Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation

Metal nanoparticles synthesized by green methods with the use of microorganisms are currently one of the most closely studied types of nanomaterials. It has accurately been shown that the characteristics of metal nanoparticles generated in the presence of different bacteria vary. For the two isogenic strains of obligate methylotrophic bacteria of the wild type (M. quaylei MTT) and its streptomycin-resistant mutant (M. quaylei SMR), the pleiotropic character of streptomycin resistance mutation in the SMR cells has been revealed. It has been shown that both cultures can generate silver nanoparticles. There is a dramatic difference in the formation of palladium nanoparticles, which are formed only in the presence of cells of the streptomycin-resistant mutant M. quaylei SMR. This study shows that closely related isogenic strains of obligate methylotrophic bacteria can be distinguished by the spectra of biogenic nanoparticles of two noble metals. While palladium nanoparticles are only generated by the cells of the streptomycin-resistant mutant M. quaylei SMR, biogenic silver nanoparticles can be generated from both cultures. Thus, the assessment of the ability of microorganisms to form biogenic nanoparticles of different metals allows the revelation of subtle metabolic differences of even close cultures.


Introduction
Methylotrophic bacteria are well known as objects of various biochemical and biotechnological studies [1][2][3][4] but not as producers of biogenic metal nanoparticles. At the same time, gram-negative aerobic methylotrophic bacteria have prospects for use in nanobiotechnology for the biogenic synthesis of metal nanoparticles. Obligate methylotrophic microorganisms are non-pathogenic because they can not receive the sole carbon and energy source for growth in a mammalian organism and it's important for production purposes. Perhaps that is why methylotrophic microorganisms have been considered as industrial, biotechnological objects in the past few decades [5][6][7][8]. The high growth rate, the low price of growth media with methanol, and the plasticity of the regulation of growth parameters make it possible to consider methylotrophs as promising universal producers.
Methylotrophic cells can use soluble periplasmic methanol dehydrogenase, which is an enzyme containing the prosthetic pyrroloquinoline quinone (PQQ) group. The methanol dehydrogenase of methylotrophs catalyzes the first reaction in an unusual periplasmic electron transport chain responsible for the oxidation of methanol to formaldehyde, utilizing the mechanism of the direct transfer of a hydride ion from the methyl group of methanol to PQQ [1]. Alternative electron acceptors in such a chain can be cations that are present in the medium, which are the source material for the formation of nanoparticles of the corresponding metals [9][10][11][12].
In cases of excessive content of toxic cations in the medium, the formation of nanoparticles from reduced atoms as a less toxic product is observed as a natural protective reaction of microbial cells [13][14][15]. It is believed that the ability to form nanoparticles of metals from sterile salt solutions artificially introduced into cell suspensions can be considered to be a complex characteristic of the natural ability of the microorganisms for self-preservation [16,17]. The possibility of using the process of the formation of biogenic silver nanoparticles for a comparative integral evaluation of the biological properties of microorganisms was first shown by the example of different microbial communities in the horizons of the Antarctic subglacial Lake Untersee [18]. In all the samples studied, the formation of Ag°NPs from an artificially introduced sterile silver salt solution occurred only in the presence of microbial cells. In the control analogs, specifically in the corresponding water samples that were released from the bacterial cells, the generation of nanoparticles was not observed because of the absence of cation reductants in the oligotrophic medium of the samples. The authors named this method of studying the cells of microorganisms the DBNG (detection of biogenic nanoparticles generation) [18,19].
It has been proposed to use the ability of microorganisms to generate metal nanoparticles from reduced cations as an indicator of the presence of metabolically active cells in test samples [19]. Since it is known that the external components of microbial cells play the primary role in the recovery of cations [20][21][22][23], this study compared the ability of closely related analogs of cultures of microorganisms that differ in the characteristics of the surface structures of their cells to generate biogenic nanoparticles of noble metals.
To compare closely related microorganisms using the DBNG method, a widely used approach in genetics was chosen. The approach consists of a comparative analysis of an isogenic pair of cultures. One of the cultures differs from the other in that it contains a single additional mutation leading to some change in the properties of the original culture. In this study, the DBNG method used the cells of an isogenic pair of the obligate methylotrophic bacterium M. quaylei, the wild type, and an isogenic streptomycin-resistant mutant, to compare the ability to form biogenic nanoparticles. Previously, the ability to compare an isogenic pair of cultures to synthesize metal nanoparticles had never been the subject of nanobiotechnological research.
It should be noted that, similar to the oligotrophic microorganisms of Lake Untersee, the cultures of the obligate methylotroph M. quaylei require simple synthetic media for growth that do not contain high-molecular-weight organic compounds that can participate in the chemical reduction of silver cations during the formation of nanoparticles. The absence of abiogenic reductants makes it possible to more accurately assess the details of the interaction of the salts with the cells of closely related methylotrophic bacteria.

