Surface-Mediated Chromate-Resistant Mechanism of Enterobacter

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Surface-Mediated Chromate-Resistant Mechanism of Enterobacter Cloacae Bacteria Investigated by Atomic Force Microscopy Chunpeng Yang,†,# Yangjian Cheng,†,# Xiaoyan Ma,† Ying Zhu,† Hoi-Ying Holman,§ Zhang Lin,*,† and Chen Wang‡ State Key Laboratory of Structural Chemistry, Laboratory of Materials Chemistry and Physics, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, 350002, People’s Republic of China, National Center for NanoScience and Technology, Beijing 100080, People’s Republic of China, and Center for EnVironmental Biotechnology, Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed October 5, 2006. In Final Form: February 5, 2007 The Enterobacter cloacae CYS-25 strain isolated from a chromate plant shows a strong capability for chromate resistance instead of chromate reduction in aerobic conditions. In this study, atomic force microscopy (AFM) was used for studying the morphology characteristics of bacterial properties during the chromate resistance process. The average length of E. cloacae bacteria in the stationary phase is about 2.3 ( 0.6 µm, while under the stimulation of 400 mg/L CrO42-, the length of bacteria increases to 3.2 ( 0.7 µm. Height and phase images showed that, with the addition of CrO42-, the smooth surface of bacteria changed into one with discontinuous features with characteristic dimension of 40-200 nm. Analysis reveals that these compact convex patches are organic components stimulated by CrO42-. A chromate resistance mechanism relating to the overexpression of extracellular biologic components for preventing the permeability of CrO42- into the cell is proposed as the survival strategy of E. cloacae in chromate situation.

Introduction Trivalent chromium is an essential trace element for organisms, while hexavalent chromium is highly toxic, mutagenic, and carcinogenic.1-3 With the increased usage of chromate in the leather, tanning, alloy, and electroplating industries, Cr(VI)containing wastes are considered to be severe pollutants. Traditional physical-chemical remediation methods have been widely adopted to tackle the problem of chromate-related pollutants. Recently, growing interest has been given to the intracellular reduction of Cr(VI) to Cr(III) as a possible detoxification mechanism in a wide variety of microorganisms.4 Since the discovery of chromium resistance and reduction capability of Pseudomonas bacteria in the late 1970s,5-6 more chromate-reducing bacteria have been found.7-15 It has been * To whom correspondence should be addressed. E-mail: [email protected]. † Fujian Institute of Research on the Structure of Matter. ‡ National Center for NanoScience and Technology. § Lawrence Berkeley National Laboratory. # These authors contributed equally to this article. (1) Toxicological ReView of HexaValent Chromium; U.S. Environmental Protection Agency: Washington, DC, 1998. (2) Katz, S. A.; Salem, H. J. Appl. Toxicol. 1993, 13, 217-224. (3) Costa, M. Crit. ReV. Toxicol. 1997, 27, 431-442. (4) Cervantes, C.; Campos-Garcı´a, J.; Devars, S.; Gutiee´rrez-Corona, F.; LozaTavera, H.; Torres-Guzma´n, J. C.; Moreno-Sa´nchez, R. FEMS Microbiol. ReV. 2001, 25, 335-347. (5) Romanenko, V. I.; Koren’kov, V. N. Mikrobiologiya 1977, 46, 414-417. (6) Summers, A. O.; Jacoby, G. A. Antimicrob. Agents Chemother. 1978, 13, 637-640. (7) McLean, J. S.; Beveridge, T. J.; Phipps. D. EnViron. Microbiol. 2000, 2, 611-619. (8) Lovley, D. R. Annu. ReV. Microbiol. 1993, 47, 263-290. (9) Brown, S. D.; Thompson, M. R.; VerBerkmoes, N. C.; Chourey, K.; Shah, M.; Zhou, J.; Hettich, R. L.; Thompson, D. K. Mol. Cell. Proteomics 2006, 5, 1054-1071. (10) Regine, H. S. F.; Volesky, V. B. Int. Microbiol. 2000, 3, 17-24. (11) Srinath, T.; Verma, T.; Ramteke, P. W.; Garg, S. K. Chemosphere 2002, 48, 427-435. (12) Bopp, L. H.; Ehrlich, H. L. Arch. Microbiol. 1998, 150, 426-431. (13) Fude, L.; Harris, B.; Urrutia, M. M.; Beveridge, T. J. Appl. EnViron. Microbiol. 1994, 60, 1525-1531.

