JOURNAL OF BACTERIOLOGY, Sept. 2009, p. 5859–5864 Vol. 191, No. 180021-9193/09/$08.00�0 doi:10.1128/JB.00724-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Pneumococcal LytR, a Protein from the LytR-CpsA-Psr Family,Is Essential for Normal Septum Formation in

Streptococcus pneumoniae�†Ola Johnsborg* and Leiv Sigve Håvarstein

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Ås, Norway

Received 4 June 2009/Accepted 29 June 2009

Proliferation of the human-pathogenic bacterium Streptococcus pneumoniae is fundamentally linked to thebacterial proteins that function in cell division. Here, we show that LytR, a pneumococcal protein from theLytR-CpsA-Psr family, is essential to this process.

Recently, we and others have described a mechanism foractive acquisition of homologous DNA that increases the effi-ciency of gene exchange between pneumococci and their closerelatives (2, 5, 6, 8, 9, 19, 20). This mechanism, termed “frat-ricide,” enables competent Streptococcus pneumoniae cells toattack and lyse noncompetent bacteria from closely relatedspecies, resulting in the release of chromosomal DNA from thetarget cells. In liquid cultures, competent pneumococci triggerlysis of the target cells by production and secretion of themurein hydrolase CbpD (10). CbpD represents a double-edged sword, since this enzyme also has an intrinsic ability toattack the cell wall of the producers. In order to protect theirown cell wall, competent pneumococci therefore produce acompetence-induced immunity protein, ComM (13). ThecomM operon also includes lytR (Fig. 1A), which clearly en-codes a protein that belongs to the LytR-CpsA-Psr family (1, 7,11, 17). Although LytR-CpsA-Psr family members are omni-present in gram-positive bacteria, their cellular function re-mains unknown. Interestingly, such proteins are generally notpresent in gram-negative bacteria. The family consists of pro-teins that carry a LytR-CpsA-Psr domain with a hitherto un-identified function. Typically, these proteins are composed ofan N-terminal part comprising one or several transmembranehelices and an extracellular C-terminal part that comprises theLytR-CpsA-Psr domain (7). Transcriptional analyses have pre-viously demonstrated that transcription of pneumococcal lytRincreases upon binding of ComE to the comM promoter duringcompetence induction, indicating that LytR perhaps couldserve a specific function in fratricide immunity (3, 14). How-ever, it has also been speculated that a putative extended �10promoter element (5�-TNTGNTATAAT-3�) situated immedi-ately upstream of spr1760 could drive expression of lytR innoncompetent cells (6) (Fig. 1A). To investigate the basalexpression of lytR in noncompetent cells, we inserted a lucif-erase reporter gene downstream of lytR in strain R704, giving

rise to strain RH424 (Table 1; and see the supplemental ma-terial for a full description of methods). As shown in Fig. 1B,RH424 displayed high luciferase activity during the early log-arithmic growth phase. In comparison, a strain carrying theluciferase reporter gene fused to the coding region of comM(RH21) displayed little or no luciferase activity. These resultsdemonstrate that the genes downstream of comM are regu-lated separately from comM during normal growth. To furtherconfirm the results from the luciferase reporter study, we nextexchanged the wild-type lytR gene with a mutant lytR geneencoding a C-terminal polyhistidine tag. In a previous studyaimed at identifying fratricide immunity factors in S. pneu-moniae, it was demonstrated that lytR could not be deleted bymariner mutagenesis, suggesting that lytR is an essential gene inS. pneumoniae (6). To facilitate the exchange of wild-type lytRwith a His-tagged version of the gene, we therefore used thecounterselectable Janus system to insert an additional copy oflytR into the genome of RH426, resulting in strain RH428. Theectopic lytR copy (pEXT1::lytR), which upon integration sub-stitutes for a Janus cassette introduced into the truncatedIS1167 element located downstream of the ami operon, isexpressed from a strong synthetic version of the dpnM ex-tended �10 promoter previously described by Sabelnikov et al.(16). Next, a Janus cassette (lytR::Janus) was inserted into thewild-type lytR locus. The resulting strain, RH429, did not dis-play any altered growth characteristics compared to the parentstrain, demonstrating that pEXT1::lytR fully compensates fordeletion of the wild-type lytR locus (data not shown). Next, thelytR::Janus cassette in RH429 was exchanged with a His-taggedcopy of lytR (RH439). Subsequent deletion of pEXT1::lytRresulted in strain RH440. Samples of strain RH440 were sub-sequently harvested at different stages during growth and an-alyzed by Western blotting (Fig. 1C). The immunoblottingresults confirmed that LytR is constitutively synthesized duringexponential growth. Taken together with the fact that a previ-ous study suggested that pneumococcal lytR is essential inwild-type cells, the above data strongly support a housekeepingfunction of LytR in dividing cells. In our experience, whenkanamycin is used as a selection marker for gene deletion,mutant cells normally emerge on agar plates after 12 to 16 h ofincubation at 37°C. During the current work, we discoveredthat small colony-sized kanamycin-resistant mutants could berescued at a low frequency when pEXT1::lytR in strain RH430

* Corresponding author. Mailing address: Department of Chemis-try, Biotechnology, and Food Science, Norwegian University of LifeSciences, P.O. Box 5003, N-1432 Ås, Norway. Phone: 47-64965887.Fax: 47-64965901. E-mail: ola.johnsborg@umb.no.

