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Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location.

Dryselius R, Izutsu K, Honda T, Iida T - BMC Genomics (2008)

Bottom Line: Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species.The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small.For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Genomic Research on Pathogenic Bacteria, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. rikard@biken.osaka-u.ac.jp

ABSTRACT

Background: Replication of bacterial chromosomes increases copy numbers of genes located near origins of replication relative to genes located near termini. Such differential gene dosage depends on replication rate, doubling time and chromosome size. Although little explored, differential gene dosage may influence both gene expression and location. For vibrios, a diverse family of fast growing gammaproteobacteria, gene dosage may be particularly important as they harbor two chromosomes of different size.

Results: Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species. We also investigated locations for specific gene types within the genome. The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small. Accordingly, large chromosome gene expression levels were generally higher and showed an influence from gene dosage. This was reflected by a higher abundance of growth essential and growth contributing genes of which many locate near the origin of replication. In contrast, small chromosome gene expression levels were low and appeared independent of gene dosage. Also, species specific genes are highly abundant and an over-representation of genes involved in transcription could explain its gene dosage independent expression.

Conclusion: Here we establish a link between replication dynamics and differential gene dosage on one hand and gene expression levels and the location of specific gene types on the other. For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity.

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Replication pattern for exponentially growing V. parahaemolyticus determined by gDNA/gDNA microarrays. Genomic DNA (gDNA) from V. parahaemolyticus in exponential phase (OD600 ~ 0.5) grown in LB with 3% NaCl at 37°C (A) or 20°C (B) or in M9 with 3% NaCl supplemented with 0.4% glucose (C) was compared against gDNA from non-replicating cells on microarrays spotted with DNA from all ORFs. Large and small chromosomes are linearised from the origins over the termini and back to the origins. Grey diamonds represent individual data points and black trend-lines show a sliding average for 50 data points. Scales on the x-axes are an approximate illustration of the respective lengths of the large and small chromosome.
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Figure 2: Replication pattern for exponentially growing V. parahaemolyticus determined by gDNA/gDNA microarrays. Genomic DNA (gDNA) from V. parahaemolyticus in exponential phase (OD600 ~ 0.5) grown in LB with 3% NaCl at 37°C (A) or 20°C (B) or in M9 with 3% NaCl supplemented with 0.4% glucose (C) was compared against gDNA from non-replicating cells on microarrays spotted with DNA from all ORFs. Large and small chromosomes are linearised from the origins over the termini and back to the origins. Grey diamonds represent individual data points and black trend-lines show a sliding average for 50 data points. Scales on the x-axes are an approximate illustration of the respective lengths of the large and small chromosome.

Mentions: A weakness in the above determinations is that they are built on assumptions about an equal and bi-directional replication rate for both chromosomes. To avoid this and get a more detailed and quantitative view of the replication dynamics, we next performed microarray analyses comparing gDNA from exponentially growing V. parahaemolyticus in rich media at 37 and 20°C and poor nutrient broth at 37°C against gDNA from non-replicating cells. The resulting replication patterns are shown in Figure 2 and display gene dosage as a decrease in DNA copy numbers when moving away from the origins of replication. For cells grown in rich broth at 37°C, smooth and similar slopes indicate an even replication progress, both within and between the two chromosomes, which lead to terminations at locations diametrically opposite to the origins of replication (Figure 2A). Also seen is an increase of nearly two orders of magnitude for origin over terminus proximate DNA quantities for the large chromosome, while a similar comparison for the small shows an increase of ~1.2 (Figure 2A). These values correspond to a large chromosome ori/ter ratio slightly below 4 and a small chromosome ori/ter ratio of ~2.3, which is in agreement with the RT-qPCR results (cf. with Figure 1Ab). Moreover, the replication patterns show a higher abundance of large over small chromosome origins while there are approximately equal amounts of termini for both chromosomes. Again, this indicates that termination rather than initiation occurs at a similar time in the cell cycle which is in agreement with previous results from V. cholerae [15]. For cells grown in rich media at 20°C (Figure 2B), ori/ter ratios and quantities are also confirmed (cf. with Figure 1A) and the overall replication patterns are very similar to those obtained for cells incubated at the higher temperature (cf. with Figure 2A). However, a closer comparison reveals a slightly more stuttered pattern which suggests that replication is temporarily arrested at 20°C. Although this could partly explain why large gene dosage differences are maintained despite longer doubling times, the relatively continuous decrease in DNA copy numbers emphasises slowed replication kinetics as the major contributor. Indeed, such temperature dependent change in replication speed has previously been detected in E. coli where cultures incubated at 14°C maintained similar replication time/doubling time ratios as cultures grown at 37°C [31]. Therefore, this suggests that the maintained gene dosage differences at a lower growth temperature are due to a slower replication kinetics that compensates for the less frequent initiation events. For cultures grown in minimal broth, only very small differences in DNA quantities along and between the chromosomes were seen and no clear locations for replication termination were discernible (Figure 2C). It therefore seems like the assay lack sensitivity for reliable determinations of replication dynamics for cells grown under these conditions. Nevertheless, the RT-qPCR results (Figure 1) and also previous analyses on V. cholerae cells grown in minimal media [15] imply a similar replication dynamics as for cells grown in rich broth.


Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location.