revealed that the process for bacteria dealing with chromate are diverse, such as (1) diminishing intracellular accumulation through either direct obstruction of the ion uptake system or active chromate efflux,7-9 (2) biosorption,10-11 and (3) reduction of Cr(VI) to less-toxic Cr(III) using chromate as the terminal electron acceptor in their respiratory chains12-13 or utilizing enzymes to catalyze the reduction of chromate.14-15 Currently, most of the mechanisms are proposed on the basis of the macroscopic experimental results of microbiology or molecular biology. Direct micro- or nanoscale observations of single bacterial surfaces are keenly needed in order to obtain comprehensive information related to the mechanisms, such as the structural basis for permeability of chromate on the bacterial surfaces. AFM is a very helpful tool for imaging biological process both ex situ and in situ at the nanometer scale.16-17 It has been used to probe a widely variety of biosystems such as eukaryotic cells,18 proteins,19 viruses,20-21 etc. AFM has been performed extensively in bacteria imaging,22-26 such as Escherichia coli (14) Ackerley, D. F.; Gonzalez, C. F.; Park, C. H.; Blake, R.; Keyhan, M.; Matin, A. Appl. EnViron. Microbiol. 2004, 70, 873-882. (15) Michel, C.; Brugna, M.; Aubert, C. et al. Appl. Microbiol. Biotechnol. 2001, 55, 95-100. (16) Ubbink, J.; Scha¨r-Zammaretti, P. Micron 2005, 36, 293-320. (17) Binning, G.; Quate, C. F.; Gerber. Phys. ReV. Lett. 1986, 56, 930-933. (18) Hoh, J. H.; Hansma, P. K. Trends Cell Biol. 1992, 2, 208-213. (19) Li, B. S.; Li, G. P.; Wang, C.; Wang, M.; Wang, D. C.; Bai, C. L. Langmuir 2002, 18, 6723-6726. (20) Drygin, Y. F.; Bordunova, O. A.; Gallyamov, M. O.; Yaminsky, I. V. FEBS Lett. 1998, 425, 217-221. (21) Ohnesorge, F. M.; Horber, J. K.; Haberle, W.; Czerny, C. P.; Smith, D. P.; Binning, G. Biophys. J. 1997, 73, 2183-2194. (22) Kasas, S.; Fellay, B.; Cargnello, R.; Celio, M. R. Surf. Interface Anal. 1994, 21, 400-401. (23) Amro, N. A.; Kotra, L. P.; Wadu-Mesthrige, K.; Bulychev, A.; Mobashery, S.; Liu, G. Y. Langmuir 2000, 16, 2789-2796. (24) Peng, L.; Yi, L.; Lu, Z. X.; Zhu, J. C.; Dong, J. X.; Pang, D. W.; Shen, P.; Qu, S. S. J. Inorg. Biochem. 2004, 98, 68-72. (25) Camesano, T. A.; Natan, M. J.; Logan, B. E. Langmuir 2000, 16, 45634572. (26) Chen, A. Y.; Guo, X. P.; Huang, J. Y.; Hong, Y. L.; Zhang, Q. Anaerobe 2006, 12, 106-109.