Materials and Methods
The Cultures of the Microorganisms The obligate methylotrophic bacteria deposited in the all-Russian collection of microorganisms (VKM), specifically, M. quaylei (VKM B-2338) of the wild type [24] and its isogenic streptomycin-resistant SM R mutant, were used.
The genetic characterization of the cultures of an isogenic pair of obligate methylotrophic M. quaylei strains was conducted using a standard sequence analysis of their 16S rRNA genes [25].

Cultivation of M. quaylei
The methylotrophic cultures were grown on MQ medium, including a mineral source of nitrogen and phosphates to stabilize the pH level, with the following composition (g L −1 ): K 2 HPO 4 1.5, KH 2 PO 4 0.7, NaNO 3 1.5, and EDTA 250. The components were sterilized in solution at 121°C for 25 min. The phosphates and trace element solutions were sterilized separately. All the solutions cooled quickly and were mixed with the trace element solution and methanol (0.5%, v/v) as the sole carbon and energy source under sterile conditions. Cultivation was carried out at 28°C and 180 rpm using a Unimax 2010 orbital platform shaker (Heidolph, Germany) within 3 days. The volume of the media was 50 mL, with cultivation in 250 mL conical flasks. Experiments of the NP generation and sensitivity of bacteria to silver and palladium salts were conducted using cultures in the exponential phase at 9 × 10 8 CFU/ml.

ζ-Potential Measurement
Measurement of cell surface ζ-potential was performed with a Delsa Nano Submicron Particle Size Analyzer (Beckman Coulter Inc., USA) that uses electrophoretic light scattering for the ζ-potential determination with the cells which are washed and placed in a buffer of a predetermined ionic strength and pH value.

Materials
As the source of ionic silver, an aqueous solution of Ag(NH 3 ) 2 NO 3 synthesized from AgNO 3 and ammonia according to a modified Tollens protocol was used to form the Ag°NPs [19,26]. The sterile Ag(NH 3 ) 2 NO 3 solution was added directly to the samples to achieve a final concentration of 0.1 mM.
The source of the palladium ions for the formation of Pd°NPs was an aqueous solution of Na 2 PdCl 4 synthesized from PdCl 2 in a reaction with excess NaCl [27][28][29]. Sterile Na 2 PdCl 4 solution was added directly to the samples to achieve a final concentration of 0.5 mM. The sterility of the aqueous solutions of Ag(NH 3 ) 2 NO 3 and Na 2 PdCl 4 was confirmed by the absence of growth on the complex agar medium LB.

Sensitivity of Bacteria to Silver and Palladium Salts
The sensitivity of fresh cell suspensions of the methylotrophic bacteria M. quaylei to silver and palladium salts was conducted in 1 mL liquid medium at room temperature (24-26°С) without mixing. After 20-min incubation, the cell suspension was plated on solid agar medium. The sensitivity level was determined by the decrease in the number of CFU of the treated samples compared to the CFU of the control suspensions.

NPs Formation
The generation of the biogenic AgNPs and PdNPs from the sterile salt solutions in all the experiments was conducted in 50-μL reaction mixtures with aliquots of cell suspension samples within 20 min, at pH 7-7.2 and temperature of 24-26°C.
Five microliters of the reaction mixture was transferred to the grids for transmission electron microscopy. After 5 min of sorption of the cells and NPs, the liquid was removed, and the grids were washed five times with double-distilled water and dried for 15 h. Aliquots of the samples from which the microorganisms were removed by centrifugation (10,000×g, 25 min) were used as the control [18,19]. The absence of bacteria in the centrifuged control samples was confirmed by electron microscopy and by the absence of growth on MQ and LB agar media. The absence of NPs in the solutions and samples was confirmed by transmission electron microscopy. The scheme of the experiment is shown in Fig. 1.

Transmission Electron Microscopy
The study of the morphology, linear sizes, and localization of nanoparticles was performed using transmission electron microscopy (TEM). Electron microscopy and X-ray microanalysis of the specimens were conducted using a JEM-1400 microscope (JEOL, Japan) equipped with an X-ray microanalyzer (Oxford Instruments, UK) at an accelerating voltage of 80 keV. The specimens were prepared using standard copper grids covered with Formvar and reinforced with carbon.

