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Involvement of FtsE ATPase and FtsX extracellular loops 1 and 2 in FtsEX-PcsB complex function in cell division of Streptococcus pneumoniae D39.

Sham LT, Jensen KR, Bruce KE, Winkler ME - MBio (2013)

Bottom Line: Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB).These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB.Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University Bloomington, Bloomington, Indiana, USA.

ABSTRACT

Unlabelled: The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.

Importance: Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsBCC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

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Delayed autolysis at 37°C of single mutants pcsBL78S-L219P(Ts) and ftsXV266A(Sup) and double pcsBL78S-L219P(Ts) ftsXS264L(Sup) and pcsBL78S-L219P(Ts) ftsXV266A(Sup) mutants. (A) Strains IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], IU6426 [ftsXS264L(Sup)], IU6371 [pcsBL78S-L219P(Ts) ftsXS264L(Sup)], IU6439 [ftsXV266A(Sup)], and IU6363 [pcsBL78S-L219P(Ts) ftsXV266A(Sup)] were grown in BHI broth at 32°C and diluted to an OD620 of ~0.001 in fresh BHI broth prewarmed to the temperatures indicated. Growth was monitored as described in Materials and Methods, and representative growth curves from more than 3 independent experiments are shown. ftsXS264L(Sup) and ftsXV266A(Sup) are non-allele-specific and allele-specific suppressors of pcsBCC(Ts), respectively (Fig. 1; see the text). A comparable delay in autolysis was detected for strain IU4262 [pcsBA160P(Ts)] (see Fig. S3 in the supplemental material). (B) At an OD620 of 0.7 in 37°C cultures, cells of strain IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], and IU4262 [pcsBA160P(Ts)] were stained for viability as described in Materials and Methods. Experiments were performed independently three times (n = 3), and ~1,300 cells were counted per sample (B).
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fig4: Delayed autolysis at 37°C of single mutants pcsBL78S-L219P(Ts) and ftsXV266A(Sup) and double pcsBL78S-L219P(Ts) ftsXS264L(Sup) and pcsBL78S-L219P(Ts) ftsXV266A(Sup) mutants. (A) Strains IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], IU6426 [ftsXS264L(Sup)], IU6371 [pcsBL78S-L219P(Ts) ftsXS264L(Sup)], IU6439 [ftsXV266A(Sup)], and IU6363 [pcsBL78S-L219P(Ts) ftsXV266A(Sup)] were grown in BHI broth at 32°C and diluted to an OD620 of ~0.001 in fresh BHI broth prewarmed to the temperatures indicated. Growth was monitored as described in Materials and Methods, and representative growth curves from more than 3 independent experiments are shown. ftsXS264L(Sup) and ftsXV266A(Sup) are non-allele-specific and allele-specific suppressors of pcsBCC(Ts), respectively (Fig. 1; see the text). A comparable delay in autolysis was detected for strain IU4262 [pcsBA160P(Ts)] (see Fig. S3 in the supplemental material). (B) At an OD620 of 0.7 in 37°C cultures, cells of strain IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], and IU4262 [pcsBA160P(Ts)] were stained for viability as described in Materials and Methods. Experiments were performed independently three times (n = 3), and ~1,300 cells were counted per sample (B).

Mentions: We characterized the growth of the Ts and suppressor strains described above at the permissive temperatures of 32°C and 37°C (Fig. 4A; also, see Fig. S3 in the supplemental material). The single mutants containing pcsBL78S-L219P(Ts), pcsBA160P(Ts), ftsXS264L(Sup), or ftsXV266A(Sup) (where Sup indicates that the mutation acts as a suppressor) and the suppressed double mutants containing pcsBL78S-L219PftsXS264L or pcsBL78S-L219PftsXV266A grew like the parent strain at 32°C (Fig. 4A; also, see Fig. S3). In contrast, the pcsBW335G(Ts) mutant grew more slowly and to a lower yield than the other strains, even at 32°C (see Fig. S3). At 37°C, cultures of the parent strain and the single mutants containing ftsXS264L(Sup) or pcsBW335G(Ts) showed the characteristic drop in optical density at 620 nm (OD620) in stationary phase, indicative of autolysis (Fig. 4A; also, see Fig. S3). Unexpectedly, cultures of the single mutants containing pcsBL78S-L219P(Ts), pcsBA160P(Ts), or ftsXV266A(Sup) and the suppressed double mutants containing pcsBL78S-L219PftsXS264L or pcsBL78S-L219PftsXV266A did not show this pronounced drop in OD620 in stationary phase (Fig. 4A; also, see Fig. S3). Consistent with a reduced drop in OD620, live-dead staining (Materials and Methods) showed that the pcsBL78S-L219P(Ts) and pcsBA160P(Ts) mutants retained cell viability in early stationary phase (OD620 ≈ 0.7) at 37°C compared to a drop for the parent strain (Fig. 4B).


