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Keywords:

  • cold-sensitive growth;
  • isoprenoid biosynthesis;
  • RodZ;
  • suppressor

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Suppression of cold-sensitive growth in the rodZ mutant
  4. ACKNOWLEDGMENTS
  5. DISCLOSURE
  6. REFERENCES

Cells lacking rodZ are defective not only in cell shape, but also in cell growth at low temperatures. Cold-sensitive growth was suppressed by a mutation of ispA without recovery from the round shape, and the mutation improved cell growth of the wild-type at low temperatures.

List of Abbreviations: 
E. coli

Escherichia coli

FLP

flippase recombinase

FRT

flippase recombination target

Bacterial morphology is closely related to the environment (1). A change of cell shape may result in reduced cell growth and viability. E.coli has at least three cytoskeletal proteins, MreB, FtsZ, and RodZ; their function is to maintain the organism's rod shape. Although FtsZ and MreB have been extensively studied (2), the functions and molecular mechanisms of RodZ are largely unknown. Cells lacking rodZ are round or oval and grow more slowly than the wild-type (3–5). Furthermore, Bendezu et al. found that, below 30°C, rodZ deletion mutants fail to form colonies on L plates (3). We noticed that, in a culture of rodZ deletion mutant cells (JW2500) in a deletion library (the Keio collection) (5, 6), some cells had formed colonies on L plates below 30°C. We surmised that an additional mutation had suppressed only the cold-sensitive growth of the rodZ mutant, because the cells that had formed colonies were still round. Therefore, the whole genome of a revertant (DS339) was sequenced. We found two mutations aside from the rodZ deletion mutation: a mutation in the ispA gene (ispA-A164T) and one at two base pairs downstream of rrfG, which is one of eight genes encoding 5S ribosomal RNA. However, the rrfG mutation proved not to be a suppressor mutation of cold-sensitive growth of the rodZ mutant (see below). In our experiments, only the ispA-A164T mutation suppressed cold-sensitive growth of the rodZ mutant. This does not exclude the possibility that another mutation of ispA or other mutated genes can suppress cold-sensitive growth of the rodZ mutant. Further isolation of the suppressors is necessary to confirm this.

To test whether cold-sensitive growth of the rodZ mutant was suppressed by the ispA mutation, we constructed the following strains by P1 transduction. All strains are derivatives of BW25113, which is a lineage of E. coli K-12 (6, 7). To restore the wild-type rodZ gene in JW2500 and DS339, the zff-208::Tn10 marker gene in ME8835 was used. Because zff-208::Tn10 is linked with the rodZ gene, a P1 lysate of ME8835 was infected into JW2500 and DS339, yielding DS366 (zff-208::Tn10, rrfG) and DS368 (ispA-A164T, zff-208::Tn10, rrfG). Because yfiR is linked with the rrfG gene, ΔyfiR::kan was used as a marker gene to restore the wild-type rrfG gene in these strains. A P1 lysate of JW2584 (ΔyfiR::kan) was infected into DS366 and DS368. Elimination of the rrfG mutation from DS366 and DS368 was confirmed by DNA sequencing of the transductants, resulting in DS410 (ΔyfiR::kan, zff-208::Tn10) and DS413 (ispA-A164T, ΔyfiR::kan, zff-208::Tn10), respectively. The kan gene, which is flanked with flippase recombination targets (FRT) within yfiR of DS410 and DS413, was eliminated by a site-specific recombination system using the FRT yeast recombination targets. Plasmid pCP20 (6, 7), carrying the yeast FLP was used for the elimination. The transformed cells were incubated at 42°C to segregate pCP20, yielding DS417 (ΔyfiR, zff-208::Tn10) and DS419 (ispA-A164T, ΔyfiR, zff-208::Tn10). Finally, to reintroduce the rodZ deletion mutation, DS426 (ΔrodZ::kan ΔyfiR, zff-208::Tn10) and DS428 (ΔrodZ::kan ispA-A164T, ΔyfiR, zff-208::Tn10) were constructed by P1 transduction of JW2500 (ΔrodZ::kan), respectively.

