An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.

Deletion of sigE, the structural gene for the sporulation-induced RNA polymerase sigma factor, crE prevented endospore formation by BaciUlus subtilis. The effects of integration of plasmids into the sigE region of the chromosome and the use of complementation analyses demonstrated that sigE is part of an operon that includes a promoter-proximal gene, spolIGA, that is essential for sporulation. Gene fusions to the promoter of this operon, spoliG, demonstrated that transcription from this promoter is induced at the beginning of sporulation and is dependent on several spoO genes.

The construction of allele-specific suppressor mutations has enabled us to demonstrate that a sporulationspecific transcription unit in Bacilus subtilis, the spolIG operon, is transcribed by a form of RNA polymerase associated with a A, the principal a factor in vegetative cells. The spolIG operon encodes sporulation-specific factor o E, and its transcription is directed from a promoter that is activated about 1 hr after the onset of endospore formation. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by raassociated RNA polymerase, but these A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We have found that substitution of an arginyl residue for the glutamyl residue at position 196 of aPA suppresses the deleterious effect of a thymidine-to-cytidine base substitution at position -11 in the spoIIG promoter. This suppression was allele-specific, since it did not suppress the effects of base substitutions in other positions in the spoIIG promoter or the effects of a thymidine-to-guanosine change at -11. These results support a model in which a form of RNA polymerase containing a A is utilized in an unusual manner to activate the transcription of the spoIIG operon well after the onset of endospore formation.Bacillus subtilis can respond to nutrient depletion by undergoing a remarkably complex differentiation process culminating in the production of a dormant endospore. This cell differentiation requires the expression of more than 50 genetic loci (for review, see ref. 1). Many of the genes that are essential for endospore formation are regulated at the level of transcription-i.e., these genes are not transcribed until after the onset of spore formation. Moreover, these genes are transcribed in a precise temporal sequence. This temporal pattern of transcription during development appears to result in part from the stage-specific appearance of different forms of RNA polymerase associated with different a-factors (for review, see ref.2). The principal form of RNA polymerase present in vegetative cells (Eo-A) disappears during sporulation (3-5).The RNA polymerase factor a-E is produced about 2 hr after the onset of sporulation (6) and is essential for the transcription of some genes during stage II of endospore formation (7-9). The structural gene for oaE, sigE, is part of the spoIIG operon (9, 10). Transcription of this operon is activated about 30 min to 1 hr after the onset of sporulation (10). Little is known about the activation of spoIIG transcription except that the products of several genes are required (10,11), including spoOH, the structural gene for another RNA polymerase factor, a-H. Since mutations in spoOH, the structural gene for o-H, prevent transcription of spofiG, it would be reasonable to speculate that oa H may direct transcription from the spoIIG promoter. However, several considerations make this unlikely. The sequence of the spofIG promoter is not similar to sequ...

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by cA-associated RNA polymerase (EoA), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17-or 18-bp spacing typical of promoters utilized by EcA. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by ErA. Similar suppression experiments failed to provide evidence for a direct interaction between Ecr and the "-35-like" element, however, and DNase I protection experiments suggested instead that the SpoOA protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of SpoOA in vitro paralleled their effects on transcription in vivo. We suggest that spolIE belongs to a class of early-intermediate sporulation genes whose transcription by E&-is activated by the SpoOA protein.Under conditions of nutrient limitation, Bacillus subtilis bacteria initiate a differentiation process which culminates in the formation of dormant endospores. This complex series of biochemical and morphological changes, which probably involves over 100 gene products, requires the temporally regulated activation of many transcription units (20). Changes in gene expression are driven, in part, by the sequential appearance of new sigma factors, which bind to core RNA polymerase (E) and confer on the holoenzyme (Ecr) the capacity to recognize new classes of sporulationspecific promoters (40). Known examples include qH, which participates in the activation of early genes, oE and uF, which regulate early-intermediate genes, and uG and UKK, which control the compartment-specific expression of gene sets activated at later times in the developing cell.Each class of promoters has a characteristic and distinct polymerase consensus sequence, typically centered about 10 and 35 bp upstream of the transcription start site (27), an observation which formed the basis for the model that or directs RNA polymerase to its cognate promoters by making sequence-specific contacts in these regions (19). The most convincing evidence for this model comes from experiments in which single amino acid changes in the cr protein were found to alter its promoter recognition specificity (9,37,42,49). These experiments suggest a direct interaction of cf with DNA and have identified regions of the a protein that are likely to contact the -10 and -35 regions of promoters.The spoIIE operon is a developmentally regulated, sporulation-specific transcription unit activated in the second hour of sporulation (1...

A sporulation-specific sigma factor of Bacillus subtilis ('E) is formed by a proteolytic activation of a precursor protein (P3l). Synthesis of the precursor protein is shown to be abolished in B. subtilis mutants with plasmid insertions as far as 940 base pairs upstream of the P31 structural gene (sigE), and processing of p3l to {rE is blocked by a deletion in this upstream region. These results substantiate the view that sigE is the distal member of a 2-gene operon and demonstrate that the upstream gene (spolIGA) is necessary for ifE formation.Bacillus subtilis responds to carbon, nitrogen, or phosphate deprivation with a program of sequential gene activation which transforms vegetatively growing bacteria into dormant endospores. At least a part of the mechanism for the proper activation of sporulation-specific genes is believed to occur through modifications to the DNA-dependent RNA polymerase of the developing cell (9). The bestcharacterized sporulation-specific RNA polymerase modification involves the association of a novel sigma factor (0rE, formerly a29) with the core enzyme (2,4,8 Synthesis of (rE is highly regulated. Transcription of the spoIIg operon commences only after the onset of sporulation and is dependent on a number of stage 0 sporulation gene products (5, 16). Aside from transcriptional regulation, a.E synthesis is controlled posttranslationally. The primary product of sigE translation is not C.E, but rather an inactive precursor protein (p31) (17). 0.E is derived from the precursor by a proteolytic modification which removes 29 amino acids from the P31 amino terminus (7). The activity responsible for processing p3' to a0E appears in sporulating cells between 1 and 2 h (Tl to T2) into development and is itself under spore gene control (18). We had previously noted that mutations at the spoIIE locus permitted the synthesis of p31 but not its processing (18). Recently, an analysis of the organization of the spoIIG operon provided evidence that spoIIG consists of two sporulation-essential genes: a promoter-proximal gene spoIIGA and a promoter-distal gene sigE (5).In this report we demonstrate directly that p31 synthesis does not occur if plasmid insertions interrupt the upstream region of the spoIIG operon; we also show that an upstream gene, spoIIGA, is essential for the processing of p31 to c29.The spoIIG operon therefore defines both the structural gene for 0.E and a gene which participates in its synthesis. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis SMY (trpC), JH642 (trpC2 phe-1), and SL608 (spoIIG49 leuA8 tal-J) were * Corresponding author. obtained from R. Losick, J. Hoch, and P. Piggot, respectively. BS50 (metC3 tal-J spoIIG4J) and the plasmids pGSIIG3 and pGSIIG12 were from J. Szulmajster. B. subtilis EU8722, EU8723, EU8724, EU8719, EU8739, EU8725, and EU8737 carrying integrated plasmids have been described previously (5). Expression of cloned sigE in the plasmid and bacteriophage vectors appears to depend on transcription initiating within the vector sequences. This...