The influence of Pseudomonas syringae on water freezing and ice melting.

Pseudomonas syringae is a widely spread plant pathogen known to have ice-nucleating proteins that serve as crystallization sites promoting ice growth at near-zero temperatures. Three temperatures that characterize water freezing and ice melting are (i) the freezing point of water, (ii) the temperature of coexistence of ice and water, and (iii) the melting point of ice. Here we show the influence of different concentrations of P. syringae on these three parameters. P. syringae appears to affect both the freezing point of water and the temperature of the coexistence of ice and water. Additionally, we propose a research technique for studying the freezing/melting process that is simple and requires no complex equipment.

Introduction

The ability of bacteria to initiate water freezing at near-zero temperatures was first discovered in 1974 using Pseudomonas syringae [1] and attributed to ice-nucleating proteins (INPs) attached to the bacterium’s outer membrane. INPs serve as nuclei of water crystallizing [2, 3]. The process of water freezing itself is quite complex, but its main aspects that are relevant to our work are discussed in article “Some peculiarities of water freezing at small sub-zero temperatures” [4].

The effect of different concentrations of P. syringae on water freezing has been the subject of many studies. Mostly, this process was controlled visually [5]. Aqueous or phosphate buffer was supplemented with a range of P. syringae concentrations, then droplets of the suspension were applied onto an aluminum plate, cooled from 0 to -10°C, and incubated for a few minutes, followed by visual counting of the frozen droplets. The temperature of solidifying of a certain number of droplets (e.g., 90%) was usually taken as a quantitative parameter of the process. This or similar methods are reported in [1, 6–8]. In [9], the same method was used to study the effect of lyophilized P. syringae cells on H2O and D2O freezing. In [10], individual droplets of a P. syringae solution were visually monitored and the freezing point of each droplet was recorded. In [11], freezing of P. syringae solutions was studied in microtubules under visual control. The above and other studies basically describe the temperature dependence of ice growth initiation at a certain concentration of living or lyophilized P. syringae.

The methods of visual droplet monitoring are clear but very labor-consuming and insufficiently accurate.

In our study, continuous temperature monitoring of a P. syringae suspension was used during both cooling and heating. This revealed the effect of different concentrations of P. syringae on three parameters: the freezing point of water (Tf), the temperature at which ice and water coexist in thermodynamic equilibrium (Ti-w), and the melting point of ice (Tm). As a control, the same experiments used different concentrations of E.coli cells.

Results

Fig 1 shows a typical temperature curve for a 0.5 ml water sample during freezing and melting. A test tube with room temperature buffer was placed in a thermostat pre-cooled to -11°C. A thermometer probe inside the test tube allowed continuous recording of sample temperature. As seen from Fig 1, for about 4 minutes the temperature decreases, with water remaining liquid and supercooled. At some freezing point Tf (in Fig 1, Tf = -9.7°C), the occurrence of an ice nucleator triggers water crystallization accompanied by heat release. After the 4th minute, the curve goes up dramatically reflecting the increasing temperature. From the 6th to the 9th minute, the sample is a mixture of ice and water that holds the temperature of coexistence of ice and water Ti-w (see the plateau in Fig 1, Ti-w = -0.11°C). With the entire water crystalized, the sample temperature decreases towards that of the thermostat (-11°C).

Fig 1

Time dependence of the sample temperature during cooling and subsequent heating.

A room-temperature sample (buffer, 20mM Tris-HCl, pH 7.5) was kept in a thermostat at -11°C for the time interval from zero to the 15th minute and after that transferred to a thermostat pre-heated to +25°C for further monitoring from the 15th minute onwards. The arrows show the curve stretches where the freezing point of water Tf, the temperature of coexistence of ice and water Ti-w, and the melting point of ice Tm were determined. For details of temperature determination, see Materials and Methods.

When transferred to a thermostat pre-heated to +25°C, the ice sample undergoes heating and melting at around the 16th-17th minute. In Fig 1, this process is seen as a bend in the curve which, ideally, should turn into a horizontal line whose position is determined by the ice melting point (Tm). In the case of pure water and an ideal device design, Tm and Ti-w must coincide (for details of temperature determination, see Materials and Methods).

