A reduced genome decreases the host carrying capacity for foreign DNA
© Akeno et al.; licensee BioMed Central Ltd. 2014
Received: 6 February 2014
Accepted: 24 March 2014
Published: 1 April 2014
Host-plasmid interactions have been discussed largely in terms of the influences of plasmids, whereas the contributions of variations in host genomes to host interactions with foreign DNA remain unclear. A strain with a so-called “clean genome” (i.e., MDS42) of reduced genome size has recently been generated from the wild-type strain MG1655, a commonly used host strain. A quantitative evaluation of the influence of plasmid burdens in these two Escherichia coli strains can not only provide an understanding of how a reduced genome responds to foreign DNA but also offer insights into the proper application of these strains.
The decreases in growth caused by the cost of carrying foreign DNA were similar for the wild-type and clean-genome strains. A negative correlation between the growth rate and the total amount of exogenous DNA was observed in both strains, but a better theoretical fit with a higher statistical significance was found for the strain with the clean genome. Compared to the wild-type strain, the clean-genome strain exhibited a reduced carrying capacity for exogenous DNA, which was largely attributed to its ability to restrict the replication of foreign DNA. A tendency to allocate energy and resources toward gene expression, but not DNA replication, was observed in the strain with the clean genome.
The possession of a clean genome constrained the plasmid copy number to a wild-type-equivalent load. The results indicate that the wild-type strain possesses a greater tolerance for foreign DNA, as in endosymbiosis, and that the use of strains with clean genomes will be favorable in the applications that require precise control and theoretical prediction.
Host-plasmid interactions have been intensively studied in both bacterial and yeast cells, with the aim of developing a quantitative understanding of how exogenous DNA, i.e., plasmids, contribute to host growth [1–8]. Decreases in the growth rates of host cells that carry foreign DNA (e.g., plasmids) have mainly been attributed to the additional costs associated with the expression of plasmid genes [9, 10]. Because the size of the prokaryotic genome is considered to be constrained by bioenergetics, (gene expression across an entire genome is assumed to consume approximately 75% of the total energy produced by bacterial cells ), whether changes in the size of a host genome can improve a host’s capacity for DNA replication is an intriguing question.
Furthermore, evaluations of the contributions of host genomes to host-plasmid interactions are also relevant to biotechnological applications. A strain with a clean genome of reduced size (i.e., MDS42) has recently been generated from the wild-type genome of strain MG1655 . The MDS42 genome possesses significant advantages in terms of plasmid stability [13, 14], protein production  and other applications in synthetic biology [16, 17]. These advantages for biotechnological applications are largely attributed to genetic characteristics such as a low mutation rate and limited amount of recombination [13, 14, 17].
The latest genome-wide analyses have revealed that the reduced genome of MDS42 exhibits both a higher level of gene expression and a more fixed chromosomal periodicity  compared to its mother strain, MG1655 . These novel properties of MDS42 largely result from the complete deletion of insertion sequences , which are considered to be nonessential for cellular life. Thus, MDS42 is believed to be an ideal host for plasmid production  because plasmids exhibit a greater degree of structural stability in MDS42 compared to MG1655 . However, to date, the increase in the productivity of plasmids inserted into MDS42 has not been well studied .
In this study, we investigated whether a reduction in host genome size would benefit the replication of foreign DNA by comparing two closely related strains (i.e., MG1655 and MDS42) of different genome sizes. Estimating the burden of foreign DNA (i.e., plasmids) in both MG1655 and MDS42 allows us to determine how a reduction in genome size influences the carrying capacity for foreign DNA, and this information also provides further insight into the relationships among DNA replication, host growth and the cost distribution of cellular energetics. Any novel information concerning the nature of the host-plasmid interactions in strains with a clean genome (e.g., MDS42) can be taken into consideration when choosing strains for various applications.
Results and discussion
The growth cost of foreign DNA is similar for a wild-type or a clean genome
Decreases in growth rate were detected in both host strains when they contained plasmids (Figure 1B, Additional file 1: Figure S2). A trend toward a decrease in host cell growth rate with an increase in plasmid size was observed. The decrease in growth rate caused by the presence of foreign DNA was more significant when the host cells were grown at normal and higher than normal temperatures but was insignificant at the lowest temperature. The results strongly suggest that the burden of foreign DNA was roughly equivalent for the two hosts, although the genome of MDS42 was shorter in length. The costs associated with the replication and/or expression of exogenous DNA were similar for the wild-type and the clean genomes. In addition, the results suggested not only the plasmid copy number (as previously reported [3, 4]) but also the plasmid size (Figure 1B) played a role in host growth. The total DNA amount, comprising the information of both copy number and plasmid size, might be a proper parameter to evaluate the host-plasmid relations in common.
The decrease in growth rate is dependent on the total amount of exogenous DNA
Enhanced predictability of the clean genome
Estimated parameter constants in the models
Residual sums of squares (RSS)
Interestingly, the clean genome exhibited better fitting irrespective of the regression model employed. The data for the clean genome fit the models better than the native host genome, and the RSS values were lower for MDS42 than MG1655 for both the PCN and Nt data (Table 2). Both the increased significance of the correlation between host growth and the amount of exogenous DNA (Figure 3, Table 2) and the higher correlation coefficients (Figure 2) indicate the highly precise control of either cell growth or DNA replication in MDS42. More predictable levels of performance may occur in hosts with fewer nonessential sequences in its genome (i.e., MDS42). The results from this study were consistent with previous findings that have indicated a highly regular, predictable and well-organized behavior for MDS42 at the genome-wide expression level [18, 25, 26].