X-ray Diffraction Analysis
The X-ray diffraction analysis (XRD) of NPs was fulfilled using XRD-6000 diffractometer (Shumadzu, Japan) under following conditions: Cu Kα 1 irradiation in the range of 2θ angles from 30°to 80°with a step of 0.02°and exposure of 30 s at a point. The samples were previously dried and triturated in an agate mortar. Metallic silver and palladium were identified by the peaks in accordance with JCPDS 04-0783 for Ag 0 and JCPDS 05-0681 for Pd 0 .

Determination of NP Sizes
The size distribution of NPs and their characteristics were determined using TEM with the subsequent processing and analysis of images using LabVIEW IMAQ Vision (National Instruments, USA), and the analysis took into account no less than 1000 NPs.

Results
Streptomycin Resistance of Cells from Two Strains of M. quaylei  Table 1).
The determination of the cell sensitivity of M. quaylei strains to silver and palladium salts was conducted in liquid synthetic growth medium with methanol, i.e., under conditions corresponding to those in which NP generation was performed with these metals. The survival of the bacteria in a 0.1 mM solution of Ag(NH 3 ) 2 NO 3 was 32% and 0.4% for the M. quaylei strains MT T and SM R , respectively. When 0.5 mM Na 2 PdCl 4 solution was added to the suspension of bacteria, the survival of the M. quaylei MT T and SM R strains was reduced to 5% and 59%, respectively ( Table 1).

Generation of Silver and Palladium NPs with the Obligate Methylotrophic Bacterium M. quaylei
Experiments on the synthesis of the NPs were conducted with actively growing cells, which were grown in a liquid synthetic growth medium with methanol and were taken directly from the flasks. The formation of nanoparticles was carried out by Bgreen synthesis,^that is, directly in the culture medium with cells in the logarithmic growth phase. Under such conditions, both the cells themselves (their surface biopolymers) and the compounds secreted by the cells can play the role of cation reducing agents in situ.
Both cultures of methylotrophic M. quaylei could form Ag°NPs when a sterile solution of Ag(NH 3 ) 2 NO 3 was added to the suspension. The Ag°NP sizes in the suspension of the wild-type M. quaylei MT T culture cells were from 16 to 100 nm, with the primary fraction of 45 nm. At the same time, in the presence of cells of the streptomycin-resistant mutant M. quaylei SM R , Ag°NPs from 25 to 120 nm in size with the primary fraction of approximately 70 nm were formed (Figs. 2, 3, and 4).  (Figs. 2, 5,  and 6). In the culture of the wild-type M. quaylei MT T , a significant number of optically opaque nanoscale objects were observed, which did not contain palladium according to X-ray microanalysis.

