Characterization of divIVA and other genes located in the chromosomal region downstream of the dcw cluster in Streptococcus pneumoniae - PubMed (original) (raw)

Characterization of divIVA and other genes located in the chromosomal region downstream of the dcw cluster in Streptococcus pneumoniae

Daniela Fadda et al. J Bacteriol. 2003 Oct.

Abstract

We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function. Inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division. Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene. Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization.

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Figures

FIG.1.

FIG.1.

Phase-contrast analysis of the Rx1 wild-type strain and the insertion-deletion mutants. Bacterial cells were cultured in TSB medium, sampled at selected times during the exponential phase of growth, fixed with 1% (vol/vol) formaldehyde for 15 min at room temperature, treated with DAPI as previously described (2), and examined by using an Axioskop HBO50 equipped with a Plan-Neofluar 100× oil lens. For each strain, phase-contrast (left panels) and DAPI stain fluorescence (right panels) micrographs are shown. The scale bars correspond to 3 μm.

FIG. 2.

FIG. 2.

Electronic transmission analysis of the Rx1 wild-type strain and of the insertion-deletion mutants. Cells were cultured in TSB medium to mid-exponential phase, fixed in 1% (vol/vol) glutaraldehyde for 2 h at room temperature, processed as previously described (11), and then observed and photographed in a Zeiss EM 10 electron microscope. The abnormalities of various null mutants are described in the text and summarized in Table 1. The scale bars correspond to 0.3 μm.

FIG. 3.

FIG. 3.

Transcriptional analysis of the region downstream of each insertion by RT-PCR. The random primer Pd(N)6 or the specific primer PDIVR was used to generate cDNA. Total RNA from the S. pneumoniae Rx1 strain and ylm::cat mutants was used as the template in a reverse transcriptase reaction with primers (PAF, PZR, PFF, PFR, PGF, PGR, PHF, PHR, and PDIVF) internal to each gene. Expression of the genes in the wild-type Rx1 strain and in the insertion-deletion mutants would give products of the following sizes: ylmF (F), 477 bp; ylmG (G), 202 bp; ylmH (H), 661 bp; and divIVA (DIV), 663 bp. A positive control, _ftsA_-ftsZ (AZ), 1,902 bp, was included in all reactions. Negative controls included identical reactions to which no cDNA template was added. DNA contamination of the RNA samples was ruled out by performing PCR directly on the RNAs (data not shown). The source of the respective RNA template used for each set of reactions is indicated under each panel. M, _Hin_dIII-digested λ phage.

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