faecalis, and H. influenzae and absent from humans, so this enzyme is a good antibacterial target. Indeed, there are reports of antibacterial discovery programs directed at the single enzyme in E. coli (20).

SpoIIIJ and YqjG.

Homologues of SpoIIIJ and YqjG are found in a wide range of other bacteria, usually as a single protein but as a pair in Streptococcus spp. Various studies with B. subtilis and E. coli suggest that the protein function is in secretion (100, 116). As no homologues exist in humans, this pair may have potential as a novel antibacterial target.

PolA and YpcP.

Among the identified isologous pairs, the pair of PolA and YpcP is the most novel and validates the rationale for this study. Previous functional genomics studies have suggested that DNA polymerase I (PolA) is essential in S. pneumoniae (27), E. coli (50), and S. enterica (55) but not in B. subtilis (56), S. aureus (35), or H. influenzae (2). We have found a possible explanation for this in that, in B. subtilis at least, polA becomes essential when ypcP is deleted.

YpcP is a small protein of previously unknown function, and its sequence similarity is restricted to the N-terminal domain of PolA, which contains a 5′-to-3′ exonuclease motif shown to be functional in S. pneumoniae and E. coli (26, 129) (Fig. (Fig.6).6). Standard single crossover insertions disrupting ypcP (with pMUTIN4) and polA (with pHT35) could not be combined. Similarly, complete deletions of each gene, though viable singly, could not be combined. However, a disruption that truncated PolA after the end of the N-terminal exonuclease domain (after codon 334) was viable and grew normally (data not shown). This and the depletion experiment mentioned above (Fig. (Fig.4)4) demonstrate that this putative 5′-to-3′ exonuclease domain is essential and that its function can be supplied either by the N-terminal part of PolA or by YpcP. Indeed, we have biochemical evidence, with a His tag fusion to YpcP, that confirms that exonuclease activity with a preference for double-stranded DNA is the essential function (E. Davison and H. Thomaides, unpublished data).

FIG. 6.
Sequence alignment of the PolA and YpcP proteins. Within the graphical representation of the alignment is the actual sequence alignment for residues 102 to 164 of the B. subtilis (B. sub.) PolA protein with homologues in other species (PolA of S. aureus ...

These data may explain the previous findings on the essentiality of PolA in some of the other species. They also suggest strongly that in these other organisms it is the 5′-to-3′ exonuclease domain of PolA that is essential. This domain duplication is present in various pathogens, including S. aureus, Mycobacterium tuberculosis, S. enterica, and E. coli (Fig. (Fig.6).6). Experimental evidence for some of these pathogens, such as E. coli and S. enterica (50, 55), suggests that although the domain is duplicated the single PolA remains essential. In other pathogens, only the single PolA protein is present, and in the case of S. pneumoniae, at least, it is essential (27). Importantly, this domain appears to be completely absent in humans. PolA/YpcP therefore represents another unexploited antibacterial target that needs to be characterized further.


This study opens many avenues for future work: 11 new essential genes have been identified and need characterization, and several previously uncharacterized genes have now been shown to be needed for growth in minimal medium. There is a whole new set of sporulation genes to be investigated. Some of these genes had initially been paired together through our bioinformatic work (e.g., yurU and yurX, yloU and yqhY, and tenI and thiE). Consequently, those that were found to be essential may encode proteins similar enough as to be inhibited by the same antibacterial, as is the case for gyrase and topoisomerase IV (94). Importantly, we have confirmed the status of three essential isologous pairs in B. subtilis (ThrS/ThrZ, FabI/FabL, and SpoIIIJ/YqjG) and identified four new ones (YwaA/YbgE, TopA/TopB, FabHA/FabHB, and PolA/YpcP) (Table (Table4).4). An extension of this work could be the development of inhibitors against SpoIIIJ/YqjG and PolA/YpcP, the unexploited isologous pairs that have no homologues in eukaryotes and therefore have potential as targets for antibacterials.

The isologous proteins do not appear to have any features, such as function, level of sequence similarity, and species conservation, distinguishing them from other proteins we paired. Although whole-genome bioinformatic analyses suggest a clustering of gene duplication (16, 60), we did not observe any clustering of the isologous genes on the chromosome. Interestingly, in each of four of the isologous pairs one gene has been identified as being “persistent” within the gram-positive clade of Firmicutes (31). This bioinformatic definition extends the number of essential bacterial functions that cannot be shown experimentally (31). The def ykrB essential pair identified elsewhere also has only one persistent gene (31, 40). That only one gene in an essential pair “persists” is consistent with our observation that the extent to which an essential function is protected by redundancy (e.g., via isologous pairs) is species specific. From our analysis, the two essential pairs involved in fatty acid biosynthesis (fabHA fabHB and fabI fabL) and the auxotrophic pair (ywaA ybgE) were not persistent, but then neither were many essential single genes (Table (Table2)2) (31). Similar analysis of the remaining pairs in this work (data not shown) suggests that an experimental approach is most suited to identify essential isologous proteins, though limited by chosen experimental conditions.

Why duplication in genomes is maintained is a heavily debated issue. We speculate that this systematic study has revealed the purpose of some of the redundancy within the B. subtilis genome by identifying some of the isologous pairs. Isologous proteins directly contribute to the fitness of the organism, while perhaps offering the possibility of acquiring mutations that fine-tune or extend protein functions. The experimental evaluation of pairs in other species or of triplets or quadruplets of genes would also investigate this issue; we detected such groupings in our preliminary analysis (D. Brown, unpublished data).

Our work sets a precedent for the systematic identification of essential isologous proteins. Similar studies will enhance our understanding of the biological significance of sequence duplication.

Supplementary Material

[Supplemental material]


We thank the Biotechnology and Biological Sciences Research Council and the Department of Trade and Industry, United Kingdom, for funding of this work through the award of a LINK grant in Applied Genomics.

We thank Celia Caulcott for her management of the LINK project. We thank Steve Ruston of Prolysis Ltd. for his support. We thank various colleagues, Neil Stokes in particular, for critical reading of the manuscript.


[down-pointing small open triangle]Published ahead of print on 17 November 2006.

||Supplemental material for this article may be found at http://jb.asm.org/.


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