Three temperatures that can be derived from the freezing/melting curve of the sample are important for understanding the activities of some proteins. For example, some antifreeze proteins or ice-binding proteins decrease the temperature of coexistence of ice and water Ti-w. In most publications, this phenomenon is called thermal hysteresis (see, e.g., [12-15]). Other studies show that some proteins can increase the melting point of ice Tm [16, 17]. Using the above method, here we show how different concentrations of P. syringae cells with ice-nucleating proteins attached to their outer membrane affect Tf, Ti-w, and Tm.

The effect of cells on water freezing and ice melting

Different concentrations of P. syringae cells were used in experiments on water freezing and ice melting, with E.coli cells as a control. Fig 2 shows typical freezing and melting curves for samples with different P. syringae concentrations. As seen, the presence of P. syringae triggers ice formation at higher temperatures, as compared to the buffer. E. coli cells do not affect water freezing (Fig 2C), that is, both with and without different E. coli concentrations, water freezing occurs at about -10°C. It should be noted that the freezing process of any solution is associated with the probability of occurrence of an ice nucleator, so the cooling/freezing curves are different even for identical solutions.

Fig 2

Time dependence of sample temperatures during cooling (A and C) and heating (B and D). The curve for the 20mM Tris-HCl buffer, pH 7.5, is shown on all graphs as a black solid line. On A and B, the curves for P. syringae solutions with a cell concentration of 0.19 (--), 0.03(-·-), and 0.003(-··-) optical units are shown in red. On C and D, the curves for E coli solutions with a cell concentration of 0.02(--), 0.2(-·-), and 0.001(-··-) optical units are in blue. The inserts on A and C show the curve stretch from which the freezing point of water Tf has been derived.

In contrast, ice melting is characterized by good reproducibility. Fig 2B shows melting curves for P. syringae-containing ice. As seen, the curve shape is virtually unaffected by the difference in P. syringae concentration. Similarly, E. coli cells in any concentration do not affect the melting point of ice (Fig 2D).

The experiments with P. syringae and E. coli solutions allowed calculating Tf, Ti-w, and Tm as dependent on cell concentration in the samples. Fig 3 shows these dependencies.

Fig 3

Dependence of Tf (A, A*), Ti-w (B, B*), and Tm (C, C*) on cell concentration. Figures marked with * are plotted on a logarithmic scale along the concentration axis. Red circles are for P. syringae solutions. Blue squares are for E. coli solutions. The empty circle shows Tf, Ti-w, or Tm for cell-free buffer averaged across ten replicates.

The data shown in Fig 3 allow the following conclusions:

None of the parameters Tf, Ti-w, and Tm depends on E. coli concentration in the solution.

Unlike Tm, the parameters Tf and Ti-w depend on P. syringae concentration in the solution.

Because freezing of supercooled water is a probabilistic process, the results presented in Fig 3A and 3A* are widely scattered, and a large variance is also observed for the buffer Tf data (see Materials and Methods). The probability of ice occurrence depends on the temperature of the solution and the number of ice nucleators. The growing P. syringae concentration increases the probability of water crystallizing due to INPs attached to the bacterium’s surface. Fig 3A* for P. syringae presents a linear dependence that allows calculating the relationship between Tf and the used cell concentrations from the following equation: where C is cell concentration measured in optical units (o.u.). A large error (with an average deviation of ±1.5°C and maximal of 3.4°C) is explained by the randomness of ice nucleator appearance. This error and the exponential dependence of Tf on cell concentration (Fig 3A) allow the conclusion that at a P. syringae concentration above 0.15 o.u., Tf invariably falls within the range from 0°C to -2°C. Fig 3A clearly shows that at a cell concentration above 1.5 o.u., the temperatures vary from 0°C to -2°C. In other words, it is impractical to use P. syringae concentrations above 0.15 o.u. that corresponds to 1.2·109 cell/ml.

Fig 3B and 3B* show the dependence of Ti-w on P. syringae concentration. A relatively small spread of data (± 0.03°C) results not only from the thermometer accuracy (± 0.01°C) but rather from a slight difference in ice growth and ice/water distribution observed in each individual test tube. Nevertheless, the dependence of Ti-w on P. syringae concentration is determined reliably with an average deviation of ±0.03°C and maximal of 0.05°C. What can this dependence be related to? Logically, it can be assumed that the cell surface-attached INPs display the ice-binding activity. If the ice surface is stabilized through these interactions, one can expect a shift in the ice melting/freezing equilibrium that will lead to an increase of Ti-w with the increasing number of cells in the solution. This conclusion might seem trivial if not for, e.g., ice-binding proteins whose effect is quite opposite: an increase in their concentration causes a lower Ti-w [12-15].