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

� Published ahead of print on 6 July 2009.

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was deleted by replacement with a Janus kan-rpsL� cassette(strain RH433). However, mutant cells would only emerge onthe agar plates when the incubation time at 37°C was extendedto 36 h. As compared to the parent strains RH426 and RH430,RH433 grew at a much slower rate and displayed prematureautolysis (Fig. 2). Next, pEXT1::lytR was reintroduced into thisstrain, thus replacing the Janus kan-rpsL� fragment. This back-cross resulted in cells (RH436) with significantly higher growthrate than the RH433 parent strain (Fig. 2). However, reintro-duction of lytR could not fully restore the growth rate back tothe level displayed by strain RH426 or strain RH430. The factthat wild-type growth could not be fully restored hinted at thepresence of a suppressor mutation in strain RH433. Inductionof competence in S. pneumoniae results in production of themurein hydrolase CbpD. Competent cells also produce theComM immunity protein, which by an unknown mechanismprotects the competent cells from the activity of CbpD. Inter-estingly, competence induction in the lytR-deficient strain

RH433 resulted in a drastic drop in optical density (data notshown), indicating that rapid autolysis was taking place. Au-tolysis was not observed in competent RH435 cells (�lytR�cbpD). Reversion of the RH433 lytR deletion mutant back toa lytR� phenotype could not fully restore immunity towardCbpD, as a CbpD-dependent drop in optical density was ob-served when cultures of RH436 were induced to competence(data not shown). This observation supports that a suppressormutation is formed upon lytR deletion. DNA sequencing dem-onstrated that this mutation does not locate to the region ofthe comM operon, and the genomic location of the suppressorhence remains unknown. In S. pneumoniae, stationary-phaseautolysis is known to be mediated by the murein hydrolaseLytA. Although the cells appear to constitutively synthesizeLytA during growth, the enzymatic activity of LytA normallymanifests itself some hours after the onset of stationary phase(12, 21). The observation that lytR deletion mutants undergorapid premature autolysis led us to investigate whether this

FIG. 1. LytR is constitutively expressed during growth. (A) Genetic organization of lytR. Both the ComE-dependent promoter located upstreamof comM and the constitutive extended �10 promoter located upstream of spr1761 are indicated. (B) Luciferase expression from RH424 (lytR::luc)and RH21 (comM::luc). Luciferase activity is presented as relative luminescence units (RLU)/optical density (OD). Black diamonds, RH424; whitetriangles, RH21. Growth was measured as OD at 492 nm (OD492). White diamonds, RH424; black triangles, RH21. RLU and OD492 weredetermined at 15-min intervals. (C) Detection of His-tagged LytR by Western blotting. Cells were harvested at the indicated points of growth, andwhole-cell extracts were loaded onto a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After transfer to polyvinylidenedifluoride membranes, His-tagged LytR was detected using the SuperSignal West Pico HisProbe kit (Pierce). A sample of RH425 harvested at anOD492 of 0.1 was used as the negative control (Ng). The specific bands observed upon immunodetection correspond to the calculated molecularmass of His-tagged LytR (38 kDa). Note that LytR still could be detected at an OD of 0.8 (data not shown).

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lysis was dependent upon LytA. Indeed, deletion of lytR in a�lytA background resulted in cells (RH434) that displayed agrowth rate similar to that of RH433 but did not autolyse (Fig.2). Hence, cells deleted for lytR become highly sensitized to-ward both LytA and CbpD. It has previously been demon-strated that LytA-dependent autolysis can be triggered bytreatment with a number of different compounds, such as de-tergents and penicillin, as well as other cell wall inhibitors (4,13, 15). Common for these compounds is that they damage thecell envelope. We therefore hypothesized that deletion of lytRgenerates alterations in the structure of the pneumococcal cellwall that trigger LytA activity. To investigate whether these

putative alterations resulted in a visible change in cell mor-phology, we analyzed lytR deletion mutants by scanning elec-tron microscopy (SEM) and transmission electron microscopy(TEM). The SEM analysis revealed that strains RH426 andRH430 grew as well-defined diplococci of relatively uniformsize. In contrast, lytR mutant cells displayed high variability inboth size and shape. Some of the mutant cells were two tothree times larger than wild-type cells, whereas others grew asirregularly shaped minicells. In contrast, lytR deletion mutantsthat were reverted to a lytR� phenotype by reinsertion of thepEXT1::lytR copy (RH436) displayed a morphological pheno-type similar to that of strains RH426 and RH430 (data not

FIG. 2. Deletion of lytR results in reduced growth and premature autolysis. Cells were grown at 37°C in C medium, and the optical density at492 nm (OD492) was measured at 15-min intervals. White squares, RH426 (�IS1167::Janus); black squares, RH430 (�lytR pEXT1::lytR); blacktriangles, RH433 (�lytR); black diamonds, RH436 (�lytR; backcross of pEXT1::lytR); white triangles, RH434 (�lytR �lytA).