10.1021/la062925j CCC: $37.00 © 2007 American Chemical Society Published on Web 03/20/2007

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Figure 1. Typical height and phase images of bacteria before treatment with CrO42-. Imaging size: (a, b) 3.0, (c, d) 1.0, (e, f) 2.5, (g, h) 1.0 µm.

and Bacillus Subtilis exposed to penicillin and Johansen,22 the structural basis for permeability of the Escherichia coli outer membrane,23 and the relationship between the heterogeneity and the mechanical properties on the Burkholderia cepacia G4 and Pseudomonas stutzeri KC surfaces.25 In this study, AFM was used to study the morphology characteristics of Enterobacter cloacae CYS-25 induced by CrO42- under aerobic conditions. A series of surface changes of E. cloacae corresponding to the dose concentration of CrO42are reported here. The nanoscale investigation of basic changes of bacterial surface will be beneficial for gaining insight into the chromate resistance and reduction mechanism of bacteria. Experimental Section Isolation and Preparation of Chromate-Resistant Bacteria. Chromate-resistant E. cloacae CYS-25 was isolated from a chromate plant (Changsha City, Hunan Province, China). Single colonies of E. cloacae on Luria broth (LB) agar plates were transferred into liquid LB media. The cultures were grown aerobically at 30 °C and pH 7.2 under continuous shaking at 180 rpm overnight. Bacteria were removed from the LB growth medium without washing and inoculated at a ratio of 1:100 into flasks containing LB medium and CrO42- with various concentrations (100, 250, 400 mg/L). This Cr(VI) treatment was performed at 30 °C under continuous shaking at 180 rpm. The pH of the solution will change from 7.2 into 8.0 during the first 24 h. Aliquots (3 mL) were withdrawn for growth and reduction experiments at different time intervals. The bacteria growth was monitored by measuring the optical density (OD) at 600 nm using a Perkin-Elmer Lamda-900 UV/vis spectrophotometer. Concentrations of Cr(VI) were determined colorimetrically at 540 nm by the same UV/vis spectrophotometer equipped with 1 cm cuvettes, using the diphenylcarbazide (DPC) assay.27-28 Atomic Force Microscopy. All AFM experiments were performed by using a Veeco Multimode NS3A-02NanoscopeIII atomic force microscope. Imaging was done in the tapping mode. The cantilever of the tip is a standard 115-135 µm long microlever with a force constant of 20-80 N/m and has a typical resonant frequency between 200 and 400 kHz. A 200 µL portion of the cell suspension was withdrawn from the above culture. The suspension was centrifuged at 4000g in an Eppendorf centrifuge for 2 min. The supernatant was removed, and the cell pellet was resuspended in ice cold Tris-HCl (pH 8.0, 2 mmol/L). This procedure was repeated twice. Finally, the pellet was resuspended in Tris-HCl. A 5 µL portion of the bacteria suspension was placed on a freshly cleaved mica substrate and dried under N2 gas for 3 min immediately. All AFM measurements were (27) Urone, P. Anal. Chem. 1955, 27, 1354-1355. (28) Fang, H. H. P.; Chan, K. Y.; Xu, L. C. J. Microbiol. Methods 2000, 40, 89-97.

Figure 2. Average length of stationary phase bacteria at different chromate concentrations. Each point is averaged from at least 20 bacteria observed from AFM in air. made within an hour. Extra caution should be taken for guaranteeing the consistency of sample preparation and imaging conditions. Height and phase images of E. cloacae were recorded simultaneously using tapping-mode AFM. Bacteria were scanned in both directions several times before capturing an image to help obviating tip artifacts, such as hysteresis. Tips were replaced frequently to maintain the desirable imaging resolution. Phase images were captured by measuring the phase signal difference between the phase angle of the excitation and the phase angle of the tip response. For obtaining the most useful phase images for distinguishing between relative degrees of surface stiffness, the target amplitude and amplitude setpoint were adjusted frequently during AFM imaging.25,29

Results Growth and Reduction Characteristics of E. cloacae CYS25. E. cloacae CYS-25 is one of the bacteria species that can be isolated from chromate-contaminated sites. We found that this strain was able to reproduce in a LB medium amended with CrO42- as high as 800 mg/L under aerobic conditions and the reproductive capability of the bacteria was influenced gradually by the increase of CrO42-. While at the same time, the bacteria can only reduce CrO42- partly, about 20-50 mg/L for an initial concentration of CrO42- from 100 to 800 mg/L. Moreover, the reduction process primarily occurred at an exponential phase (during the first 24 h after the addition of CrO42-), followed by a chromate resistance process at a stationary phase(24-72 h) and a death phase (96-120 h). Thus, we conclude this strain has a strong capability for chromate resistance instead of chromate reduction in the specific culture conditions. In the following, we focus on the characteristic changes of the morphology and surface mechanical properties of E. cloacae during the chromate resistance stage. (29) Magonov, S. N.; Elings, V.; Whangbo, M. H. Surf. Sci. Lett. 1997, 375, 385-391.

