1 Aerobic Respiratory Enzymes, Cbb3-type Cytochrome c Oxidases, Impact the Anaerobic Life of ...

33 Pages·2014·2.33 MB·English

JB Accepts, published online ahead of print on 2 September 2014
J. Bacteriol. doi:10.1128/JB.01978-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 Aerobic Respiratory Enzymes, Cbb3-type Cytochrome c Oxidases, Impact the Anaerobic Life of
2 Pseudomonas aeruginosa PAO1
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4 Masakaze Hamada,* Masanori Toyofuku, Tomoki Miyano and Nobuhiko Nomura#
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6 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki o
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7 305-8572, Japan. lo
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9 Running Head: Role of Cbb3 Oxidases under Anoxic Conditions o
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11 #Address correspondence to Nobuhiko Nomura, [email protected]. /jb
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12 *Present address: Masakaze Hamada, Department of Microbiology and Infectious Diseases, Toho University sm
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13 School of Medicine, Tokyo, Japan rg
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14 M.H. and M.T. contributed equally to this work. n

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24 ABSTRACT
25 In bacteria, many studies have focused on the role of respiratory enzymes in energy conservation; however, their
26 effect on cell behavior is poorly understood. Pseudomonas aeruginosa can perform both aerobic respiration and
27 denitrification. Previous studies demonstrated that Cbb3-type cytochrome c oxidases that support aerobic
28 respiration are more highly expressed in P. aeruginosa under anoxic conditions compared with other aerobic
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29 respiratory enzymes. However, little is known about their role under such conditions. In this study, it was shown o
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30 that Cbb3 oxidases of P. aeruginosa PAO1 alter anaerobic growth, denitrifying process and cell morphology under lo
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31 anoxic conditions. Furthermore, biofilm formation was promoted by the Cbb3 oxidases under anoxic conditions. e
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32 Cbb3 oxidases led to the accumulation of NO that is produced during denitrifying process. Cell elongation induced o
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33 by NO accumulation was reported to be required for robust biofilm formation of P. aeruginosa PAO1 under anoxic t
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34 conditions. Our data show that Cbb3 oxidases promote cell elongation by inducing NO accumulation during /jb
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35 denitrifying process, which further leads to robust biofilms. Our findings show that Cbb3 oxidases, which have sm
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36 been well studied as aerobic respiratory enzymes, are also involved in denitrification and influence the lifestyle of P. rg
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37 aeruginosa PAO1 under anoxic conditions. n

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47 INTRODUCTION
48 Respiration is a fundamental process for energy conservation. In eukaryotic mitochondria, Aa3-type cytochrome c
49 oxidase (terminal oxidase) catalyzes the terminal reaction of electron transport in aerobic respiration through
50 oxygen reduction. Furthermore, the generation of reactive oxygen species (ROS) and the oxidation of cytochrome
51 c by Aa3 oxidase can induce apoptosis; thus, Aa3 oxidase also plays a role in cell fate determination (1, 2). In
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52 bacteria, multiple terminal oxidases have been reported to catalyze energy conservation; however, few studies have o
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53 reported their influence on cell behavior. lo
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54 Pseudomonas aeruginosa is a ubiquitous bacterium with versatile aerobic and anaerobic respiratory systems (3). e
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55 Under oxic environments, five terminal oxidases (Cyo, CIO, Aa3, Cbb3-1 and Cbb3-2) reduce oxygen as terminal o
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56 electron acceptors. The terminal oxidases Cyo and CIO are quinol oxidases, while the terminal oxidases Aa3 and t
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57 Cbb3 are cytochrome c oxidases. Compared with other terminal oxidases, Cbb3 oxidases possess a high affinity for /jb
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58 oxygen. Cbb3-1 and Cbb3-2 oxidases are coded by the tetracistronic ccoNOQP-1 and ccoNOQP-2 operons (4). sm
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59 The ccoNOQP-1 operon is constitutively transcribed under various oxygen concentrations (5). On the other hand, rg
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60 the ccoNOQP-2 operon is highly transcribed under low-oxygen concentrations because the oxygen-responsive n