Suppression of cold-sensitive growth in the rodZ mutant

  1. Top of page
  2. ABSTRACT
  3. Suppression of cold-sensitive growth in the rodZ mutant
  4. ACKNOWLEDGMENTS
  5. DISCLOSURE
  6. REFERENCES

DS428 (ΔrodZ ispA-A164T) grew better than DS426 (ΔrodZ) at 25°C, indicating that the ispA mutation partially suppresses the cold-sensitive phenotype of rodZ mutant cells (Figures 1a and b). Importantly, the ispA mutation suppressed neither defects in growth at 37°C nor aberrant cell shape (Figures 1b and c). Improvement in cell growth was seen only at low temperatures. Next, we confirmed that the suppression was caused by the ispA mutant. To this end, we first cloned ispA or ispA-A164T into pBAD24 (8), yielding pDS1026 (ispA) and pDS1027 (ispA-A164T). We first tested whether overproduction of wild-type IspA or IspA-A164T caused an inhibition of cell growth at 25°C or 37°C in rodZ+ cells (DS410 and DS413). Overproduction of wild-type IspA or IspA-A164T did not inhibit cell growth of those strains under the conditions tested (Figure 2a). Then, we tested the effect of overproduction of wild-type IspA or IspA-A164T on cell growth in rodZ deletion mutants (Figure 2b). Induction of wild-type ispA in DS428 slightly inhibited the suppression of ispA-A164T at 25°C, while overproduction of wild-type IspA or IspA-A164T in DS428 did not inhibit cell growth at 37°C. Overproduction of wild-type IspA and IspA-A164T in DS426 did not inhibit cell growth at 25°C and 37°C (Figure 2b). These results indicate that the ispA-A164T mutation is the suppressor of the cold-sensitive phenotype of the rodZ mutant.

image

Figure 1. Suppression of the cold-sensitivity of cells lacking rodZ by an ispA-A164T mutation. (a) Colony viabilities of WT, rodZ cells, and their derivatives. Ten-fold serial dilutions of overnight cultures of DS410 (WT), DS426 (ΔrodZ), DS413 (ispA-A164T), and DS428 (ΔrodZ, ispA-A164T) were made, and 5 μL of 10−1 to 10−4 dilutions spotted on L plates. The plates were incubated at 25°C for 48 hr or 37°C for 12 hr. (b) Cell growth of DS410 (blue), DS426 (green), DS413 (magenta), and DS428 (red) in L broth at 25 or 37°C. Absorbance (OD660) was recorded automatically by a Bio-photorecorder (TVS 062CA, Advantech) every minute. (c) Phase contrast images of DS410, DS426, DS413, and DS428 grown at 37°C. Scale bar is 2 μm.

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image

Figure 2. Overproduction of IspA results in slow growth in DS428 cells at 25°C. (a) Ten-fold serial dilutions of overnight cultures of DS410 (WT) and DS413 (ispA-A164T) carrying either pBAD24, pDS1026 (pBAD24-ispA), or pDS1027 (pBAD24-ispA-A164T) were made, and 5 μL of 10−4 to 10−7 dilutions spotted on L plates supplemented with or without 0.02% arabinose. The plates were incubated at 25°C for 48 hr or 37°C for 24 hr. (B) Ten-fold serial dilutions of overnight cultures of DS426 (ΔrodZ) or DS428 (ΔrodZ ispA-A164T) carrying either pBAD24, pDS1026 (ispA), or pDS1027 (ispA-A164T) were made, and 5 μL of 10−1 to 10−4 dilutions spotted on L plates supplemented with or without 0.02% arabinose. The plates were incubated at 25°C for 48 hr or 37°C for 24 hr.

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Improvement in cell growth at low temperatures

We next tested whether the ispA mutation promoted cell growth at low temperature in rodZ+ cells. As shown in Figure 2a, we did not observe any improvement of cell growth at 25°C. We then carried out incubation at a lower temperature. Although the colony-forming abilities of DS410 (WT) and DS413 (ispA-A164T) were about the same (2.9 × 109 and 3.9 × 109) (Figure 3a), cell growth of DS413 was slightly increased on L plates at 15°C as compared with that of DS410 (Figure 3b). Indeed, when the colony sizes of both strains were measured, those of DS413 were significantly larger than those of DS410, the P-value determined by an unpaired t-test being < 0.0001 (Figures 3c and d). Thus, the ispA-A164T mutation improves cell growth at low temperatures.

