Enhancement of Hydrogen Peroxide Production by RNA Polymerase rif Mutations of Lactobacillus delbrueckii subsp. lactis MDC 9617

doi:10.21203/rs.3.rs-1955867/v1

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Enhancement of Hydrogen Peroxide Production by RNA Polymerase rif Mutations of Lactobacillus delbrueckii subsp. lactis MDC 9617

https://doi.org/10.21203/rs.3.rs-1955867/v1

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It was found out that among rifampicin resistant mutants (Rif) of Lactobacillus delbrueckii subsp. lactis MDC 9617 the frequency of occurrence of active hydrogen peroxide producers is about five times higher than in Rif + strain. Aerobically growing Rif mutants show relatively higher kinetics of H 2 O 2 production and earlier entry into the stationary phase due to auto-inhibition caused by inhibitory concentrations of H 2 O 2 . As a result, they synthesize about the same amount of H 2 O 2 but with a smaller biomass. The concentration of H 2 O 2 , which causes auto-inhibition of aerobically growing cultures is approximately 2 times lower than the MIC of exogenous H 2 O 2 for anaerobically growing cultures. The higher sensitivity of aerobically growing cultures is rather due to the existing intracellular H 2 O 2 , which together with extracellular H 2 O 2 causes an additive/cumulative effect. Lack of auto-inhibition at 5 °C allows the mutants to accumulate 50-70% more peroxide than the parental strain. Reduction of the food-borne pathogen Escherichia coli O157:H7 by selected active Rif-4 mutant during co-cultivation in LAPTg at 5 °C was significantly higher than that of the parental strain. Due to high (~40%) co-aggregation ability of LAB with E. coli , the death of the pathogen detected at an early stage of co-cultivation in the absence of inhibitory concentrations of H 2 O 2 , may occur by direct release of H 2 O 2 onto the target cell. The ability of Rif mutants to produce higher amounts of H 2 O 2 with a smaller biomass enable them to extend the shelf-life of refrigerated food without risk of altering sensory characteristics of the latter.

L. delbrueckii subsp. lactis

rif mutations

hydrogen peroxide

E. coli O157:H7

bio-preservation.

Lactic acid bacteria (LAB) which produce enough hydrogen peroxide (H₂O₂) at refrigeration temperature are widely used as bio-preservatives to extend the shelf-life of refrigerated raw food due to inhibition of food-borne pathogens and psychrophilic spoilage microorganisms (Ito et al. 2003; Amézquita and Brashears 2002; Gyawali et al. 2014; Holzapfel et al. 1995; Kostrazynska and Bachand 2006). Lactobacilli produce a wide range of inhibitory compounds, but the dominant inhibitory factor is identified as hydrogen peroxide synthesized at refrigerator temperature without growth (Ammor et al. 2006; Jones et al. 2008, Collins at al. 1980; Goodarzi et al. 2016; Ruby and Ingham 2009; Villegas et al. 1998; Yap and Gilliland 2000, Hovhannisyan et al. 2016).

Among known lactobacilli, L. delbrueckii subsp. lactis produces the most amount of H₂O₂ at low temperatures, and is most used for food preservation (Yap and Gilliland 2000). The hydrogen peroxide production varies from strain to strain, therefore, selection of lactobacilli with a consistently high H₂O₂ producing activity is becoming increasingly important. The enzymes that catalyze the H₂O₂ generation process still remain uncharacterized. It is known that NADH and flavin are involved in enzymatic reaction, and that it is catalyzed by a constitutively expressed protein, independent of availability of molecular oxygen (Martin and Suarez 2010; Yi X et al. 1998; Marty-Teysset et al. 2000).

Hydrogen peroxide may accumulate in aerobic cultures of many strains of LAB which in turn inhibit their own growth. Many reports clearly demonstrate that accumulation to auto-inhibitory levels during exposure to oxygen is widespread among LABs (Condon 1987). The accumulation of H₂O₂ is associated with a higher overall activity of systems producing H₂O₂ than those that eliminate it (Condon 1983).

The H₂O₂ forming oxidases are undoubtedly the principal mechanisms for the hydrogen peroxide production in the LAB. Several dozen genes are involved in the formation, degradation and scavenging of hydrogen peroxide and more of them are transcriptionally regulated (Gonzalez and Demple 1997; Hertzberger 2016). The multiplicity of genes involved in the formation of H₂O₂ and the phenomenon of self-inhibition create great problems for the mutational increase in the productivity of H₂O₂ in lactic acid bacteria (Hovhannisyan et al. 2010). The use of the mutations related with transcription, could change H₂O₂ associated genes expression due to characteristic pleiotropy feature of their phenotypic realization (Goldstein 2014; Jin and Gross 1989; Hovhannisyan et al. 2010; Hovhannisyan and Barseghyan 2015). It was proven that rif mutations affecting the function of RNA polymerase can cause a variant reading of genetic information at the level of transcription of almost every gene, which leads to a change in a whole spectrum of bacterial characteristics, such as colony morphology, growth rate, temperature sensitivity, needs for growth factors, etc. (Jin and Gross 1989; Hovhannisyan and Barseghyan 2015).

The aim of this study was to increase the yield of hydrogen peroxide of L. delbrueckii subsp. lactis MDC 9617 using rif mutations, to assess the growth and H₂O₂ producing behavior of Rif mutants depending on environmental conditions and their impact on food-borne pathogen E. coli O157:H7.

