Curr Microbiol DOI 10.1007/s00284-013-0392-8
Sublethal High Hydrostatic Pressure Treatment Reveals the Importance of Genes Coding Cytoskeletal Protein in Escherichia Coli Morphogenesis Atsumu Abe • Soichi Furukawa • Yuya Migita Motoharu Tanaka • Hirokazu Ogihara • Yasushi Morinaga
•
Received: 14 March 2013 / Accepted: 18 April 2013 Ó Springer Science+Business Media New York 2013
Abstract We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, DzapA and DrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.
A. Abe S. Furukawa (&) Y. Migita M. Tanaka H. Ogihara Y. Morinaga (&) Laboratory of Food Microbiology, Department of Food Science and Technology, College of Bioresource Sciences, Nihon University, 1866, Kameino, Fujisawa-shi 252-0880, Japan e-mail:
[email protected]
Introduction Morphology is one of the most important characteristics of cells. We previously reported that the morphology of growing Escherichia coli K-12 cells was temporarily changed after release from high hydrostatic pressure treatment (HPT) [1]. The growth of E. coli K-12 after release from HPT resulted in elongated cells because of an HPT-caused disorder in FtsZ ring formation, which is essential for cell division. This result indicated that the polymerization of FtsZ monomer, a cytoskeletal protein, was highly sensitive to HPT [1]. Our analogous studies of Saccharomyces cerevisiae [2] and Schizosaccharomyces pombe [3, 4] also showed that HPT inhibited the polymerization of the cytoskeletal proteins actin and tubulin, respectively. Studies of the physiological mechanism induced by HPT in E. coli have revealed that cell division and DNA replication [5, 6] and translation and transcription [7, 8] are key pressure-sensitive processes. Among these processes, cell division appears to be the most sensitive because there have been many reports of HPT-induced deficits in cell division [9–11]. ZoBell and Cobet first reported the elongation of E. coli under the pressurized condition [10, 11]. Sato et al. [9] observed that FtsZ polymerization leading to FtsZ ring formation was repressed under the high-pressure condition. FtsZ ring formation is necessary for septum formation during cell division [12–14]. Cell elongation under HPT was also reported in Pseudomonas sp. [7]. Bacterial cell morphology is maintained by the cell wall [15, 16] and cytoskeleton [17–19], and cell morphology varies slightly with the cell cycle [20], especially in the cell division phase. In the present study, we investigated the effects of HPT, especially sublethal HPT in which cell damage is not lethal, but serious growth delays occur on
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A. Abe et al.: Sublethal HPT and bacterial 1 cell morphogenesis
the morphogenesis of E. coli K-12 mutants in which genes related to the cytoskeleton, cell wall, and cell division were deleted. Lethal HPT is a promising practical method widely used for food processing and sterilization [5, 21]. In the present study, we point out that sublethal HPT could be a promising tool for investigating the factors that determine and control bacterial cell morphology.