Involvement of FtsE ATPase and FtsX extracellular loops 1 and 2 in FtsEX-PcsB complex function in cell division of Streptococcus pneumoniae D39.

Sham LT, Jensen KR, Bruce KE, Winkler ME - MBio (2013)

Delayed autolysis at 37°C of single mutants pcsBL78S-L219P(Ts) and ftsXV266A(Sup) and double pcsBL78S-L219P(Ts) ftsXS264L(Sup) and pcsBL78S-L219P(Ts) ftsXV266A(Sup) mutants. (A) Strains IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], IU6426 [ftsXS264L(Sup)], IU6371 [pcsBL78S-L219P(Ts) ftsXS264L(Sup)], IU6439 [ftsXV266A(Sup)], and IU6363 [pcsBL78S-L219P(Ts) ftsXV266A(Sup)] were grown in BHI broth at 32°C and diluted to an OD620 of ~0.001 in fresh BHI broth prewarmed to the temperatures indicated. Growth was monitored as described in Materials and Methods, and representative growth curves from more than 3 independent experiments are shown. ftsXS264L(Sup) and ftsXV266A(Sup) are non-allele-specific and allele-specific suppressors of pcsBCC(Ts), respectively (Fig. 1; see the text). A comparable delay in autolysis was detected for strain IU4262 [pcsBA160P(Ts)] (see Fig. S3 in the supplemental material). (B) At an OD620 of 0.7 in 37°C cultures, cells of strain IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], and IU4262 [pcsBA160P(Ts)] were stained for viability as described in Materials and Methods. Experiments were performed independently three times (n = 3), and ~1,300 cells were counted per sample (B).
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fig4: Delayed autolysis at 37°C of single mutants pcsBL78S-L219P(Ts) and ftsXV266A(Sup) and double pcsBL78S-L219P(Ts) ftsXS264L(Sup) and pcsBL78S-L219P(Ts) ftsXV266A(Sup) mutants. (A) Strains IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], IU6426 [ftsXS264L(Sup)], IU6371 [pcsBL78S-L219P(Ts) ftsXS264L(Sup)], IU6439 [ftsXV266A(Sup)], and IU6363 [pcsBL78S-L219P(Ts) ftsXV266A(Sup)] were grown in BHI broth at 32°C and diluted to an OD620 of ~0.001 in fresh BHI broth prewarmed to the temperatures indicated. Growth was monitored as described in Materials and Methods, and representative growth curves from more than 3 independent experiments are shown. ftsXS264L(Sup) and ftsXV266A(Sup) are non-allele-specific and allele-specific suppressors of pcsBCC(Ts), respectively (Fig. 1; see the text). A comparable delay in autolysis was detected for strain IU4262 [pcsBA160P(Ts)] (see Fig. S3 in the supplemental material). (B) At an OD620 of 0.7 in 37°C cultures, cells of strain IU1945 (parent), IU4261 [pcsBL78S-L219P(Ts)], and IU4262 [pcsBA160P(Ts)] were stained for viability as described in Materials and Methods. Experiments were performed independently three times (n = 3), and ~1,300 cells were counted per sample (B).
Mentions: We characterized the growth of the Ts and suppressor strains described above at the permissive temperatures of 32°C and 37°C (Fig. 4A; also, see Fig. S3 in the supplemental material). The single mutants containing pcsBL78S-L219P(Ts), pcsBA160P(Ts), ftsXS264L(Sup), or ftsXV266A(Sup) (where Sup indicates that the mutation acts as a suppressor) and the suppressed double mutants containing pcsBL78S-L219PftsXS264L or pcsBL78S-L219PftsXV266A grew like the parent strain at 32°C (Fig. 4A; also, see Fig. S3). In contrast, the pcsBW335G(Ts) mutant grew more slowly and to a lower yield than the other strains, even at 32°C (see Fig. S3). At 37°C, cultures of the parent strain and the single mutants containing ftsXS264L(Sup) or pcsBW335G(Ts) showed the characteristic drop in optical density at 620 nm (OD620) in stationary phase, indicative of autolysis (Fig. 4A; also, see Fig. S3). Unexpectedly, cultures of the single mutants containing pcsBL78S-L219P(Ts), pcsBA160P(Ts), or ftsXV266A(Sup) and the suppressed double mutants containing pcsBL78S-L219PftsXS264L or pcsBL78S-L219PftsXV266A did not show this pronounced drop in OD620 in stationary phase (Fig. 4A; also, see Fig. S3). Consistent with a reduced drop in OD620, live-dead staining (Materials and Methods) showed that the pcsBL78S-L219P(Ts) and pcsBA160P(Ts) mutants retained cell viability in early stationary phase (OD620 ≈ 0.7) at 37°C compared to a drop for the parent strain (Fig. 4B).

Bottom Line: Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB).These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB.Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University Bloomington, Bloomington, Indiana, USA.

ABSTRACT

Unlabelled: The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.

Importance: Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsBCC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

Show MeSH
Related in: MedlinePlus