Dryselius R, Izutsu K, Honda T, Iida T - BMC Genomics (2008)

Replication pattern for exponentially growing V. parahaemolyticus determined by gDNA/gDNA microarrays. Genomic DNA (gDNA) from V. parahaemolyticus in exponential phase (OD600 ~ 0.5) grown in LB with 3% NaCl at 37°C (A) or 20°C (B) or in M9 with 3% NaCl supplemented with 0.4% glucose (C) was compared against gDNA from non-replicating cells on microarrays spotted with DNA from all ORFs. Large and small chromosomes are linearised from the origins over the termini and back to the origins. Grey diamonds represent individual data points and black trend-lines show a sliding average for 50 data points. Scales on the x-axes are an approximate illustration of the respective lengths of the large and small chromosome.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2612033&req=5

Figure 2: Replication pattern for exponentially growing V. parahaemolyticus determined by gDNA/gDNA microarrays. Genomic DNA (gDNA) from V. parahaemolyticus in exponential phase (OD600 ~ 0.5) grown in LB with 3% NaCl at 37°C (A) or 20°C (B) or in M9 with 3% NaCl supplemented with 0.4% glucose (C) was compared against gDNA from non-replicating cells on microarrays spotted with DNA from all ORFs. Large and small chromosomes are linearised from the origins over the termini and back to the origins. Grey diamonds represent individual data points and black trend-lines show a sliding average for 50 data points. Scales on the x-axes are an approximate illustration of the respective lengths of the large and small chromosome.
Mentions: A weakness in the above determinations is that they are built on assumptions about an equal and bi-directional replication rate for both chromosomes. To avoid this and get a more detailed and quantitative view of the replication dynamics, we next performed microarray analyses comparing gDNA from exponentially growing V. parahaemolyticus in rich media at 37 and 20°C and poor nutrient broth at 37°C against gDNA from non-replicating cells. The resulting replication patterns are shown in Figure 2 and display gene dosage as a decrease in DNA copy numbers when moving away from the origins of replication. For cells grown in rich broth at 37°C, smooth and similar slopes indicate an even replication progress, both within and between the two chromosomes, which lead to terminations at locations diametrically opposite to the origins of replication (Figure 2A). Also seen is an increase of nearly two orders of magnitude for origin over terminus proximate DNA quantities for the large chromosome, while a similar comparison for the small shows an increase of ~1.2 (Figure 2A). These values correspond to a large chromosome ori/ter ratio slightly below 4 and a small chromosome ori/ter ratio of ~2.3, which is in agreement with the RT-qPCR results (cf. with Figure 1Ab). Moreover, the replication patterns show a higher abundance of large over small chromosome origins while there are approximately equal amounts of termini for both chromosomes. Again, this indicates that termination rather than initiation occurs at a similar time in the cell cycle which is in agreement with previous results from V. cholerae [15]. For cells grown in rich media at 20°C (Figure 2B), ori/ter ratios and quantities are also confirmed (cf. with Figure 1A) and the overall replication patterns are very similar to those obtained for cells incubated at the higher temperature (cf. with Figure 2A). However, a closer comparison reveals a slightly more stuttered pattern which suggests that replication is temporarily arrested at 20°C. Although this could partly explain why large gene dosage differences are maintained despite longer doubling times, the relatively continuous decrease in DNA copy numbers emphasises slowed replication kinetics as the major contributor. Indeed, such temperature dependent change in replication speed has previously been detected in E. coli where cultures incubated at 14°C maintained similar replication time/doubling time ratios as cultures grown at 37°C [31]. Therefore, this suggests that the maintained gene dosage differences at a lower growth temperature are due to a slower replication kinetics that compensates for the less frequent initiation events. For cultures grown in minimal broth, only very small differences in DNA quantities along and between the chromosomes were seen and no clear locations for replication termination were discernible (Figure 2C). It therefore seems like the assay lack sensitivity for reliable determinations of replication dynamics for cells grown under these conditions. Nevertheless, the RT-qPCR results (Figure 1) and also previous analyses on V. cholerae cells grown in minimal media [15] imply a similar replication dynamics as for cells grown in rich broth.

Bottom Line: Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species.The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small.For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Genomic Research on Pathogenic Bacteria, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. rikard@biken.osaka-u.ac.jp

ABSTRACT

Background: Replication of bacterial chromosomes increases copy numbers of genes located near origins of replication relative to genes located near termini. Such differential gene dosage depends on replication rate, doubling time and chromosome size. Although little explored, differential gene dosage may influence both gene expression and location. For vibrios, a diverse family of fast growing gammaproteobacteria, gene dosage may be particularly important as they harbor two chromosomes of different size.

Results: Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species. We also investigated locations for specific gene types within the genome. The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small. Accordingly, large chromosome gene expression levels were generally higher and showed an influence from gene dosage. This was reflected by a higher abundance of growth essential and growth contributing genes of which many locate near the origin of replication. In contrast, small chromosome gene expression levels were low and appeared independent of gene dosage. Also, species specific genes are highly abundant and an over-representation of genes involved in transcription could explain its gene dosage independent expression.

Conclusion: Here we establish a link between replication dynamics and differential gene dosage on one hand and gene expression levels and the location of specific gene types on the other. For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity.

Show MeSH
Related in: MedlinePlus