If P. syringae cells are assumed to be bound to the ice surface, Tm would also be expected to depend on their concentration. But Fig 3C shows that Tm is totally unaffected by either P. syringae or E. coli present in the solution. This result is surprising and difficult to explain. It is possible that the reason is the technical peculiarities of the experiment.

When ice grows in supercooled water, the test tube contains a mixture of small ice crystals and water. The thermometer probe is immersed in this mixture, so it measures the average Ti-w of the sample accurately enough.

Whereas in the melting experiment, the thermometer probe is surrounded by a large piece of ice whose melting begins from the outer surface close to the test tube walls that are in contact with the thermostat. Therefore, it is not the temperature of a mixture of water and ice that is measured but rather the temperature of the probe-surrounding ice. Perhaps this is the reason why we were unable to detect the dependence of Tm on the P. syringae concentration.

Conclusions

One of the results of this work is the proposed measurement technique. It turned out that using a solid-state thermostat maintaining a certain temperature and a high precision digital thermometer, it is possible to obtain well-reproducible data on peculiarities of water freezing and ice melting. This technique yielded Eq (1) linking the P. syringae concentration with the freezing point of a voluminous aqueous solution.

An important result is that this study has first revealed the dependence of Ti-w on the P. syringae concentration (Fig 3B), which is evidence for P. syringae binding to the ice surface. This result might seem trivial since surface INPs of P. syringae obviously must interact with the ice surface, but importantly, the obtained dependence means that only free-floating cells interact with ice, unlike those where ice has been already formed.

How to assess whether the interaction of cells with ice is strong or weak? In our experiments, P. syringae cells at a concentration of 0.3 o.u. (~2·108 cell/ml) increased Ti-w by Δ = +0.13 оС. The equal Ti-w decrease (Δ = -0.13 оС) will be observed if the solution is supplemented, e.g., with 70 mM of a low molecular weight substance or 0.25% of ethyl alcohol [18, 19]. Another comparison example is ice-binding proteins which at high concentrations change Ti-w by more than 1оС [20]. Thus, despite INPs, the P. syringae binding to the ice surface is weak.

Materials and methods

Cell culture experiments

E. coli BL21 (DE3) cells were grown on LB growth medium (VWR Life Science AMRESCO) at a temperature of 37°C. P. syringae cells (Pseudomonas syringae pv.syringae) grown on medium L (yeast extract 5.0 g/l; peptone 15.0 g/l; NaCl 5.0g/l) at a temperature of 26°C. All cells were grown in a liquid medium up to cell density OD600 = 1.0OU, then precipitated on a centrifuge at 6000g, washed twice with a solution of 20mM Tris-HCl, pH 7.5. The initial cell solution was diluted with a buffer solution of the same composition to the desired concentration. The concentration of cells was controlled by absorption at 600 nm.

Measurement of water freezing and ice melting

This study used a Biosan CH-100 (Latvia) solid-state thermostat with a fixed temperature of -11°C to measure water freezing or +25°C to measure ice melting. 0.5 ml samples in plastic 1.7 ml test tubes were placed in the thermostat. Temperatures were measured with a digital thermometer LT-300 (Russia) having an absolute accuracy of ±0.05°C and a relative accuracy of ± 0.01°C. The metal probe of the thermometer was fixed in a holder and positioned in the center of the tube. The temperature from the thermometer was recorded in real time on a computer. Fig 4 presents a schematic diagram of the described devise.

Fig 4

The device used in the work.

Schematic diagram of a device for water freezing and ice melting studies.

Peculiarities of Tf, Ti-w, and Tm determination

From the graph of the temperature dependence of the sample on time (an example is shown in Fig 1) we determined three temperatures Tf, Ti-w and Tm.

The freezing point Tf was determined as the minimum value of the temperature at the time of the beginning of water crystallization in the sample.

