The core set of 16 genes involved in DNA replication from the B. subtilis essential gene list are all scored as essential in S. aureus using TMDH. Of the 9 essential B. subtilis genes involved in DNA packaging and segregation, 6 were essential in S. aureus. The exceptions were smc, ypuG (scpA) and ypuH (scpB). The products of these three genes form a complex that is involved in chromosome condensation and segregation . The reason for their apparent dispensability in S. aureus is unclear.
The essential B. subtilis DNA methylation genes ydiO and ydiP do not have homologues in S. aureus. Five additional genes in this category that were not essential in B. subtilis were identified as putatively essential in S. aureus: recU, ruvA, ruvB, yrrK and yvcI. These are all associated with recombination and repair, and could be essential to survive the experimental procedure, and thus may be an artefact of the particular methodology that has been used to generate the transposon library.
The four components of the basic transcription machinery (rpoA, rpoB, rpoC and sigA) are all essential in both B. subtilis and S. aureus. There are eight essential genes involved in RNA modification in B. subtilis: cca, cspR, rnpA, rnc, trmU, trmD, ykqC and yqjK. All of these are also essential in S. aureus, with the exception of rnc, which encodes a ribonuclease, and cspR, which encodes a putative RNA methylase. The trmU gene is split into two parts in the NCTC 8325 genome sequence, but this is due to a frameshift that occurs in a run of five consecutive adenine residues and most likely represents a sequencing error, or a consequence of slipped strand mis-pairing; the gene is intact in all other available S. aureus genome sequences. Three additional essential RNA modification genes were identified in S. aureus, rimM, ymfA and thdF. Insertions in rimM may not be tolerated due to polar effects on the essential trmD gene. ymfA is a paralogue of ykqC that is non-essential in B. subtilis, but is evidently more important in S. aureus. The ThdF protein (also known as TrmE) is a tRNA modification GTPase that is conserved across bacteria and eukaryotes, and is essential in some Escherichia coli genetic backgrounds . The three essential B. subtilis RNA regulation genes, yycF (vicR), yycG (vicK) and nusA that are conserved in S. aureus are all essential, together with eight additional putative regulators: glnR, sarA, rnr, yvhJ (msrR), lexA, greA, yjbD and fur.
There is no direct experimental evidence that the B. subtilis complement of 52 ribosomal proteins is essential, but they are considered as such since the ribosome itself is essential . Most of the ribosomal genes appear essential in S. aureus. However, the rpmG genes, of which there are two copies in both genomes, can each be mutated and are therefore not individually essential in S. aureus. The two S. aureus copies are more similar to each other than the two B. subtilis genes, both showing more similarity to B. subtilis rpmGA than to rpmGB. rpmE is non-essential in B. subtilis, due to the presence of two paralogous copies , but essential in S. aureus, which possesses only a single copy. The rplI and rpsT genes are essential in B. subtilis but not in S. aureus. The reason for this is unclear. rplK is not essential in B. subtilis but was identified as essential by TMDH, possibly due to its involvement in the heat shock response . rpsJ is not listed as essential as it is not annotated in the NCTC 8325 genome, but an ORF is present that is predicted to encode a protein identical to RpsJ from other S. aureus strains. It is likely that this is an oversight in annotation and rpsJ is identified by TMDH as essential in S. aureus NCTC 8325.
Of 24 tRNA synthetases listed as essential in B. subtilis, 20 are similarly essential in S. aureus. Two others, glyS and glyQ, which are adjacent in the B. subtilis genome and encode the α- and β-subunits of a glycyl-tRNA synthetase, are not conserved at the sequence level between B. subtilis and S. aureus. However, at the equivalent position within a syntenic region, S. aureus has an alternative glyS gene that is essential. This encodes a class-II glycyl-tRNA synthetase, similar to that encoded by Bacillus cereus . The thrS and thrZ genes of B. subtilis can compensate for the absence of each other , but only one copy (thrS) is present in S. aureus, and as may be predicted this is essential. The asnS gene is essential in B. subtilis but not in S. aureus. yacA, a gene originally classified as essential but of unknown function in B. subtilis , is involved in the modification of tRNA-Ile  and is also essential in S. aureus
The ten genes identified as essential translation accessory factors in B. subtilis are all essential in S. aureus. The S. aureus list includes three additional genes, homologues of efp, yrvI and smpB. The SmpB protein is associated with tmRNA-mediated rescue of stalled ribosomes, and is important for B. subtilis growth at high temperatures . B. subtilis efp mutants are viable but defective in sporulation , however this gene is essential for protein synthesis in E. coli . The yrvI (dtd) gene in B. subtilis lacks a start codon, and cell extracts have not shown any D-Tyr-tRNATyr deacylase activity , suggesting that the gene is non-functional in B. subtilis. The gene is intact in other Bacillus genomes, so the inactivation may have occurred during the adoption of B. subtilis as a laboratory strain. The gene is conserved across most bacteria and eukaryotes, and has been suggested to counteract the toxiCity of D-tyrosine .