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61 transcriptional regulator ANR upregulates its promoter activity. These enzymatic and transcriptional characteristics y

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62 of Cbb3 oxidases enable P. aeruginosa to grow under low-oxygen environments. In addition, under anoxic 2
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63 conditions where oxygen is depleted, P. aeruginosa can use N-oxides as alternative electron acceptors in a process
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64 known as denitrification. During this process, nitrate (NO3-) is reduced subsequently to nitrite (NO2-), nitric oxide gu
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65 (NO), nitrous oxide (N2O) and finally to dinitrogen (N2). Each reduction step is performed by NO3- reductase t
66 (NAR), NO2- reductase (NIR), NO reductase (NOR) and N2O reductase (NOS). The denitrifying process is
67 triggered by physicochemical factors, such as oxygen and N-oxides, via ANR and other transcriptional regulators,
68 such as NarXL and DNR (6, 7). Moreover, quorum sensing (QS) molecules, such as N-acyl-L-homoserine lactone
69 (AHL) and Pseudomonas quinolone signal (PQS), fine-tune the metabolic activity to control NO concentration (8,
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70 9).
71 Interestingly, Kawakami et al. reported that Cbb3 oxidases of P. aeruginosa are more highly expressed under
72 anoxic denitrifying conditions compared with other terminal oxidases, suggesting that Cbb3 oxidases may have a
73 unique role under anoxic conditions (5). However, their role has not yet been elucidated. In this study, we showed
74 that Cbb3 oxidases of P. aeruginosa PAO1 lead to NO accumulation during denitrifying process under anoxic
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75 conditions. This effect resulted in the promotion of biofilm formation via cell elongation. Such a morphological o
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76 change and complex formation are helpful to survival under many environmental stresses (10, 11). Thus, it is lo
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77 expected that Cbb3 oxidases are key enzymes involved in regulating these cell behaviors in response to anoxic e
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78 environments. Our findings indicate a new role of Cbb3 oxidases, emphasizing their importance in bacterial o
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79 physiology. t
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80 /jb
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93 MATERIALS AND METHODS
94 Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. For routine
95 cultures, bacterial strains were grown at 37 °C in Lysogeny broth (LB) medium (Lennox; Nacalai, Kyoto, Japan) or
96 on LB agar plates. When necessary, gentamicin was added at the concentration of 10 μg ml-1 for Escherichia coli
97 and 100 μg ml-1 for P. aeruginosa. Anoxic planktonic cultures were prepared using a method described previously
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98 (9). P. aeruginosa was grown oxically in 24-ml test tubes containing 4 ml of LB medium for 10 h and inoculated o
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99 into butyl-rubber-sealed 17-ml Hungate tubes containing 5 ml of LB medium supplemented with 100 mM KNO3 lo
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100 (LBN medium) at a starting optical density of 0.01 at 660 nm (OD660). The air of the Hungate tubes was replaced ed

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101 with argon by flushing gas through a needle. Anoxic planktonic incubation was performed at 37 °C with shaking at o
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102 200 rpm. By using a negative control mutant of denitrification, ΔnarG (12), it was confirmed that this experimental t
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103 condition is anoxic (Fig. S1). Hypoxic incubation was carried out by using butyl-rubber-sealed 17-ml Hungate /jb
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104 tubes containing 5 ml of LB medium without replacing the air with argon. sm
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105 Construction of P. aeruginosa mutants. The plasmids used in this study are listed in Table 1. The PCR primers rg
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106 used in this study are listed in Table S1. pG19ccoN1 and pG19ccoN2 plasmids carrying deletion cassettes of ccoN1 n

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107 and ccoN2 were constructed based on a previous method (13). The pG19II-derived plasmids (pG19ccoN1 and y