The temperature of coexistence of ice and water Ti-w was defined as the temperature of the horizontal section of the curve after the moment of freezing of water. For example, in Fig 1, such a section of the curve is between the 6th and 10th minute.

To determine the melting temperature of ice Tm a graph of the first derivative of the temperature dependence curve was plotted. In Fig 5, the blue dotted line shows the section of the temperature dependence curve where ice melting occurs. The black solid line shows the graph of the first derivative of this section of the curve. The arrows in Fig 4 show how the temperature Tm is determined. Obviously, with such a definition of Tm, this parameter will always differ from the actual melting temperature of the TmReal sample. If the sample had a sufficiently large volume and the thermometer could correctly measure its average temperature, then the curve would asymptotically tend to the TmReal temperature. In Fig 4, the red dotted line shows the possible shape of the curve with a large sample volume. Moreover, a very accurate determination of the sample temperature is not possible due to the device. The temperature measurement takes place in the middle of the sample (ice), and its melting begins on the walls of the tube (which are in contact with the thermostat). Therefore, the temperature readings are always underestimated (in the case of melting) compared to the average temperature of the sample. Nevertheless, the temperature determination method shown in Fig 5 allows the Tm temperature to be determined in the same way for different samples and to compare the effect of different substances on the melting temperature of ice.

Fig 5

Determination of the melting point of ice, Tm.

The blue dashed line is an example of the time dependence of sample temperature during heating. The black solid line is the first derivative of the blue curve. The minimum of the first derivative graph allows determining the inflection point of the blue dashed curve (shown by the black arrow) from the position of which Tm is determined (shown by the blue arrow). The red dashed line shows a probable change in the experimental curve (blue) for the case when the sample is voluminous and the thermometer can precisely measure the average temperature of the sample. The red dashed line asymptotically tends to the real melting point of ice, TmReal. The red arrow shows the real melting point of ice.

More than 10 measurement replicates of buffer (20mM Tris-HCl, pH 7.5) freezing and melting have been done to obtain the following three parameters: Tf = -9.8±0.5оС, Ti-w = -0.12±0.01оС, and Tm = -0.2±0.01оС. Fig 1 shows only one curve as an example. Fig 6 shows three buffer freezing curves: one of them with a minimum deviation from the average Tf = -9.8°C and two others with a maximum deviation. The curves for buffer melting are not plotted here because they are identical to the curves shown in Fig 2B and 2C.

Fig 6

An example of the time dependence of buffer (20mM Tris-HCl, pH 7.5) temperature during cooling.

For each curve, Tf values are indicated at the bottom of the graph. The Ti-w values are indicated at the top of the graph near the arrow. The average values of Tf and Ti-w across more than 10 replicates are -9.8 ±0.5оС and -0.12±0.01оС, respectively.

20 Dec 2021

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Reviewer #1: The language level of the manuscript must be increased. There is also a need to explain the nature of the phenomenon under study much better. Why and how the P. syringae affect the freezing point of water and the temperature of the coexistence of ice and water should be answered on more detailed level. The conditions of the liquid/solid phase transition must be more precisely discussed in the context of physical background of the complex freezing-thawing processes: I. Kratochvilova et al. Theoretical and experimental study of antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability, RSC Advances 7 (2017) 352-360.

Reviewer #2: These authors have built a simple apparatus to measure three temperatures in the presence of a bacterium (Pseudomonas syringae) that has surface displayed ice nucleation proteins (INPs). These temperatures are i) the freezing point of water; ii) the temperature at which ice and water co-exist; and iii) the melting point of ice. They have measured these parameters for a wide range of bacterial optical densities in 0.5-mL volumes in a home-made apparatus.

However, it is not clear from the paper what has been discovered. For example, the brief (8-line) Abstract states: “P. syringae appears to affect both the freezing point of water and the temperature of the co-existence of ice and water.” But it does tell us the direction and magnitude of the effects and the scientific basis for them. The last sentence of the Abstract states: “…we propose a novel research technique that is simple and requires no complex equipment.” but it does not say what the technique is for, or what it can accomplish towards solving a scientific problem. These points are also not answered in the paper.