The two genes essential for protein folding in B. subtilis, groES and groEL, are also essential in S. aureus. Three other protein folding genes, dnaJ, dnaK and grpE, are scored as essential in S. aureus, however dnaK mutants are temperature sensitive  so may be viable but unable to survive the procedure for generating the transposon library. A homologue of the essential B. subtilis post-translational modification gene map is also essential in S. aureus, however, it shows more similarity to the non-essential B. subtilis gene yflG. Deformylation is essential in B. subtilis, but the genome contains two genes associated with this process (def and ykrB), and either can be inactivated singly . In S. aureus only one deformylation gene, ykrB (pdf1) is present, and it is essential. Five of the six B. subtilis genes essential for protein translocation are also essential in S. aureus. The exception is prsA, which encodes a protein involved in extracellular folding of secreted proteins. Three additional protein translocation genes, sipT (spsB), secDF and yqjG, are essential in S. aureus. In B. subtilis, yqjG has a paralogue, spoIIIJ, that is absent from S. aureus; in B. subtilis the double knockout is lethal . sipT encodes one of four closely related type I signal peptidases in B. subtilis, whereas only two are present in S. aureus (spsA and spsB). spsB, which is most closely related to B. subtilis sipT, is essential , and has been targeted for the development of novel antimicrobial agents . SpsA, however, lacks residues essential for catalytic activity and is not essential .
Cell Envelope/Cell Wall and Associated Proteins
Sixteen genes involved in the production of the cell membrane are essential in B. subtilis . Of these, only gpsA, which encodes an NAD-dependent glycerol-3-phosphate dehydrogenase, is not essential in S. aureus. Mutants in this gene in other bacteria are auxotrophic for glycerol . Four additional cell envelope genes are essential in S. aureus: fabH, fabI, ywpB and yuxO. The first three of these are non-essential in B. subtilis due to the presence of a second gene with an overlapping function, fabHB, fabL and ycsD, respectively [42, 43, 4]. The reason for the essential nature of yuxO, a possible thioesterase , is unclear. Three additional genes that are thought to encode membrane-associated proteins, pbpX (fmtA), yfiX (fmtC) and ypbE (ebpS), are also essential in S. aureus but not B. subtilis. pbpX may be involved in resistance to high temperatures. EbpS in S. aureus is an elastin binding protein  and although a mutant can be isolated it shows a growth defect . The multi-subunit sodium-hydrogen antiporter encoded by the mrp (mnh) operon is essential in B. subtilis but not in S. aureus, most likely due to the presence of a paralogous system in the S. aureus genome. A transposon-insertion mutant of mnhD in S. aureus retains halotolerance and loses transmembrane potential during postexponential growth , leading to the suggestion that the mnh operon is involved in electron transport. It is possible that the two systems have diverged following a duplication event, but retain the ability to functionally compensate for one another.
The essential B. subtilis genes involved in amino sugar biosynthesis prior to peptidoglycan polymerization are all also essential in S. aureus, with the exception of yvyH, which is present as three copies (capG, capP and mnaA) in the S. aureus genome. Diaminopimelate biosynthesis is not important in S. aureus as it uses lysine in its peptidoglycan. Of the six B. subtilis essential genes involved in the process, only four (asd, dapA, dapB and ykuQ) are retained in S. aureus. These are required for lysine biosynthesis  but are not essential. Of the two racemases that convert L-glutamate and L-alanine into the corresponding D-isomers, racE is essential in S. aureus, but alr is present as two copies neither of which is individually essential. The other genes essential for the synthesis of peptidoglycan precursors in B. subtilis (ddl, murAA, murB, murC, murD, murE, murF, murG and mraY) are all similarly essential in S. aureus. Three S. aureus genes absent from B. subtilis, femA, femB and fmtB (femX), are essential in the formation of the pentaglycine interpeptide bridge, which is characteristic of S. aureus peptidoglycan . Also essential in S. aureus but not B. subtilis are ponA (pbp2), yrvJ (lytH) and SAOUHSC_02107. The essential genes involved in teichoic acid biosynthesis in B. subtilis are all essential in S. aureus. The genes dltA, B, C and D are also scored as essential, but this may be because they are part of the sigma X regulon which is associated with survival at high temperatures in B. subtilis .
Perhaps unsurprisingly, given the morphological differences between the two species, the orthologues of the essential B. subtilis cell shape determining genes, mreC and rodA, are not essential in S. aureus, and there is no orthologue of the mreB gene. The essential B. subtilis gene ylaN is involved in the determination of cell shape, with a similar phenotype to rodA , and is included in this category. Like rodA it is not essential in S. aureus. The seven essential cell division-related genes described for B. subtilis are all essential in S. aureus, together with five others: ylmF, ezrA, yyaA, gidA and ypsB. YlmF can complement the activity of the FtsZ-binding protein FtsA in B. subtilis, to the extent that overexpression of YlmF complemented the otherwise lethal ftsA null mutant . These overlapping roles seem to have diverged in S. aureus, since both genes are essential. ezrA encodes a negative regulator of FtsZ ring formation, which can be knocked out in B. subtilis resulting in multiple FtsZ rings . The nucleoid occlusion protein YyaA (also known as Noc) is essential for cell division in the absence of the Min system in B. subtilis; the genes encoding the Min system are not present in the S. aureus genome. gidA mutants affect cell division in E. coli cells grown on glucose , and GidA may be involved in tRNA modification . It is also essential in Helicobacter . YpsB localizes to the cell division site in B. subtilis, but its role is unclear .