Discussion
The study of the mechanisms of the antibiotic resistance of certain microorganisms remains a topic of medicine, molecular biology, and microbiology [16,[30][31][32]. The fact that the surface structures of the cells play a key role in the resistance to most antibiotics has been revealed for many types of microorganisms. In addition, studies devoted to the comparative study of cell resistance to the toxic effects of salts and the corresponding metal NPs on the pure cultures of various microorganisms were conducted [33][34][35][36]. It should be noted that one of the most informative techniques of the functional comparative studies of microbial cultures is the comparison of the properties of the isogenic pairs of microbial cultures. Isogenic pairs of cultures differ only by one feature, for example, modification by a mutation such as increased resistance to an antibiotic. However, this approach is generally not applied to the ability of isogenic cultures to form NPs [37,38].
It was shown that the resistance to streptomycin in most isolates (up to 70-80%) with such a phenotype (SM R ) is determined by mutations affecting the bacterial ribosomal protein S12 and 16S rRNA, encoded by rpsL or rrs and 7-methylguanosine (m 7 G) methyltransferase gidB, respectively [30,39,40]. From the earliest studies, it was known that streptomycin-resistant derivatives are characterized by the pleiotropic nature of the manifestation of the mutations. In addition, streptomycin-resistant mutants often also showed additional unpredictable resistance to other antibiotics [41]. In this study, we compared for the first time the ability of a pair of isogenic cultures of the obligate methylotrophs M. quaylei (one of which is a streptomycin-resistant derivative of the other) to protect against the biocidal influence of noble metal salts in a growth medium by the formation of biogenic nanoparticles. We compared the effect of silver Ag(NH 3 ) 2 NO 3 and palladium Na 2 PdCl 4 salts on the cells and observed the formation of biogenic nanoparticles of reduced silver and palladium. The pleiotropic nature of the streptomycin resistance mutation in M. quaylei SM R was known previously [24]. The most noticeable differences were in the characters affecting the surface biostructures of the cells. These aspects include a fivefold decrease in the secretion of the exopolysaccharides (up to 0.2 g L −1 ) and an extremely low hydrophobicity of the cell surface (5.5%) compared to the hydrophobicity of the cells of the original M. quaylei MT T strain, which is increased to 39% (Table 1)  of microorganisms, noble metals were chosen, primarily silver and palladium, which differ in the valency of the cations [42][43][44]. The use of the pair of obligate methylotrophic M. quaylei strains in the study appeared to be highly rational, since a simple synthetic medium was used to cultivate the bacteria. This medium does not contain a variety of organic compounds that may have reducing abilities and further affect the process of generating nanoparticles.
In preliminary experiments with methylotrophs, it was shown that at salt concentrations of 0.1 mM Ag(NH 3 ) 2 NO 3 and 0.5 mM Na 2 PdCl 4 , a sufficiently pronounced biocidal effect is achieved while maintaining the cell viability level acceptable for producing biogenic nanoparticles (Table 1). Under these conditions, for the first time, significant differences in the features of the formation of reduced silver and palladium nanoparticles in the presence of a pair of isogenic cultures of obligate methylotrophs directly in synthetic growth media with methanol were observed. Thus, in the streptomycin-resistant mutant M. quaylei SM R , the intensive formation of palladium nanoparticles occurred. The average size of the Pd°NPs was 70 nm. Most of these nanoparticles were adsorbed on cells or occurred in the form of chains that connected the individual cells together (Fig. 5a). In contrast, in the bacterial suspension of the original M. quaylei MT T strain, with a 20-min contact with a solution of the palladium salt, there was a complete absence of biogenic palladium nanoparticles. This result occurred with the background decrease in the cell survival to 5%. At the same time, a significant number of small structures were observed on the TEM image, in which there was no detectable number of atoms of the reduced palladium (Fig. 5f). Taking into account the pleiotropic nature of the streptomycinresistant mutation in M. quaylei SM R bacteria, it can be assumed that the actively synthesized exopolysaccharides of M. quaylei MT T very quickly come into contact with palladium ions Pd and therefore selectively block the formation of Pd°NPs. This statement is supported by data on the high reactivity of Pd 2+ cations towards biopolymers [45][46][47]. Thus, from the two isogenic cultures of methylotrophic bacteria, the ability to generate Pd°NPs manifests only in the streptomycinresistant M. quaylei SM R .
Both cultures of methylotrophic bacteria can generate sufficiently large silver nanoparticles after the addition of 0.1 mM Ag(NH 3 ) 2 NO 3 solution for several minutes. Analysis of the size distribution showed that in the presence of wild-type cells, Ag°NPs have a size ranging from 16 to 100 nm with a bimodal distribution (with the primary  Table 1). It is significant that most of the silver NPs are adsorbed on the cells or somehow associated with them. It can also be noted that the reduced secretion of exopolysaccharides and the extremely low hydrophobicity of the cell surface in the culture of M. quaylei SM R promote the formation of both silver and palladium nanoparticles ( Table 1). The XRD patterns are shown in Fig. 7 and also confirmed the formation of Ag 0 and Pd 0 NPs with face centered cubic (fcc) crystalline structure.
In general, this study shows for the first time the ability of obligate methylotrophic bacteria of the species M. quaylei to form noble metal nanoparticles; unlike the culture of the wild type (M. quaylei MT T ), the culture of the isogenic streptomycin-resistant mutant (M. quaylei SM R ) reduces and forms nanoparticles from both silver and palladium cations. Such a clear difference in the formation of biogenic silver and palladium nanoparticles (noble metals of differing cationic valences) indicates that the DBNG method allows researchers using several metals under similar conditions to distinguish very closely related cultures. This method would be an analog of a Bvariegated series^in the comparison of microbial species or cultures. It can be postulated that the assessment of the interaction of microbial cells with the salts of several metals may be useful for more complete characterization of closely related drug-resistant cultures of clinical isolates.
It has been shown that both cultures of the obligate methylotrophic bacteria M. quaylei (wild-type MT T culture and streptomycin-resistant mutant SM R ) can reduce silver salts and form Ag°NPs directly in a minimal synthetic medium with methanol as the only organic carbon source. In contrast to the wild-type culture, the isogenic streptomycinresistant mutant M. quaylei SM R can generate nanoparticles from reduced palladium atoms.
It can be expected that the assessment of the interaction of the microbial cells with various metal salts may be useful for more complete characterization of closely related drugresistant cultures of clinical isolates.