The technique described seems to be a crude version of a manually operated differential scanning calorimeter. This is not a useful instrument for measuring the activity of ice-nucleating bacteria on the freezing point of water because ice nucleation is stochastic event. This parameter is typically measured on small sample volumes (nL to uL) in a multiplexed apparatus where 102 droplets are simultaneously studied and the mid-point where 50% of the droplets are frozen is taken as the nucleation temperature. The authors say: “The methods of visual droplet monitoring are clear but very labor-consuming and insufficiently accurate.” This is simply not true because the appearance of the 102 droplets can be simultaneously recorded through a polarizing filter during the freezing process, and the data can be tabulated in minutes on replay of the video. Another rapid method is to record the exotherms of 102 individual droplets and tabulate the freezing process this way.

The relatively large volume (0.5 mL) of the sample is a problem when measuring the melting process as described in the last paragraph prior to the conclusions. Ice remains around the thermometer probe while the ice melts at the sample edges. This prevented measuring the dependence of the melting temperature on the amount of ice nucleating bacteria present, which was one of the objectives of the study. This seems like a cumbersome technique and it effectiveness has not been demonstrated here.

One way to assess the potential of this system is to do a parallel analysis with ice-binding proteins (IBPs). These proteins have well-documented effects on the temperature at which ice and water co-exist, and the melting point of ice. The former is a temperature range for IBPs that is called the thermal hysteresis value and can be as high as 5-6 C for some insect IBPs. The latter is elevated by a fraction of a degree for most IBPs but would be again a good test of the sensitivity of the apparatus.

This manuscript has been inadequately edited and would have benefitted from a peer review by a colleague. There are many issues to address. It talks about the ‘concentration’ of bacteria. This term applies to something in solution, whereas the bacteria are in a suspension. Cell density is a more appropriate term. E. coli needs a space between E. and coli, as does 20 mM. Elsewhere mM is written as mm.

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24 Feb 2022

Reviewer #1: The language level of the manuscript must be increased. There is also a need to explain the nature of the phenomenon under study much better. Why and how the P. syringae affect the freezing point of water and the temperature of the coexistence of ice and water should be answered on more detailed level. The conditions of the liquid/solid phase transition must be more precisely discussed in the context of physical background of the complex freezing-thawing processes: I. Kratochvilova et al. Theoretical and experimental study of antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability, RSC Advances 7 (2017) 352-360.

We have made a lot of corrections, which, in our opinion, have significantly improved the English. The main changes are marked in the text. You can see that we have tried to work on almost every sentence.

As for the detailed discussion of the transition of water from the liquid phase to the solid phase. Unfortunately, this is a very complicated process. Just mentioning the process of water crystallization will not help the understanding of the article, and detailed explanations require some mathematical calculations that are not relevant in this article. We have inserted a reference to article AV. Finkelstein “Some particularities of water freezing at small sub-zero temperatures”, which discusses the process of freezing water and the role of nuclei in ice formation.

Reviewer #2: These authors have built a simple apparatus to measure three temperatures in the presence of a bacterium (Pseudomonas syringae) that has surface displayed ice nucleation proteins (INPs). These temperatures are i) the freezing point of water; ii) the temperature at which ice and water co-exist; and iii) the melting point of ice. They have measured these parameters for a wide range of bacterial optical densities in 0.5-mL volumes in a home-made apparatus.

However, it is not clear from the paper what has been discovered. For example, the brief (8-line) Abstract states: “P. syringae appears to affect both the freezing point of water and the temperature of the co-existence of ice and water.” But it does tell us the direction and magnitude of the effects and the scientific basis for them. The last sentence of the Abstract states: “…we propose a novel research technique that is simple and requires no complex equipment.” but it does not say what the technique is for, or what it can accomplish towards solving a scientific problem. These points are also not answered in the paper.

The whole article is devoted to explaining what technique we use can give. In particular, this method made it possible to continuously monitor the freezing/melting process and made it possible to reveal the influence of PSeringae cells on the temperature of the coexistence of ice and water. Our method allows us to study three temperature parameters (on one installation) and, accordingly, compare them with each other.

Reviewer #2 The technique described seems to be a crude version of a manually operated differential scanning calorimeter.

Our setup does not look like a bad calorimeter, but a good cryosmometer - an instrument that is usually used to study IBP proteins.

