Genetic analysis of the Staphylococcus epidermidis macromolecular synthesis operon: Serp1129 is an ATP binding protein and sigA transcription is regulated by both sigma(A)- and sigma(B)-dependent promoters

Abstract

Background: The highly conserved macromolecular synthesis operon (MMSO) contains both dnaG (primase) and sigA (primary sigma factor). However, in previously evaluated gram-positive species, the MMSO is divergent upstream of dnaG. The MMSO of Bacillus subtilis contains three open reading frames (ORFs) that are differentially regulated by multiple promoters. In conjunction with studies to determine the expression profile of dnaG, the MMSO of Staphylococus epidermidis was characterized.

Results: The ORFs of S. epidermidis were compared to the previously described MMSO of B. subtilis and two additional ORFs in S. epidermidis, serp1129 and serp1130, were identified. The largest transcript, 4.8 kb in length, was expressed only in exponential growth and encompassed all four ORFs (serp1130, serp1129, dnaG, and sigA). A separate transcript (1.5 kb) comprising serp1130 and serp1129 was expressed in early exponential growth. Two smaller transcripts 1.3 and 1.2 kb in size were detected with a sigA probe in both exponential and post-exponential phases of growth. Western blot analysis correlated with the transcriptional profile and demonstrated that Serp1129 was detected only in the exponential phase of growth. Computational analysis identified that Serp1130 contained a CBS motif whereas Serp1129 contained an ATP/GTP binding motif. Functional studies of Serp1129 demonstrated that it was capable of binding both ATP and GTP. Comparisons with a sigB:dhfr mutant revealed that the 1.3 kb sigA transcript was regulated by a sigma(B)-dependent promoter.

Conclusions: These studies demonstrated that the S. epidermidis 1457 MMSO contains two ORFs (serp1129 and serp1130) not described within the B. subtilis MMSO and at least three promoters, one of which is sigma(beta)-dependent. The transcriptional regulation of sigA by sigma(B) provides evidence that the staphylococcal sigma(B)-dependent response is controlled at both the transcriptional and post-transcriptional level. The conservation of serp1129 across multiple gram-positive organisms and its capability to bind ATP and GTP support the need for further investigation of its role in bacterial growth.

Figure 1

Schematic diagram demonstrating the conservation of the MMSO region in four gram-positive bacteria. Genes contained within the S. epidermidis MMSO and their equivalents in Bacillus subtilis, Listeria monocytogenes, and Streptococcus pyogenes are highlighted in red. Orthologues that were identified in B. subtilis, L. monocytogenes, or S. pyogenes that are not found in S. epidermidis (between rpsU 5' of the MMSO and rhe 3' of the MMSO) are highlighted in green.

Figure 2

Growth analysis of S. epidermidis 1457. S. epidermidis was grown aerobically in tryptic soy broth over a 18 hour time period. Growth was assessed by measuring the optical density at 600 nm.

Figure 3

Northern blot analysis of the S. epidermidis MMSO using a sigA and dnaG DNA probe. The number above each lane in panels A (hybridized with a sigA probe) and B (hybridized with a dnaG probe) represents the time in hours of growth before each RNA sample was processed. A picture of the ethidium bromide stained gel is shown beneath each blot to serve as a loading control and verify RNA integrity. Arrows in panels A and B denote transcripts A, C through F as discussed in text. Panel C: Schematic depiction of the S. epidermidis MMSO. Small arrows above and below the schematic represent primer sets used in RT-PCR reactions and other cloning experiments. Arrows below the schematic correspond to transcripts A, B, C, and D as discussed in text.

Figure 4

Northern blot analysis of the S. epidermidis MMSO using a serp1129 and serp1130 DNA probe. The number above each lane in panels A (hybridized with a serp1129 DNA probe) and B (hybridized with a serp1130 DNA probe) represents the time in hours of growth before each RNA sample was processed. A picture of the ethidium bromide stained gel is shown beneath each blot to serve as a loading control and verify RNA integrity. Arrows in panels A and B denote transcripts A, B, E and F as discussed in text.

Figure 5

Primer extension analysis of the S. epidermidis MMSO. Primer extension showing the +1 transcriptional start site (denoted by small arrow) of the (A) P1 promoter upstream of serp1130 using primer 1178, (B) σ^B-dependent P2 promoter upstream of sigA using primer 1222, and (C) P3 promoter upstream of sigA using primer 1194. WT above each panel represents wildtype S. epidermidis 1457, whereas σ^Bdenotes 1457 sigB::dhfr. (D) Schematic diagram showing the position of proposed promoters (P1, P2, and P3) in the MMSO of S. epidermidis. Small arrows depict the position of the primer extension and RACE primers used to detect the three transcriptional initiation sites. Sequence of putative -35 and -10 boxes, defined transcriptional start site (+1) and ATG start site of (E) P1 promoter, (F) σ^B-dependent P2 promoter, and (G) P3 promoter.

Figure 6

Northern blot analysis of 1457 and 1457 sigB::dhfr using a sigA probe. The number above each lane represents the time in hours of growth before each RNA sample was processed. WT above each lane represents wildtype S. epidermidis 1457, whereas σ^Bdenotes 1457 sigB::dhfr. Small arrows denote transcripts C and D as discussed in text.

Figure 7

Western blot analysis to demonstrate Serp1129 expression. Western blot analysis showing the expression of Serp1129 from 2 to 12 hours of growth. Number above each lane represents the hour (growth) at which the protein sample was collected. The arrow on the left of the figure notes the expression of the 30.8 kDa native Serp1129 throughout growth of S. epidermidis 1457. The "+" lane is the positive control containing the 35.6 kDa recombinant His- tagged Serp1129 protein and is denoted by an arrow on the right.

Figure 8

ATP and GTP Competition Assays for Serp1129. (A) ATP and GTP binding assay. The lane marked "0" indicates that no unlabeled ATP or GTP was added to the reaction and increasing levels (5, 10, 20, and 30 μM) of unlabeled ATP or GTP are indicated by the triangle above the appropriate lanes. The lanes marked as "-" are the negative control containing CidA [38], which does not bind ATP or GTP. B. SDS-PAGE loaded with the same protein concentration of Serp1129 as in Figure 6A and stained with Coomassie Blue; shown as a loading control.