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Figure 3. Typical height and phase images of bacteria treated with 100 mg/L CrO42- at certain time intervals. Imaging size: (a, b) 3.0, (c, d) 1.0, (e, f) 3.0, (g, h) 1.0, (i, j) 3.0, (k, l) 1.0 µm.

Figure 4. Typical height and phase images of bacteria treated with 250 mg/L CrO42-. Imaging size: (a, b) 3.0, (c, d) 1.0 µm.

Morphology of E.cloacae Grown Without CrO42-. Figure 1 shows the AFM images of bacteria in LB medium without CrO42-. As shown in Figure 1a and c, height images reveal that typical bacteria grown without CrO42- for 24 h are rod-shaped and with smooth surfaces, only some of the bacteria are still attached with pili, owing to the centrifuging treatment. Panels b and d of Figure 1 are the corresponding phase images of the bacteria. It shows that though the heights of the bacterial bodies and the pili are different, the phase contrast of the bacterial body and pili are almost the same, indicating that the homogeous distribution of the bacterial surfaces (including pili). Panels e-h of Figure 1 are typical height and phase images of bacteria grown without CrO42- for 48 h. No differences can be identified for both bacterial size and surface fine structures with time from 24 to 48 h. Morphology Changes of E.cloacae Induced by CrO42-. Figure 2 shows the average length of E. cloacae bacteria at different initial concentrations of CrO42-. It reveals that the bacterial size is dependent on the initial chromate concentrations rather than cultivation time. For example, the length of control bacteria at the beginning of stationary phase is about 2.3 ( 0.6 µm. In contrast, after the addition of 100 and 400 mg/L CrO42- for 24 h, the average length have increased to 2.7 ( 0.5 and 3.2 ( 0.7 µm, respectively. Moreover, the whole bacterial volume have also increased as shown in Table S1 in the Supporting Information. Accordingly, AFM height and phase images show that with the addition of 100 mg/L of CrO42-, the bacteria have series surface morphology changes in addition to the above-mentioned size changes. As shown in Figure 3a-d, at 24 h, the bacterial surface is still smooth, the characteristics in phase images shows more features than in Figure 1a-d. With the extension of the

incubating time (48-72 h), the roughness of bacterial surface increases and the bacterial surface and the pili have a lighter color in the phase images, revealing the increase of hardness of some surface components (Figure 3e-h). At 120 h, there were fine features on the bacterial surfaces and some of the bacteria were found damaged (Figure 3i-l). Actually, we found that most of the bacteria grown in the exponential phase or stationary phase (0-72 h) can be characterized with limited heterogeneity, which is greatly enhanced for bacteria in the death stage phase (96-120 h), owing to the different growth situations. Since the AFM sample preparation and imaging conditions are identical for these studies, the heterogeneity characteristics in Figure 3i should be real for bacteria in the death phase, not caused by the dehydration. When the initial concentration of CrO42- increases to 250 mg/L, as shown in Figure 4, there are more patches appeared on the bacterial surfaces. These fine features are enhanced and highlighted in the phase image. Thus, these nanopatches are more condensed as compared to the bacterial surface treated in 100 mg/L CrO42- at the same time duration (Figure 3e-h). When the initial concentration of CrO42- further increases to 400 mg/L, as shown in Figures 5a-d, the roughness of the bacterial surfaces have already increased apparently at the duration time of 24 h. With time increased, it reveals that the surfaces of the bacteria are fully covered with these condensed patches, like a crust wrapped on the surfaces (Figure 5e-h). One can further observe that such patches can also fall off of the bacterial surface and leave vacant positions on the bacterial surface in the phase image. We also noticed that the pili are attached with bright dots in AFM phase imaging. We hypothesize that during the sample treatment, the pili might be the preferential sites to adsorption of the nanoparticles of biological components produced from