image

Figure 3. IspA-A164T promotes growth of rodZ+ cells at 15°C. (a) Comparison of colony-forming units (CFU/mL) on L plates incubated at 15°C for 120 hr or 37°C for 24 hr. (b) Cell growth of DS410 (WT) and DS413 (ispA-A164T). Ten-fold serial dilutions of overnight cultures of DS410 (WT) and DS413 (ispA-A164T) were made, and 5 μL of 10−1 to 10−5 dilutions spotted on L plates. The plates were incubated at 15°C for 72 hr or 37°C for 24 hr. (c, d) Colony sizes of DS410 and DS413 on L plates incubated at 15°C for 120 hr or 37°C for 24 hr. Average size and standard deviation are shown in (d). (e, f) Colony sizes of DS1186 (WT) and DS1187 (ispA-A164T) on L plates. The plates were incubated at 15°C for 144 hr or 37°C for 24 hr. Average size and standard deviation are shown in (f).

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To investigate whether the ispA-A164T mutation improves cell growth at low temperatures generally or only in this particular strain, the ispA-A164T mutation was introduced into strain W3110 by P1 transduction as follows. We used ΔyajO::cat as a selection marker for P1 transduction of the ispA-A164T mutation, because yajO is a non-essential gene for cell growth and is only 3 kb away from ispA. To construct DS1173 (ΔyajO::cat) and DS1174 (ispA-A164TΔyajO::cat), a DNA fragment including the cat gene was inserted into yajO in DS410 and DS413 according to the method of Datsenko and Wanner (7). P1 lysates of the resulting strains were infected to W3110, and chloramphenicol-resistant colonies isolated. The ispA gene of chloramphenicol-resistant cells was sequenced to confirm the ispA-A164T mutation of the transductants. Then, the P1 tranductants derived from W3110 were identified as DS1186 (W3110 ΔyajO::cat) and DS1187 (W3110 ispA-A164TΔyajO::cat).

Growth of the resulting strains was examined at 15°C or 37°C. Colony sizes of W3110 carrying ispA-A164T (DS1187) were significantly larger than those of the control strain (DS1186) (Figures 3e and f). This result suggests that the ispA-A164T mutation generally improves cell growth at low temperatures.

IspA is essential for cell growth in E. coli and is involved in the isoprenoid biosynthesis pathway (9). This pathway conducts the formation of several essential lipids involved in the biosynthesis of peptidoglycan and lipopolysaccharides, and hence maintenance of the cell wall (10). It is plausible that E. coli cells with the ispA-A164T mutation have altered the compositions and/or structures of the membrane and peptidoglycan to adapt to cold environments. Furthermore, it has been reported that L-forms of Bacillus subtilis, which is a wall-deficient cell, have a mutation in ispA (11). It is thought that the mutation of ispA allows bacterial cells to grow under environmental stresses affecting the cell wall.

The three-dimensional structure of IspA with its substrate reveals conformational changes of the α4-α5 and α9-α10 loops compared with the apo-structure (12). The A164 residue is located in the α7-α8 loop which faces the α9-α10 loop. Therefore, it is possible that the A164T mutation affects the conformational change of the α9-α10 loop. Although it is uncertain how the A164T mutation affects the properties of IspA, the improvement of cell growth by ispA is interesting because RodZ is involved in the synthesis of peptidoglycan (5, 13, 14).

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. Suppression of cold-sensitive growth in the rodZ mutant
  4. ACKNOWLEDGMENTS
  5. DISCLOSURE
  6. REFERENCES

We are grateful to Drs. Atsushi Toyoda, Tomoyuki Aizu, Fumio Eshima, Asao Fujiyama, Osamu Arai, and Yuji Kohara for genomic sequencing. We thank all members of the Niki Lab for helpful comments and suggestions. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. N., and a Grant-in-Aid for Young Scientists (Start-up) and Young Scientists (B), and an National Institute of Genetics Postdoctoral Fellowship to D. S. Strains were supplied by the National Bioresource Project, Japan: E. coli (NIG, Mishima).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. Suppression of cold-sensitive growth in the rodZ mutant
  4. ACKNOWLEDGMENTS
  5. DISCLOSURE
  6. REFERENCES

We certify that there is no conflict of interest with any financial organization regarding the material discussed in this manuscript.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. Suppression of cold-sensitive growth in the rodZ mutant
  4. ACKNOWLEDGMENTS
  5. DISCLOSURE
  6. REFERENCES