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108 pG19ccoN2) were transferred into P. aeruginosa PAO1 wild type (WT) and the ΔccoN1 strains by conjugating 2
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109 with the mobilizer E. coli S17-1 (14), followed by homologous recombination as previously described (13). Target
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110 gene deletions were confirmed by PCR analysis. In addition, to construct the ΔccoN1 ΔccoN2 ΔnirS-N mutant, g
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111 which is devoid of NIR and does not produce NO, pG19nirS-N (15) was transferred into the ΔccoN1 ΔccoN2 strain t
112 by conjugating with E. coli S17-1, followed by homologous recombination.
113 Catechol 2,3-dioxygenase (C23O) specific activity assay. The ccoN1 and ccoN2 transcriptional fusion
114 plasmids were constructed by cloning the promoter regions of ccoN1 and ccoN2 (4, 5) into the pMEX9 reporter
115 plasmid upstream of xylE. These plasmids were introduced into P. aeruginosa PAO1 WT (16). The specific
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116 activities of C23O (the xylE gene product) in the strains carrying reporter plasmids were determined by monitoring
117 the absorbance at 375 nm (A375), following a method described previously (13).
118 Differential Interference Contrast (DIC) and bright-field observation. For DIC observation of cells in
119 planktonic shaking cultures, 5 μl of the cultures were mounted onto micro cover glasses (VWR International, USA)
120 following a method described previously (17). For the bright-field observation of cells in biofilms, biofilms formed
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121 in 6-well plates were washed with phosphate buffered saline (PBS). After scraping the biofilms with PBS, 10 μl of o
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122 the suspensions were mounted onto micro cover glasses. A Carl Zeiss Laser Scanning Microscope (LSM 710) lo
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123 equipped with a 63x/1.40 numerical aperture Plan-Apochromat objective (Carl Zeiss, Oberkochen, Germany) was e
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124 used to acquire DIC and bright-field images. The images were captured using an AxioCam MRm digital camera o
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125 and analyzed with the AxioVision software (Version 4.8; Carl Zeiss). t
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126 Denitrification activity assay. Denitrification activities were measured by analytical methods described /jb
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127 previously (9, 12). NO3- concentrations were determined by the brucine-sulfanilic acid method (18). NO2- sm
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128 concentrations were determined by the sulfanilamide-naphthylethylene diamine method (18). N2O and N2 rg
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129 concentrations were measured with a gas chromatograph (model GC-8AIT; Shimadzu). n

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130 Diphenylamine (DPA) assay. Quantification of the cellular levels of deoxynucleoside triphosphates (dNTPs) in y

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131 cell extracts was performed based on a previous method (19). Anoxic incubation of P. aeruginosa was performed 2
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132 using butyl-rubber-sealed 500-ml Erlenmeyer flasks containing 80 ml of LBN medium at 37 °C with shaking at
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133 200 rpm. Anoxically grown cells were washed in 100 mM potassium phosphate buffer (pH 7.5) and sonicated. g
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134 Lysed cell extracts containing equal amounts of protein were mixed with DPA reagent, consisting of 2 g of DPA t
135 (Wako, Osaka, Japan) dissolved in 100 ml of pure acetic acid and 2.75 ml of concentrated sulfuric acid. After
136 incubation at 37 °C for 4 h, the A595 was measured.
137 NO detection. Cellular NO levels were measured based on a previous method (20) with a NO detection reagent,
138 diaminofluorescein-2 diacetate (DAF-2 DA) (Sekisui Medical, Tokyo, Japan). 1 ml of planktonic shaking cultures
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139 grown under anoxic conditions were incubated with 10 μM of DAF-2 DA. After incubation at 37 °C for 1 h and
140 washing with PBS, the green fluorescence of a reaction product, DAF-2 T, was measured using a Varioskan flash
141 fluorometer (Thermo Scientific, Waltham, MA, USA) at an excitation wavelength of 495 nm and emission
142 wavelength of 515 nm. The fluorescent intensity was normalized to OD660.
143 Biofilm formation assay. Overnight cultures of P. aeruginosa grown under oxic conditions were diluted to an
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144 OD660 of 0.1 in LBN medium. Aliquots (100 μl) of the cultures were inoculated into 900 μl of LBN medium in o
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145 24-well polystyrene plates to achieve an OD660 of 0.01. The plates were incubated at 37 °C under anoxic conditions. lo
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146 For anoxic incubation, an AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan) was used (17). After e
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147 incubation, quantification of biofilms was performed with the crystal violet staining method, as previously o
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148 described (17, 21). t
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149 Biofilm visualization with Continuous-Optimizing Confocal Reflection Microscopy (COCRM). For /jb
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150 biofilm visualization, 6-well polystyrene plates were used. Overnight cultures of P. aeruginosa grown under oxic sm
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151 conditions were diluted to an OD660 of 0.5 in LBN medium. Aliquots (0.1 ml) of the cultures were inoculated into rg
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152 4.9 ml of LBN medium in 6-well plates to achieve an OD660 of 0.01. The plates were anoxically incubated at 37 °C n
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153 with an AnaeroPack system. After incubation for 24 h, biofilms that had formed at the bottom of the 6-well plates y