Materials and Methods Bacterial Strains and Growth Conditions The bacteria used were E. coli K-12 BW25113 ((DaraDaraB)567, DlacZ4787 (::rrnB-4), lacIp-4000 (lacIq)) and gene-related mutants derived from BW25113 that were obtained from the National Institute of Genetics (Shizuoka, Japan) supported by the National BioResource Project (NIG, Japan): E. coli. The E. coli strains used in this study are described in Table 1. We used E. coli K-12 BW25113 and its corresponding deletion mutants in which genes related to the cytoskeleton, cell wall, and cell division were deleted (Table 1). Escherichia coli cells were grown in LB broth (Difco, USA) overnight at 37 °C with shaking. The fully grown cells were used as stationary phase cells. Unless otherwise stated, all reagents were reagent grade and obtained from Kanto Chemical Co., Inc. (Tokyo, Japan), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), or Sigma-Aldrich Corp. (St. Louis, MO, USA). Viable Cell Counting After High Hydrostatic Pressure Treatment Cultured cells were collected by centrifugation at 5,500 rpm for 10 min, and the pellet was washed twice with 1/15 M phosphate buffer (pH 7.0) and then suspended in the buffer. The suspension, approximately 2 ml, was transferred to a cryovial (Greiner Japan, Tokyo, Japan) and carefully sealed with a cap excluding air from the filled tube. Samples were treated in a pressure vessel (model HPV-80C20-S; Sugino Machine Ltd., Toyama, Japan). Pressurization was performed at 75 MPa for 30 min at 37 °C. Surviving cells were measured by plating 100 ll of appropriately diluted samples onto LB broth plates. Colonies were counted after incubation at 37 °C for 24 h. Growth After High Hydrostatic Pressure Treatment Cells were pressurized similarly in the pressure vessel as described above. Pressurization was performed at sublethal conditions, such as 75 MPa for 30 min at 37 °C [1]. The sublethal HPT condition used was the highest pressure
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treatment condition under which the viable cell count of each strain was decreased by less than one tenth after HPT [1]. After HPT, samples were transferred to LB broth and cultured at 37 °C with shaking. The absorbance of the cultures at 600 nm was assessed with a Spectronic 20 spectrophotometer every 30 min. Microscopic Observation Samples were observed and photographed with an Olympus BX60 equipped with a UPlanFl oil immersion lens (Olympus, Tokyo, Japan) and PM-C35DX camera (Olympus). Statistical Analysis All experiments were performed at least twice. The data presented are the means of three or more experiments. Significant differences were determined by the Student’s t test (P \ 0.05).
Results and Discussion HPT and Cell Growth The effects of the deletion of genes related to the cytoskeleton, cell wall, and cell division on the morphologic changes in growth after release from sublethal HPT (75 MPa for 30 min at 37 °C) of E. coli K-12 were investigated. Surviving cells significantly decreased by less than one tenth just after sublethal HPT (data not shown); however, all deletion mutants showed significantly slower growth rates after HPT compared with wild-type bacteria (Fig. 1). The ratios of morphologically changed cells in all strains were increased by sublethal HPT (Table 2). This result indicates that sublethal HPT slightly injured some parts of the cells. HPT-Induced Morphologic Changes in E. coli Mutants There were various types of morphologically changed cells after sublethal HPT (Fig. 2), and we classified them as filamentous, swelling, spherical, branched, or collapsed cells. Some long filamentous and swelling cells were observed in wild-type bacteria (Figs. 2, 3), and these results corresponded with our previous study [1]. It was previously reported that cell filamentation was induced by insufficient formation of FtsZ rings [1]. On the other hand, sublethal HPT induced swelling cells in E. coli K-12 wild-type bacteria as well as in all mutant strains (Figs. 2, 3). Swelling is usually observed among protoplast cells, and it was deduced that sublethal HPT-treated cells lost rigid
A. Abe et al.: Sublethal HPT and bacterial 1 cell morphogenesis Table 1 Strains used in this study Types
