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MioC and GidA proteins promote cell division in E. coli.

Lies M, Visser BJ, Joshi MC, Magnan D, Bates D - Front Microbiol (2015)

Bottom Line: The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function.Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress.Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Human Genetics, Baylor College of Medicine Houston, TX, USA.

ABSTRACT
The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

No MeSH data available.


Related in: MedlinePlus

Promoter mutations do not impair DNA replication or chromosome segregation. (A) Rifampicin runoff DNA histograms in PmioC and PgidA mutants indicate synchronous replication initiations. Numbers represent the number of chromosomes at the major peaks. (B) DNA/mass in exponentially growing cultures was measured by flow cytometry of DAPI stained cells (values are relative to wildtype). (C) The ratio of oriC sites to ter sites in above cells was measured by qPCR, indicating the rate of DNA replication and whether replication forks were blocked between oriC and ter. Values in B,C are means of 3 independent experiments (±1 SD). (D) The positions of oriC (green foci) and ter (red foci) in elongating triple mutant PmioC PgidA fis cells was analyzed by FISH (left panel). Bright extracellular foci (appearing as red halos) are multi-fluorescent beads added for image alignment. DAPI staining (right panel) indicates ter foci are located between segregated nucleoids, consistent with normal chromosome segregation (Text). See Material and Methods for details.
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Figure 4: Promoter mutations do not impair DNA replication or chromosome segregation. (A) Rifampicin runoff DNA histograms in PmioC and PgidA mutants indicate synchronous replication initiations. Numbers represent the number of chromosomes at the major peaks. (B) DNA/mass in exponentially growing cultures was measured by flow cytometry of DAPI stained cells (values are relative to wildtype). (C) The ratio of oriC sites to ter sites in above cells was measured by qPCR, indicating the rate of DNA replication and whether replication forks were blocked between oriC and ter. Values in B,C are means of 3 independent experiments (±1 SD). (D) The positions of oriC (green foci) and ter (red foci) in elongating triple mutant PmioC PgidA fis cells was analyzed by FISH (left panel). Bright extracellular foci (appearing as red halos) are multi-fluorescent beads added for image alignment. DAPI staining (right panel) indicates ter foci are located between segregated nucleoids, consistent with normal chromosome segregation (Text). See Material and Methods for details.

Mentions: We previously reported that promoter mutations in mioC and gidA did not adversely affect the timing or efficiency of chromosomal replication (Bates et al., 1997). Even a subtle delay in replication could create an unbalanced cell cycle that results in delayed cell division. For example, blocking replication elongation by depleting dNTPs with hydroxyurea results in incomplete replication intermediates that block division independently of both SOS and SlmA (Cambridge et al., 2014). To confirm and extend our previous analysis of DNA replication, we measured DNA content and cell mass by flow cytometry in the mutant set. As expected, promoter single and double mutants in a fis+ background exhibited a normal number of chromosomes and synchronous replication initiation, as shown by rifampicin runoff histograms (Figure 4A). In this method, which involves blocking replication initiation with rifampicin and cell division with cephalexin and allowing ongoing replication forks to complete, cells accumulate a number of fully replicated chromosomes equal to the number of origins present at the time of drug treatment. Under our growth conditions (LB/37°C), wild-type cells mostly contained either 4 or 8 origins (Figure 4A, left). This indicates that replication initiation occurred synchronously, with all origins in a cell firing ~simultaneously, and thus containing 2n origins where n is equal to any positive integer. Double PmioC PgidA mutants exhibited slightly more 8-origin cells than wildtype, but this difference was not statistically significant. Rifampicin runoff in fis− cells did not produce discernable chromosome peaks in our flow cytometry analysis, likely owing to asynchronous replication (Flatten and Skarstad, 2013) and uneven DAPI staining due to abnormal chromosome structure (Skoko et al., 2006).


MioC and GidA proteins promote cell division in E. coli.

Lies M, Visser BJ, Joshi MC, Magnan D, Bates D - Front Microbiol (2015)

Promoter mutations do not impair DNA replication or chromosome segregation. (A) Rifampicin runoff DNA histograms in PmioC and PgidA mutants indicate synchronous replication initiations. Numbers represent the number of chromosomes at the major peaks. (B) DNA/mass in exponentially growing cultures was measured by flow cytometry of DAPI stained cells (values are relative to wildtype). (C) The ratio of oriC sites to ter sites in above cells was measured by qPCR, indicating the rate of DNA replication and whether replication forks were blocked between oriC and ter. Values in B,C are means of 3 independent experiments (±1 SD). (D) The positions of oriC (green foci) and ter (red foci) in elongating triple mutant PmioC PgidA fis cells was analyzed by FISH (left panel). Bright extracellular foci (appearing as red halos) are multi-fluorescent beads added for image alignment. DAPI staining (right panel) indicates ter foci are located between segregated nucleoids, consistent with normal chromosome segregation (Text). See Material and Methods for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446571&req=5

Figure 4: Promoter mutations do not impair DNA replication or chromosome segregation. (A) Rifampicin runoff DNA histograms in PmioC and PgidA mutants indicate synchronous replication initiations. Numbers represent the number of chromosomes at the major peaks. (B) DNA/mass in exponentially growing cultures was measured by flow cytometry of DAPI stained cells (values are relative to wildtype). (C) The ratio of oriC sites to ter sites in above cells was measured by qPCR, indicating the rate of DNA replication and whether replication forks were blocked between oriC and ter. Values in B,C are means of 3 independent experiments (±1 SD). (D) The positions of oriC (green foci) and ter (red foci) in elongating triple mutant PmioC PgidA fis cells was analyzed by FISH (left panel). Bright extracellular foci (appearing as red halos) are multi-fluorescent beads added for image alignment. DAPI staining (right panel) indicates ter foci are located between segregated nucleoids, consistent with normal chromosome segregation (Text). See Material and Methods for details.
Mentions: We previously reported that promoter mutations in mioC and gidA did not adversely affect the timing or efficiency of chromosomal replication (Bates et al., 1997). Even a subtle delay in replication could create an unbalanced cell cycle that results in delayed cell division. For example, blocking replication elongation by depleting dNTPs with hydroxyurea results in incomplete replication intermediates that block division independently of both SOS and SlmA (Cambridge et al., 2014). To confirm and extend our previous analysis of DNA replication, we measured DNA content and cell mass by flow cytometry in the mutant set. As expected, promoter single and double mutants in a fis+ background exhibited a normal number of chromosomes and synchronous replication initiation, as shown by rifampicin runoff histograms (Figure 4A). In this method, which involves blocking replication initiation with rifampicin and cell division with cephalexin and allowing ongoing replication forks to complete, cells accumulate a number of fully replicated chromosomes equal to the number of origins present at the time of drug treatment. Under our growth conditions (LB/37°C), wild-type cells mostly contained either 4 or 8 origins (Figure 4A, left). This indicates that replication initiation occurred synchronously, with all origins in a cell firing ~simultaneously, and thus containing 2n origins where n is equal to any positive integer. Double PmioC PgidA mutants exhibited slightly more 8-origin cells than wildtype, but this difference was not statistically significant. Rifampicin runoff in fis− cells did not produce discernable chromosome peaks in our flow cytometry analysis, likely owing to asynchronous replication (Flatten and Skarstad, 2013) and uneven DAPI staining due to abnormal chromosome structure (Skoko et al., 2006).

Bottom Line: The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function.Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress.Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Human Genetics, Baylor College of Medicine Houston, TX, USA.

ABSTRACT
The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

No MeSH data available.


Related in: MedlinePlus