TABLE 1. Characteristics of the strains used in this study

Strain or plasmid Genotype/relevant feature(s)a Source or reference

StrainsR704 R6 derivative; comA::ermAM Eryr 6CP 1200 Rx, but malM511 str-1 Smr 18RH21 R704, but comM::luc (pRcomM) comM Eryr Cmr 8RH424 R704, but lytR::luc (pRlytR) lytR� Eryr Cmr This studyRH425 R704, but Smr by transformation with CP1200 chromosomal DNA, Eryr Smr This studyRH426 RH425, but � IS1167::Janus Eryr Kanr This studyRH428 RH426, but substitution of Janus cassette with pEXT1::lytR, Eryr Smr This studyRH429 RH428, but deletion of wild-type lytR locus by insertion of a lytR::Janus cassette, Eryr Kanr This studyRH430 RH429, but �lytR by transformation with a PCR fragment substituting for the lytR::Janus

insertion, Eryr SmrThis study

RH431 RH430, but �lytA::spc Eryr Smr Spcr This studyRH432 RH430, but �cbpD::spc Eryr Smr Spcr This studyRH433 RH430, but �pEXT1::lytR by insertion of �IS1167::Janus PCR fragment, Eryr Kanr This studyRH434 RH431, but �pEXT1::lytR by insertion of �IS1167::Janus PCR fragment, Eryr Kanr Spcr This studyRH435 RH432, but �pEXT1::lytR by insertion of �IS1167::Janus PCR fragment, Eryr Kanr Spcr This studyRH436 RH433, but substitution of Janus cassette with pEXT1::lytR, Eryr Smr This studyRH437 RH436, but �cbpD::Janus Eryr Kanr This studyRH438 RH436, but �comM::Janus Eryr Kanr This studyRH439 RH429, but lytR::his by transformation with a PCR fragment substituting for the

lytR::Janus insertion, Eryr SmrThis study

RH440 RH439, but deletion of pEXT1::lytR by transformation with a �IS1167::Janus PCRfragment, Eryr Kanr

This study

PlasmidpRlytR pR424 derivative, Cmr, carries a lytR targeting fragment and luc gene, insertion duplication

in S. pneumoniae generates a lytR::luc (lytR�) fusionThis study

a Cm, chloramphenicol; Ery, erythromycin; Kan, kanamycin; Spc, spectinomycin; Sm, streptomycin. “Janus” indicates the presence of a kan::rpsL cassette.

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FIG. 3. TEM of S. pneumoniae cells. Thin sections of early exponential phase cells were prepared as described in Materials and Methods.(A) RH426; (B and C) RH433 (�lytR); (D) RH436 (�lytR; backcross of pEXT1::lytR). Anomalous division sites in RH433 are indicated by arrows.Note that although normal septum formation was restored in RH436, some of the RH436 cells appeared to inherit a slightly elongated or“stretched” shape compared to the cells of strain RH426.

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shown). The basis for the altered morphology of lytR mutantswas identified in the TEM analysis. As expected, thin sectionsof strain RH426 displayed cells with symmetrical septum for-mation that divided into evenly shaped progeny of the ex-pected size (Fig. 3A). In contrast, a significant number of theRH433 cells displayed nonsymmetrical septum formation inwhich several septa often were observed to be distributed atmultiple sites within the same cell. In contrast to the perpen-dicular septa found in strain RH426, the septa in RH433 werefrequently found to form at odd angles with respect to the longaxis of the cell (Fig. 3B and C). Thus, the abnormal morphol-ogy of RH433 cells results from lack of nonsymmetrical celldivision and frequent formation of multiple septa. As shown inFig. 3D, symmetrical septum formation was restored uponreversion of the RH433 lytR deletion mutant back to a lytR�

phenotype by insertion of pEXT1::lytR (RH436). This resultconfirmed that the observed defect in control of septum place-ment in RH433 is due to the deletion of lytR.

The present work was originally initiated to investigatewhether LytR participates in the CbpD immunity mechanismduring competence-induced fratricide in S. pneumoniae. Al-though lytR mutants appeared to be sensitized toward mureinhydrolase activity in general, the data do not support that lytRis expressed to perform a specific role in fratricide immunity.Rather, the data suggest that the main role of LytR is per-formed during exponential growth, where the protein is essen-tial for proper septum placement. Further work to define theexact role of LytR in pneumococcal cell division is in progress.

We thank H. R. Kolstad, E. Ørmen, and T. Krekling at the IPMimaging laboratory for technical assistance with the SEM and TEManalyses.

This work was supported by the Research Council of Norway.

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