Strain
Description
Reference
WT
E. coli K-12 BW25113
Cytoskeleton-relatedgene
E. coli K-12 JW5060 DbolA:kanr
Stationary-phase morphogene, transcriptional repressor for MreB; also regulates dacA, dacC, and ampC transcription; shape determinant
Keio collection
E. coli K-12 JW2500 DrodZ:kanr
Cytoskeletal protein required for MreB assembly; required for swarming phenotype; transmembrane protein
Keio collection
E. coli K-12 JW4111 DampC:kanr
Intrinsic weak beta-lactamase activity; penicillin resistance; affects morphology; penicillin-binding protein
Keio collection
E. coli K-12 JW5052 DampH:kanr
Probable role in peptidoglycan, cell wall synthesis, and cell morphology; penicillin-binding protein
Keio collection
E. coli K-12 JW0627 DdacA:kanr
D-alanine D-alanine
Keio collection
E. coli K-12 JW3149 DdacB:kanr
D-alanine D-alanine
Cell wall-relatedgene
Keio collection
carboxypeptidase PBP5, cell morphology; penicillin-binding protein 5; betalactamase activity carboxypeptidase PBP4; penicillin-
Keio collection
carboxypeptidase PBP6; penicillin-
Keio collection
binding protein 4 E. coli K-12 JW0823 DdacC:kanr
D-alanine D-alanine
binding protein 6
Cell division-related gene
E. coli K-12 JW5329 DdacD:kanr
D-alanine D-alanine
Keio collection
E. coli K-12 JW3116 DlpoA:kanr
OM lipoprotein stimulator of MrcA transpeptidase
Keio collection
E. coli K-12 JW5157 DlpoB:kanr
OM lipoprotein stimulator of MrcB transpeptidase
Keio collection
E. coli K-12 JW2325 DmepA:kanr E. coli K-12 JW3359 DmrcA:kanr
Murein DD-endopeptidase, penicillin-insensitive Murein polymerase, PBP1A; bifunctional murein transglycosylase and transpeptidase; penicillin-binding protein 1A; dimeric
Keio collection Keio collection
E. coli K-12 JW0145 DmrcB:kanr
Murein polymerase, PBP1B; bifunctional murein transglycosylase and transpeptidase; penicillin-binding protein 1B; dimeric
Keio collection
E. coli K-12 JW2503 DpbpC:kanr
Penicillin-binding protein PBP1C murein transglycosylase
Keio collection
E. coli K-12 JW5355 DpbpG:kanr
Murein D-alanyl-D-alanine endopeptidase, periplasmic PBP7 and PBP8; uniquely binds beta-lactam penems, lysing nongrowing cells
Keio collection
E. coli K-12 JW5395 DyfeW:kanr
Weak penicillin binding protein PBP4B, predicted periplasmic esterase
Keio collection
E. coli K-12 JW4110 Dblc:kanr
Outer membrane lipoprotein, cell division and growth function; prokaryotic lipocalin
keio collection
E. coli K-12 JW1720 DcedA:kanr
DNA-binding protein that modulates cell division; relieves inhibition of cell division after over-replication of chromosome in dnaAcos mutants
Keio collection
E. coli K-12 JW3351 DdamX:kanr
Cell division protein, binds septal ring;bile salts resistance
Keio collection
E. coli K-12 JW1566 DdicB:kanr
Control of cell division, activates MinC, Qin prophage
Keio collection
E. coli K-12 JW1561 DdicC:kanr
Transcriptional repressor for dicB, Qin prophage
Keio collection
E. coli K-12 JW0506 DfdrA:kanr
Multicopy suppressor of dominant negative ftsH mutations
Keio collection
r
Membrane bound protease complex with FtsH and HflK; regulates lysogeny; nonessential
Keio collection
E. coli K-12 JW4132 DhflK:kanr
Membrane bound protease complex with FtsH and HflC; regulates lysogeny; nonessential
Keio collection
E. coli K-12 JW1165 DminC:kanr
Inhibition of FtsZ ring polymerization; forms membraneassociated coiled arrays, more concentrated at the poles
Keio collection
E. coli K-12 JW2878 DzapA:kanr
FtsZ-associated protein; coiled-coil protein
Keio collection
E. coli K-12 JW3899 DzapB:kanr
Septal ring assembly factor, stimulates cell division; coiled-coil protein
Keio collection
E. coli K-12 JW4133 DhflC:kan