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154 were washed with PBS and the structures were visualized with COCRM (22, 23). A Carl Zeiss LSM 5 PASCAL 2
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155 equipped with a 63x/0.9 numerical aperture W N-Achroplan water immersion objective was used to acquire
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156 COCRM images. Biofilms were illuminated with a 514-nm argon laser, and the reflected light was collected. g
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157 COCRM images were analyzed with the LSM 5 PASCAL software (Version 3.5; Carl Zeiss). t
158 LIVE/DEAD bacterial viability assay. The bacterial viability in planktonic shaking cultures was determined
159 by using a Baclight LIVE/DEAD bacterial viability staining kit (Molecular Probes, Eugene, OR, USA), following
160 a previous method (24). A Carl Zeiss LSM 5 PASCAL equipped with a 63x/1.4 numerical aperture
161 Plan-Apochromat objective was used to acquire LIVE/DEAD images. Live SYTO9-stained cells and dead
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162 propidium iodide-stained cells were excited with an argon laser and a helium neon laser, respectively, and detected
163 with a 560-nm long-pass filter and a 505- to 530-nm band-pass filter, respectively. LIVE/DEAD images were
164 analyzed with the LSM 5 PASCAL software (Version 3.5; Carl Zeiss).
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185 RESULTS
186 Cbb3 oxidases alter cell morphology under anaerobic denitrifying growth. To investigate the effect of two
187 Cbb3 oxidases on cell growth, P. aeruginosa PAO1 WT and ΔccoN deletion mutants that are deficient in Cbb3-1
188 and/or Cbb3-2 oxidases were incubated oxically in LB medium or anoxically in LBN medium. Under anoxic
189 denitrifying planktonic conditions, the ΔccoN1 ΔccoN2 mutant grew better than the WT (Fig. 1A). Growth of
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190 ΔccoN single mutants was similar to that of the WT. CFU value of the ΔccoN1 ΔccoN2 mutant was higher than that o
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191 of the WT and the ΔccoN single mutants at 14 h (Fig. S2). These results show that Cbb3-1 and Cbb3-2 oxidases lo
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192 alter anaerobic denitrifying growth. Cbb3-1 subunits of P. aeruginosa have a high degree of amino acid similarity e
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193 to Cbb3-2 subunits (4). Therefore, two Cbb3 oxidases may complement each other under anoxic denitrifying o
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194 conditions. On the other hand, aerobic planktonic growth of ΔccoN1 and ΔccoN1 ΔccoN2 mutants was slightly t
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195 delayed compared with that of the WT and the ΔccoN2 mutant (Fig. S3A). In addition, the ΔccoN1 ΔccoN2 mutant /jb
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196 showed less growth than the WT under hypoxic conditions in the absence of nitrate (Fig. S4), which was consistent sm
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197 with a previous report (25). rg
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198 Kawakami et al. examined the expression levels of Cbb3 oxidases under different oxygen concentrations (5). To n