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Figure 5. Typical height and phase images of the bacteria treated with 400 mg/L CrO42- at certain time intervals. Imaging size: (a, b) 3.0, (c, d) 1.0, (e, f) 4.0, (g, h) 1.0, (i, j) 0.5 µm.

the surface of bacteria. Thus, the difference of the pili and bacterial bodies are apparent at the beginning (Figure 5b). With more nanoparticles produced, the bacterial bodies are entirely covered with these nanostructures; thus, the features of the pili and bacterial bodies appear similar in the phase contrast image (Figure 5j). Detailed Analysis of the Surface Convexes Induced by 400 mg/L CrO42-. As we observed above, with the increasing concentrations of CrO42-, there are significant changes in bacterial surface roughness. As to the bacteria grown in 400 mg/L CrO42for 24 and 48 h, it reveals that there is no obvious difference of surface roughness in height images, while the corresponding phase image characteristics are totally different. As illustrated in the white circles in Figure 6, at an incubation time of 24 h, the surface convex parts are not homogeneous in the phase image. With incubating time increasing to 48 h, all the surface convexes become relatively compact in the phase image. These observations imply that, under the stimulation of chromate, disperse components appeared on the bacteria surface first and, with the increasing stimulation time, initial dispersed features are grown into compact structures on bacteria surfaces. In order to investigate the characteristics of the surface crust structures, bacteria grown in 400 mg/L CrO42- for 48 h were centrifuged for over 20 min at 4000g. As shown in Figure 7a, we captured a bacteria with half of the surface crust being peeled off. Though there is no obvious difference in the height image of the bacteria, the phase image reveals that half of the bacterial surface is very homogeneous (part I) and half of the bacterial surface is covered with a compositionally different layer (part II). In fact, we found that at the boundary the height of part II is about 20-30 nm higher than that of part I. A 3D image of the bacteria (Figure 7b) shows that there is an extrusive center stripe on the bacteria, which corresponds to the beginning of part II in Figure 7a. On the left of the extrusive stripe, the bacterial surface is extremely featureless. Thus, we propose that the extrusive center stripe represents the folding back surface crust layer from the left. Figure 7c is the sectional analysis of the bacteria height and phase images, as indicated in the arrows in Figure 7a. It reveals that the section line in the phase image is suddenly changed at the boundary of the extrusive stripe.

Figure 6. Detailed analysis of phase images for E.cloacae with 400 mg/L CrO42- for (a, b) 24 and (c, d) 48 h, showing the growing behavior from dispersed particles into compact structures on bacterial surface. Imaging size: 750 nm.

Discussion In order to exclude the dehydration effect on bacterial surface characteristics during the ex situ imaging process, typical AFM images of bacteria in buffer solution were collected. As shown in Figure 8, the surfaces of the control bacteria are very smooth and without any detectable fine structures. For bacteria treated with 400 mg/L CrO42- for only 24 h, nanosized patches were observed on the bacterial surface, confirming the observation obtained ex situ. Additionally, the bacterial length is also observed to increase from 1.6 ( 0.5 (no chromate) to 2.2 ( 0.7 µm (400 mg/L for 24 h) (more details about the size variation are provided in the Supporting Information), consistent with the behavior observed in ex situ. The difference of the heterogeneity of phase images in liquid imaging situation are relatively reduced, possibly owing to the difference of imaging medium, while the amplitude mode is helpful for providing information relating to the surface fine structures. It was reported that E. cloacae HO1 strains could tolerate high concentrations of chromate under both aerobic and anaerobic conditions and develop the capability to reduce chromate under anaerobic conditions.30 This is very similar to our finding that (30) Wang, P. C.; Mori, T.; Komori, K.; Sasatsu, M.; Toda, K.; Ohtake, H. Appl. EnViron. Microbiol. 1989, 55, 1665-1669.

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Figure 7. AFM analysis of a single E.cloacae bacterium with half of the surface crust being peeled off.

Figure 8. AFM analysis of E.cloacae in liqid cell. (a-c) Without chromate for 24 h and (d-f) with 400 mg/L CrO42- for 24 h. Imaging size: 1.5 µm.