Carbon Metabolism and Respiration
The core essential genes involved in glycolysis in B. subtilis are also essential in S. aureus. Three additional components of the glycolysis pathway are essential in S. aureus: pgi, gap, and pykA. The essentiality of these three genes means that all components of the pathway from glucose to pyruvate are essential in S. aureus, with the exception of the initial conversion of glucose to glucose-6-phosphate. The pentose phosphate pathway also appears to be more important in S. aureus than B. subtilis, with the component genes rpe (cfxE), zwf, yqiI (gnd) and SAOUHSC_02612 all scored as essential. A number of genes that play an intermediary or regulatory role in metabolism are also essential. These are: sucC, which encodes the beta subunit of succinyl-CoA synthetase, glnA, which encodes glutamine synthetase, yvcK, which is of unknown function but is required for growth on Krebs cycle intermediates and carbon sources metabolized by the pentose phosphate pathway , the HPr (Ser) kinase/phosphatase gene hprK, and the phosphotransferase system component ptsH. None of these is essential in B. subtilis, although glnA is essential in Salmonella enterica serovar Typhimurium, Mycobacterium tuberculosis and Haemophilus influenzae . Interestingly, glnR, which encodes a repressor of glnA, is also scored as essential. The genes are within an operon, so the essentiality of glnR could be due to polar effects on glnA.
Perhaps the most striking differences between the essential gene complements of S. aureus and B. subtilis are in the respiratory pathways. The menaquinone biosynthesis pathway, which is essential in B. subtilis, seems to be dispensable in S. aureus, with the exception of the final step catalyzed by the menA gene product. The isoprenoid pathway is essential in B. subtilis but in S. aureus it is replaced by the mevalonate pathway, where it is itself essential, as it is in other Gram positive cocci .
Nucleotides and Cofactors
There are 10 essential genes involved in nucleotide biosynthesis in the B. subtilis list. Of these 8 are essential in S. aureus. guaB was described as essential in the B. subtilis study, but this was a likely consequence of a lack of guanine in the growth medium : it is not required in S. aureus, but the downstream gene that encodes GMP synthase, guaA, is essential. hprT, which was a surprising inclusion in the B. subtilis list, does not play an essential role in S. aureus. Three other genes, relA, which encodes GTP pyrophosphokinase, pyrH (smbA), which encodes uridylate kinase, and thyA, which encodes thymidilate synthase, were all essential in S. aureus. relA, which mediates the stringent response, has previously been reported as being essential for in vitro growth of S. aureus .
Of the genes involved in cofactor biosynthesis, those required for the production of NAD and SAM are essential in both S. aureus and B. subtilis. The genes required for the iron-sulphate cluster in B. subtilis are also essential in S. aureus, with an additional requirement for ferredoxin encoded by fer, which is not essential in B. subtilis. The pathways to produce CoA and folate are more divergent. In B. subtilis, only the last gene in the CoA biosynthesis pathway was essential, suggesting that the immediate precursor (dephospho-CoA) could be scavenged from the medium , but that process does not seem to occur in S. aureus under our experimental conditions, since three other genes involved in CoA biosynthesis were essential, ylbI, yloI and SAOUHSC_02371. These allow the synthesis of CoA from the precursor pantothenate, which is presumably obtained from the growth medium. Similarly, folate is not obtained from the growth medium, so all the genes required for its synthesis (yciA, folB, folK, folP, folC and dfrA) are essential. There are two non-homologous genes in B. subtilis, yciA and mtrA, that encode a type I GTP cyclohydrolase (FolE) that catalyses the initial stages of the pathway from GTP to folate . The S. aureus genome contains an orthologue of only one of these, yciA, which is essential. The riboflavin biosynthesis gene ribC was not identified as essential in B. subtilis in Kobayashi et al. , but had been in an earlier study , and is essential in S. aureus.
Genes of unknown function
Sixty-six putatively essential genes without a clear indication of their role have been identified in S. aureus. Of these, 14 are also essential in B. subtilis. Six members of the family of low molecular mass GTP binding proteins, proposed to have ribosome-associated functions , are indispensible in B. subtilis and we have found them also to be essential in S. aureus. Two of the genes, yneS and yfnI have recently been functionally characterised. YneS (subsequently renamed to PlsY) is a glycerol-3-phosphate acyl transferase required for fatty acid and phospholipid biosynthesis and is essential in B. subtilis . YfnI (renamed to LtaS) is essential for lipoteichoic acid biosynthesis in S. aureus but not in B. subtilis due to the presence of numerous paralogues . Elucidation of the function of further unknowns will shed light on important facets of cellular physiology and form the basis for potential new antibiotic targets.