carboxypeptidase PBP6b; penicillinbinding protein 6b
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A. Abe et al.: Sublethal HPT and bacterial 1 cell morphogenesis 0.16 0.14 0.12
OD 600
0.10 0.08
WT ΔcedA
0.06
ΔbolA
ΔmrcA
0.04
ΔpbpG ΔzapA
0.02
ΔzapB ΔrodZ
0.00
30
60
90
Time (min) Fig. 1 Growth of E. coli BW25113(wild-type) and mutants after sublethal HPT at 75 MPa for 30 min at 37 °C
cell walls or the cytoskeletal proteins necessary to support the correct morphology. Previous studies of E. coli K-12 swelling cells [22] found that swelling cells were induced by injuries to RodZ and MreB. Therefore, RodZ and MreB were also injured in HPT-treated E. coli K-12 wild-type swelling cells. There were some spherical, branched, or collapsed cells in almost all the used various deletion mutants (Figs. 2, 3). Spherical cells were observed in some mutants after sublethal HPT (Figs. 2, 3), indicating that the cytoskeletal proteins necessary for maintaining rod cell shape were defective in these mutants. Rod cell shape is usually controlled by RodZ, and most DrodZ cells were spherical before sublethal HPT (Figs. 2, 3). It was previously shown that E. coli K-12 spherical cells [22] were induced by dysfunction of RodZ or MreB because of gene deletion. Therefore, dysfunction of RodZ or MreB seemed to occur in HPT-treated E. coli K-12 wild-type spherical cells. However, most DrodZ cells were branched after sublethal HPT (Fig. 3). RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are
Table 2 Effect of sublethal HPT on the cell morphology of Escherichia coli K-12 mutants Intact DeterioElongated Elongated Normal Swelling Spherical Branched rated >5 >2 WT 96.9 0.0 2.1 0.0 1.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 0.0 Cytoskeleton bolA related gene rodZ 16.7 0.0 0.0 3.6 76.6 0.0 3.1 ampC 100 0.0 0.0 0.0 0.0 0.0 0.0 ampH 98.9 0.0 0.0 1.1 0.0 0.0 0.0 dacA 98.9 0.0 0.0 0.0 0.0 1.1 0.0 dacB 99.0 0.0 1.0 0.0 0.0 0.0 0.0 dacC 98.9 0.0 0.0 0.0 0.0 1.1 0.0 dacD 100 0.0 0.0 0.0 0.0 0.0 0.0 100 0.0 0.0 0.0 0.0 0.0 0.0 Cell wall lpoA related gene lpoB 100 0.0 0.0 0.0 0.0 0.0 0.0 mepA 100 0.0 0.0 0.0 0.0 0.0 0.0 mrcA 100 0.0 0.0 0.0 0.0 0.0 0.0 mrcB 100 0.0 0.0 0.0 0.0 0.0 0.0 pbpC 100 0.0 0.0 0.0 0.0 0.0 0.0 pbpG 100 0.0 0.0 0.0 0.0 0.0 0.0 yfeW 100 0.0 0.0 0.0 0.0 0.0 0.0 blc 100 0.0 0.0 0.0 0.0 0.0 0.0 cedA 100 0.0 0.0 0.0 0.0 0.0 0.0 damX 100 0.0 0.0 0.0 0.0 0.0 0.0 dicB 100 0.0 0.0 0.0 0.0 0.0 0.0 dicC 100 0.0 0.0 0.0 0.0 0.0 0.0 Cell divisio fdrA 100 0.0 0.0 0.0 0.0 0.0 0.0 related gene hflC 95.9 1.1 1.1 1.9 0.0 0.0 0.0 hflK 98.1 0.0 1.9 0.0 0.0 0.0 0.0 minC 97.0 0.0 1.9 0.0 1.1 0.0 0.0 zapA 97.8 1.1 1.1 0.0 0.0 0.0 0.0 zapB 99.0 0.0 1.0 0.0 0.0 0.0 0.0 Inreased more than 1.0%. Inreased more than 5.0%. Inreased more than 10%.
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Treated Elongated Elongated DeterioNormal Swelling Spherical Branched >5 >2 rated 93.4 1.0 3.7 1.9 0.0 0.0 0.0 86.4 3.1 3.9 1.9 0.0 3.6 1.1 2.2 0.0 0.0 6.1 4.3 79.6 7.8 96.9 1.0 1.0 1.1 0.0 0.0 0.0 95.1 0.0 1.9 1.9 0.0 0.0 1.1 84.6 4.1 6.1 2.1 0.0 3.1 0.0 77.7 2.1 3.1 15.0 0.0 1.0 1.1 91.1 2.1 2.7 1.9 0.0 1.1 1.1 92.4 1.9 1.1 1.9 2.7 0.0 0.0 90.9 1.9 2.7 1.9 0.0 1.6 1.0 85.0 1.0 8.0 6.0 0.0 0.0 0.0 91.2 1.9 3.7 2.1 1.1 0.0 0.0 90.9 1.0 6.2 1.9 0.0 0.0 0.0 91.7 1.1 2.1 4.1 0.0 1.0 0.0 92.3 2.7 3.1 1.9 0.0 0.0 0.0 90.1 2.7 1.3 5.9 0.0 0.0 0.0 90.4 1.7 5.2 2.7 0.0 0.0 0.0 97.1 0.0 1.0 1.9 0.0 0.0 0.0 96.1 1.0 1.9 1.0 0.0 0.0 0.0 93.3 1.9 1.9 1.9 0.0 1.0 0.0 95.0 0.0 1.9 2.1 0.0 1.0 0.0 95.8 0.0 2.1 2.1 0.0 0.0 0.0 88.1 2.6 6.1 3.2 0.0 0.0 0.0 95.0 1.0 1.9 2.1 0.0 0.0 0.0 90.9 1.0 2.1 1.9 3.1 1.0 0.0 87.7 1.0 1.9 5.6 2.7 0.0 1.1 76.6 7.4 13.4 2.6 0.0 0.0 0.0 88.4 3.9 3.7 4.0 0.0 0.0 0.0