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199 monitor whether Cbb3 oxidases were expressed under our experimental conditions, promoter fusion plasmids with y

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200 the xylE gene fused with promoter regions of ccoN1 and ccoN2 genes were used. The specific activities of C23O, 2
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201 the xylE gene product, were determined by a previous method (13). As expected, promoter expression of Cbb3-1
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202 and Cbb3-2 oxidases in P. aeruginosa PAO1 WT was confirmed during the logarithmic to stationary phase (Fig. g
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203 1B), suggesting that these respiratory enzymes have a physiological role under anoxic denitrifying conditions. In t
204 the study of Yoon et al., it was demonstrated that P. aeruginosa PAO1 elongates and aggregates in anoxic
205 planktonic shaking cultures (26). Similar phenomena were observed in the WT by DIC observation. In contrast, the
206 ΔccoN1 ΔccoN2 mutant did not elongate under anoxic denitrifying conditions (Fig. 1C). Both strains were
207 rod-shaped under oxic conditions (Fig. S3B). These results show that Cbb3 oxidases alter cell morphology under
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208 anaerobic denitrifying growth.
209 Cbb3 oxidases alter denitrifying process. Under anoxic conditions where NO3- is present, P. aeruginosa can
210 grow through denitrification, in which NO3- is reduced subsequently to NO2-, NO, N2O and finally to N2 (3). Cell
211 elongation under anoxic conditions is attributed to the accumulation of NO that is produced during such
212 denitrifying process (26). Our data showed that Cbb3 oxidases affect cell morphology; hence, the effect of Cbb3
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213 oxidases on denitrifying process was investigated. As shown in Fig. 2A and B, the ΔccoN1 ΔccoN2 mutant reduced o
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214 more NO3- and NO2- than the WT. N2O production was higher in the ΔccoN1 ΔccoN2 mutant than in the WT (Fig. lo
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215 2C). N2 production of the ΔccoN1 ΔccoN2 mutant was similar to that of the WT (Fig. 2D). These results show that ed

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216 Cbb3 oxidases alter denitrifying process. As shown by our data, the amounts of NO2- and N2O were most affected rom

217 by the Cbb3 oxidases, suggesting that Cbb3 oxidases alter denitrifying process in between the reduction of NO2- to htt
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218 N2O. /jb
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219 Cbb3 oxidases alter cell morphology through NO. Because NO accumulation induces cell elongation (26), it m
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220 can be hypothesized that Cbb3 oxidases affect NO accumulation, which leads to cell elongation. To test this rg
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221 hypothesis, NIR mutants that do not produce NO were used. Compared with the growth difference between the n

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222 WT and the ΔccoN1 ΔccoN2 mutant, ΔnirS-N and ΔccoN1 ΔccoN2 ΔnirS-N mutants had similar but not identical y

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223 growth (Fig. 1A and 4B, Fig. 3A and B), suggesting that NO is one of the main causes of the growth difference. 2
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224 Consistent with other reports (26), the ΔnirS-N mutant did not elongate as the WT and was rod-shaped similar to
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225 the ΔccoN1 ΔccoN2 mutant (Fig. 1C and 3C). When planktonic shaking cultures were observed by LIVE/DEAD g
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226 staining, the WT culture comprised dead or membrane-damaged cells, while the NIR mutant culture comprised t
227 intact cells (Fig. S5). This result was consistent with previous observations (27, 28). The ΔccoN1 ΔccoN2 mutant
228 culture comprised intact cells, similar to NIR mutants. These results support the hypothesis that Cbb3 oxidases
229 affect NO accumulation during denitrification.
230 NO derived from denitrification inhibits DNA synthesis by ribonucleotide reductase (RNR), especially class
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