E. cloacae CYS-25 has obvious chromate resistance capability instead of chromate reduction ability under aerobic conditions. As to the survival mechanism of E. cloacae HO1 strains under aerobic conditions, it was suggested that it is related to the decreased uptake of chromate.31 In this work, we found that the reduction process of the bacteria mainly happened during the first 24 h after the addition of chromate. Subsequently, bacteria can still survive while no obvious reduction process happened. Correspondingly, AFM investigation reveals that (1) the average sizes of bacteria increase appreciably with the addition of chromate. The higer the initial concentration of chromate, the larger the bacterial size. And the increase of size is happened during the reduction period. (2) Prior to the exposure to chromate, the surfaces of E.cloacae CYS-25 were homogenuous, while surface heterogeneity was developed on the bacterial surface under the stimulation of chromate. (3) These nanosize surface features become compact with extended stimulation time under the chromate solution. Above results indicate that the dimension and compactness change are directly related to the chromate resistance mechanism of bacteria. Previous reports show that when the E.cloacae HO1 are induced by chromate, many particles could be found on the surface of (31) Ohtake, H.; Komori, K.; Cervantes, C.; Toda, K. FEMS Microbiol. Lett. 1990, 67, 85-88.

bacteria by TEM.32 The study considered those particles were chromic hydroxide. However, only trace chromium was found on the surface of E.cloacae CYS-25 treated with chromate by analyzing with energy-dispersive X-ray spectroscopy (EDS) (data not shown), which indicated that the heterogeneities on the surfaces were not caused by the precipitation of chromic composition on the E.cloacae CYS-25 surfaces. When stimulated by chromate, it was found that many exopolysaccharide pellets can be formed on the surface of marine E. cloacae bacteria, and the stimulating effect was increased with the increase of chromium concentration.33 The increase of organic components was found in our previous work for basaltinhabiting bacteria, where both surface nodules and bacteria cell wall thickness increase under the stimulation of chromate.34 In this investigation, analysis relating to Figure 6 indicates the micrometer scale size changes of the bacteria dimension under the stimulated of Cr(VI) is not caused by the formation of the surface crust layer with a thickness of tens of nanometers. Thus, we proposed that there are at least two kinds of components are produced on the bacterial surfaces under the stimulation of chromate, one is the soft envelope layer and the other is the (32) Wang, P. C.; Mori, T.; Toda, K.; Ohtake, H. J. Bacteriol. 1990, 172, 1670-1672. (33) Iyer, A.; Mody, K.; Jha, B. Mar. Pollut. Bull. 2004, 49, 974-977. (34) Lin, Z.; Zhu, Y.; Kalabegishvili, T. L.; Tsibakhashvili, N. Y.; Holman, H. Y. Mater. Sci. Eng. C 2006, 26, 610-612.

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condensed nanocrust layer. It is possible that the overexpression of biocomponents such as exopolysaccharide and protein under the stimulation of chromate should be the reason for the dimensional changes of the bacteria. We propose these condensed patches, together with the soft envelope, prevent the chromate to pass into the cell membrane, providing a survival route via avoiding the contact of chromate with the cell interior instead of reducing the chromate. Systematic work is keenly needed for clarifying the composition of the surface components and the function of these biomolecules.

developed on the bacterial surface under the stimulation of chromate. We propose that biological components produced outside of the cell wall contribute to these patches and size enlargement, which prevent the chromate to pass into the cell interior and avoid direct contact to the cells. Acknowledgment. Financial support for this study was provided by the President foundation of CAS, Foundation for Overseas Scholar Fellowship, the National Natural Science Foundation of China (20501020), and the Nanoscience Foundation of China (90406024).

Conclusion We have investigated the changes in morphology characteristics of chromate-resistant bacteria E. cloacae CYS-25. AFM images clearly reveal that the sizes of bacteria vary appreciably with the increase of chromate concentration and a compact crust layer is

Supporting Information Available: Systematic statistic data and more details about the size variation in situ and ex situ. This material is available free of charge via the Internet at http://pubs.acs.org. LA062925J