Reduced carrying capacity of the clean genome
Because the linear regression model (model 1) examines the relationship between cell growth rate and the amount of exogenous DNA, the slope (a) represents the magnitude of the growth cost of exogenous DNA to the host. A greater decrease in growth was clearly observed in MDS42 compared to MG1655 (Table 1), indicating that the difference in the carrying capacity for exogenous DNA depended on the host genome size. For example, when the linear regression was normalized by the genome size (Additional file 1: Figure S5), in the case of a 50% decrease in fitness, MG1655 could bear an amount of exogenous DNA that was 1.5-fold greater than its genome size, whereas MDS42 could only bear an amount of exogenous DNAs that was equivalent to its genome size. The clean genome might save cellular energy and/or resources for its own genome and restrict energy and/or resources spent on foreign DNA.
Restricted replication allows for higher expression levels in the clean genome
Taken together, the plasmid carrying capacity was lower in MDS42, independent of the growth temperature or the plasmid size (Figure 5), whereas gene expression was definitely higher in MDS42 (Figure 6C), a finding in agreement with previous reports [15, 18]. Compared to the wild-type genome, the clean genome assigned a higher priority to the gene expression of foreign DNA than to its replication. Such trade-off like phenomena of increased protein production with decreased plasmid carrying capacity in the reduced genome could be also detected when using the rich medium, despite of the highly accelerated cell growth (Additional file 1: Figure S6). This finding indicates that removing the redundant genes from a host genome can save energy and resources for their direction toward both host genome expression  and the expression of foreign genes (Figure 6C), but not DNA replication.
In summary, the burden of foreign DNA was evaluated in two E. coli strains with different genome sizes. The growth rates of both host strains were more negatively correlated with the total amount of exogenous DNA than the plasmid copy numbers. The growth decrease, which was mediated by plasmid replication, was more significant and more theoretically predictable in the strain with the clean genome (MDS42) than that with the wild-type genome (MG1655). The greater decrease in growth that occurred in MDS42 was caused by a slower replication rate of foreign DNA. The results indicate that the MG1655 strain can bear more exogenous DNA but experiences an equivalent loss in growth fitness, whereas MDS42 can restrict the amplification of foreign DNA to maintain its level of growth fitness. Plasmids that are inhibited by constraints in the cellular conditions for replication might benefit from the highly induced level of gene expression found in MDS42. These results indicate that the wild-type strain MG1655 is more suitable for an endosymbiotic application and possesses an advantage in terms of adaptation and evolution, whereas the engineered, clean-genome strain MDS42 more closely resembles a type of living machinery (a controllable cell) and is more suitable for the precise control and production of synthetic materials.
The lacZ region of pUC19 was removed using the In-Fusion HD Cloning Kit (Clontech) and the pUC19_del_lacZ_Fw and pUC19_del_lacZ_Rv primers. Both colony PCR and blue-white selection were performed to verify the deletion. The resultant plasmid had a length of 2,334 bp and was named pUC-S. A DNA sequence (4,092 bp) of pSC101 was amplified with the pSC101_seq_Fw and pSC101_seq_Rv primers. Using the In-Fusion HD Cloning Kit, the resultant fragment was inserted into plasmid pUC19 in the same region where pUC-S was inserted, by using the pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv primers. This increased the plasmid length of pUC-M to a total length of 6,426 bp. A longer fragment (6,986 bp) from pSC101 that included the tetracycline-resistance gene (Tcr) was amplified using the pSC101_seq_Fw2 and pSC101_seq_Rv2 primers and then inserted into pUC19 as described above to create the pUC-preL plasmid. Subsequently, a 198-bp fragment was removed from the translation initiation region for Tcr in pUC-preL using the primers pSC101_del_TcR_Fw and pSC101_del_TcR_Rv, which formed a 9,573-bp-long plasmid, pUC-L. Thus, all three plasmids that were produced had a common site of initiation for replication derived from pUC19, and carried a single gene for ampicillin resistance.
A GFP sequence that includes the promoter Ptet was amplified from the pBRgalKGR plasmid (a pBR322 derivative) [26, 27] using the pBR_gfp_kanR_Fw and pBR_gfp_kanR_Rv primers. The lacZ region of pUC19 was also removed using the In-Fusion HD Cloning Kit (Clontech) but with a different primer set (pUC19_del_lacZ_linearize_Fw and pUC19_del_lacZ_linearize_Rv2), resulting in a 2,334-bp-long fragment, the same length as pUC-S. The two fragments were ligated using the In-Fusion HD Cloning Kit. The kan R gene sequence (2,866 bp) was removed using the pUC_del_kanR_Fw and pUC_del_kanR_Rv primers, which finally created the pUC-G plasmid. ECOS™ Competent E. coli DH5α cells (Nippon Gene) were used for the cloning and amplification of the plasmids. All the primers used in this study are listed in Additional file 1: Table S1.
Cell culture and FCM analysis
Plasmid copy number and total amount of exogenous DNA
Evaluation of various models
In all three models (equivalent to Eqs. 1, 2 and 3), y is the normalized growth rate and x indicates either the copy number of plasmids or the total amount of plasmid DNA. The parameter c represents the maximum possible number of plasmids per cell, and the exponent m was set to 0.5 according to the recommendation of a previous study .
This work was partially supported by a Grant-in-Aid for Scientific Research on Innovative Areas No. 25111715 (to BWY) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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