A. Abe et al.: Sublethal HPT and bacterial 1 cell morphogenesis
Intact
WT
Intact
Treated
Δ blc
Δ bolA
Δ cedA
Δ ampC
Δ dicB
Δ ampH
Δ dicC
Δ dacA
Δ damX
Δ dacB
Δ dacC
Δ dacD
Treated
Δ fdrA
Δ hflC
Δ hflK
Δ lpoA
Δ minC
Δ lpoB
Δ zapB
Δ rodZ
Fig. 2 Morphologic changes of E. coli WT and mutant cells after sublethal HPT at 75 MPa for 30 min at 37 °C
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A. Abe et al.: Sublethal HPT and bacterial 1 cell morphogenesis
Intact
Treated A
WT
Δ zapA
E
Δ rodZ
B C
D
Fig. 3 Morphologic changes of E. coli WT, DzapA, and DrodZ cells after sublethal HPT at 75 MPa for 30 min at 37 °C. A: elongated, B: swelling, C: spherical, D: branched, and E: deteriorated
necessary for maintaining the cellular rod shape. Here, MreB is essential for E. coli K-12. After sublethal HPT, the formation of FtsZ rings, which are necessary for cell division, was impaired. Therefore, the disorder of cytoskeletal proteins necessary for the formation of the rod cell shape and FtsZ rings would induce branched cells. DbolA cells were also branched as well as DrodZ cells after sublethal HPT (Fig. 3). BolA is a transcriptional repressor of MreB. Thus, increased MreB production and disordered FtsZ rings both cause branched cells. Studies of E. coli K-12 branched cells [16, 23] have shown that branched cells were induced by injury to peptidoglycan. Therefore, peptidoglycan is also injured in HPT-treated E. coli K-12 wild-type branched cells. Some of the DrodZ cells were collapsed after HPT (Fig. 3), and it was deduced that collapsed cells came from branched cells. Previous studies of E. coli K-12 branched cells [16, 24] showed that branched cells were induced by injury to the cytoskeleton and peptidoglycan. Therefore, the cytoskeleton and peptidoglycan were also injured in HPT-treated E. coli K-12 wild-type branched cells. Morphologic Changes of E. coli Cells by HPT and Cytoskeletal Protein Sublethal HPT especially affected the cell morphology of DzapA and DrodZ (Figs. 2, 3). ZapA supports FtsZ ring formation with ZapB [25]. As mentioned above, RodZ interacts with MreB, and both factors are necessary for maintaining rod shape of cells [26–28]. These results show that disordered
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FtsZ rings induce cell elongation and that disordered MreB bacterial cytoskeleton induces the branched and collapsed cell shape. Thus, the formation of FtsZ rings and MreB bacterial cytoskeleton are important for keeping normal cell shape during growth after sublethal HPT, and the polymerization of cytoskeletal proteins is a critical target of HPT. DdacA, B, C, and D also showed morphologic changes after sublethal HPT. D-alanine carboxypeptidase is penicillin-binding protein, and they appear to be related to the cell wall structure [29]. These results indicate that cell wall structure significantly contributes to cell morphology after sublethal HPT. Our results indicate that sublethal HPT induced E. coli cell morphologic changes, which could provide important information regarding morphology-related genes. Therefore, sublethal HPT may be a good tool for studying the importance of genes related to bacterial cell morphogenesis. We believe that this technology could be used with other microorganisms and also with plant and animal cells to study their physiology from the view point of their morphology. Acknowledgments The authors would like to thank the National Institute of Genetics (Shizuoka) and the National BioResource Project (NIG, Japan): E. coli for providing the E. coli strains used in this study.
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