Reviewer #2 This is not a useful instrument for measuring the activity of ice-nucleating bacteria on the freezing point of water because ice nucleation is stochastic event. This parameter is typically measured on small sample volumes (nL to uL) in a multiplexed apparatus where 102 droplets are simultaneously studied and the mid-point where 50% of the droplets are frozen is taken as the nucleation temperature. The authors say: “The methods of visual droplet monitoring are clear but very labor-consuming and insufficiently accurate.” This is simply not true because the appearance of the 102 droplets can be simultaneously recorded through a polarizing filter during the freezing process, and the data can be tabulated in minutes on replay of the video. Another rapid method is to record the exotherms of 102 individual droplets and tabulate the freezing process this way.

We discuss all of the above in the introduction and refer to articles that explore droplets. But when examining droplets, it is impossible to investigate temperature parameters with an accuracy of 0.01 °C. In addition, droplets technique does not allow one to determine the temperature of the coexistence of ice and water.

In our work, we measure temperature parameters with high accuracy, and this is what allows us to draw the main conclusions.

Which technique is more difficult or easier is a moot point. Perhaps there are researchers who will decide that a polarizing microscope should be considered a simple technique, and a thermometer that continuously measures temperature is difficult. We think the opposite.

Reviewer #2 The relatively large volume (0.5 mL) of the sample is a problem when measuring the melting process as described in the last paragraph prior to the conclusions. Ice remains around the thermometer probe while the ice melts at the sample edges. This prevented measuring the dependence of the melting temperature on the amount of ice nucleating bacteria present, which was one of the objectives of the study. This seems like a cumbersome technique and it effectiveness has not been demonstrated here.

One way to assess the potential of this system is to do a parallel analysis with ice-binding proteins (IBPs). These proteins have well-documented effects on the temperature at which ice and water co-exist, and the melting point of ice. The former is a temperature range for IBPs that is called the thermal hysteresis value and can be as high as 5-6 C for some insect IBPs. The latter is elevated by a fraction of a degree for most IBPs but would be again a good test of the sensitivity of the apparatus.

We are discussing the problem of measuring the melting of ice. And we are discussing the fact that the point is not in the installation and its accuracy, but in the fact that it is impossible to start the process of ice melting in the entire volume of ice. Melting always starts at the surface. Therefore, the large volume of the sample (0.5ml) allowed us to somehow measure this parameter. Small droplets melt quickly and it is not possible to fix the exit of the curve to a plateau. We have an idea to make these measurements more accurately, but for this it is necessary to modernize our installation or do joint work with a laboratory that has a “simple” technique for measuring solutions in droplets. In this case, their installation will also have to be changed. Simply put, these measurements cannot be taken quickly.

Regarding the comparison of the results of our experiments with ice binding proteins. That most IBPs are examined using cryo-osmometers. The cryo-osmometer differs from our installation only in the presence of a vibrator, which "starts" the process of water crystallization at relatively high temperatures (-5 oC). We do not need it, because we have studied the influence of cells on the process of ice nucleation. That is, to put it simply, our work is carried out in exactly the same way as most IBP studies. And, therefore, the results of the study can be compared with works in which a cryo-osmometer is used. We used our facility to study IBP proteins The article in print, but, the results are available in BioRxiv (Glukhova K.A., Okulova J.D., Melnik B.S. (2020) Designing and studying a mutant form of the ice-binding protein from Choristoneura fumiferana. BioRxiv. https://doi.org/10.1101/2020.08.31.275651)

Reviewer #2 This manuscript has been inadequately edited and would have benefitted from a peer review by a colleague. There are many issues to address. It talks about the ‘concentration’ of bacteria. This term applies to something in solution, whereas the bacteria are in a suspension. Cell density is a more appropriate term. E. coli needs a space between E. and coli, as does 20 mM. Elsewhere mM is written as mm.

As for typos. You are absolutely right. Minor typos, such as mm instead of mM, greatly interfere with the understanding of the article. We tried to fix it.

Submitted filename: Response_to_rev12e.docx

Click here for additional data file.

7 Mar 2022

The influence of Pseudomonas syringae on water freezing and ice melting.

PONE-D-21-36407R1

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2 May 2022

PONE-D-21-36407R1

The influence of Pseudomonas syringae on water freezing and ice melting.

Dear Dr. Melnik:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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