Bacterial Cultures

Lactobacillus delbrueckii subsp. lactis MDC 9617 (GenBank accession no. HQ379117 and HQ379180) and Escherichia coli O157:H7 MDC 5003 are from Microbial Depository Center (MDC) of Republic of Armenia.

Media

The following media have been used during the research work: LAPTg medium (10 g/l yeast extract, 15 g/l Bacto peptone, 10 g/l Bacto tryptone, 10 g/l glucose, 1 g/l Tween 80), Nutrient broth (NB) (Condalab, Spain), Tryptose agar (T-agar) (Merck, Germany), MacConkey agar (Sigma-Aldrich, Germany), N-methyl-N′-nitro-N-nitrosoguanidine (NG) (Sigma-Aldrich, Germany), Quantofix Peroxide Test strips with measuring ranges 0.5-25 mg/L and 1-100 mg/L (Sigma-Aldrich, Germany), Horseradish peroxidase (Roche, Germany).

NG induced mutagenesis and isolation of rifampicin resistant mutants

The culture of L. delbrueckii was subjected to NG mutagenesis with concentration of 300 μg/ml for 30 min as described earlier (Hovhannisyan et al. 2010). Appropriate dilution of NG treated cultures was plated on LAPTg agar with (100µg/ml) and without rifampicin and incubated for 48 hours at 37 °C. An equal number of randomly selected resistant and sensitive colonies were purified on appropriate media at least three times.

Hydrogen Peroxide Assay

For total assessment of hydrogen peroxide concentration Quantofix Peroxide Test strips were used, according to the manufacturer instruction. In other cases, hydrogen peroxide was assayed according to the methods of Villegas and Gilliland (Villegas and Gilliland 1998).

Selection of active producers of H₂O₂

Overnight cultures (per 0.1 ml) were inoculated in test tubes with 2 ml LAPTg and incubated with intensive agitation (200 rpm) at 37 °C for 5 hours and the H₂O₂ amount was measured with Quantofix Peroxide Test strips.

Bacteriological assay

In mixed cultures, for separate enumeration of E. coli and Rif LAB, 10-fold serial dilutions were prepared and plated on MacConkey agar for E. coli and 100 μg/ml rifampicin containing LAPTg agar for Rif LAB.

Minimum inhibitory concentration (MIC) determination

Two-fold serial dilutions of H₂O₂in tubes with 1 ml LAPTg broth for LAB and NB broth for E. coli were prepared, then inoculated with 0.1 ml ~10⁵ CFU/ml of test cultures and incubated for 24 h at 37 °C. The MIC endpoint is the lowest concentration of H₂O₂ where no visible growth is seen in the tubes.

Evaluation of Rif mutants’ cultural liquids antimicrobial activity with agar disk diffusion method

The culture of E. coli O157: H7, grown in LB broth for 18 h at 37 °C, was diluted to a concentration 10⁷cell/ml and 0.1 ml was spread over the surface of NB agar. Sterile Whatman paper disks (diameter 5 mm) were soaked with LAB culture liquids and placed on the pathogen lawn. After 2 h of exposition to refrigeration condition for antimicrobial’s diffusion the plates were transferred to 37 °C, incubated for 18 h and growth inhibitory zones were measured.

Evaluation of Rif mutant antagonism against E. coli O157: H7 at 5 °C

The overnight culture of E. coli was harvested via centrifugation, re-suspended in fresh LAPTg and diluted in 500 ml LAPTg at a concentration of approximately 10⁵ CFU/ml (Brashears et al. 1998). The suspension was divided into four equal parts, where one served as a control, and the rest were infected by LAB in different proportions. All samples were stored at 5°C for 5 days in static condition and periodically subjected to microbiological analysis. The viable counts of E. coli O157: H7 cells in mixed culture was determined by plating of the 10-fold serial dilutions onto MacConkey agar non permissive for lactobacillus growth.

Co-aggregation assay

Co-aggregation between L. delbrueckii MDC 9602 and E. coli O157: H7 was performed according to Handley P. et al. (1987).

Statistical analysis

Statistical analysis was performed using SPSS program (Version 16). Standard deviation (SD) of mean was used to describe data. Fisher’s range test was used to determine differences between tested groups. P value < 0.05 is considered as significant.

NG mutagenesis and isolation of Rif mutants’ active producers of hydrogen peroxide

The selection of mutants with increased production of H₂O₂ is difficult to overcome due to the phenomenon of "auto-inhibition", therefore, mutants were tested for H₂O₂ activity before the beginning of auto-inhibition. The experiment showed that the NG mutagenesis increased the frequency and the diversity of pleiotropic rif mutations, which in turn increased the yield of H₂O₂. Out of three independent experiments, among 150 rifampicin-resistant cultures assessed per hydrogen peroxide synthesis, 3.3 % was found to accumulate relatively high amount of hydrogen peroxide, while among the same amount of rifampicin sensitive NG-treated cultures only 0.67% was active. Thus, the frequency of occurrence of active peroxide producers among Rif mutants was about five times higher than among cultures sensitive to rifampicin.

The activity of selected Rif mutants was examined and confirmed also by microbiological agar disk diffusion method (Fig. 1).

Relatively larger inhibitory zones on the lawn of the pathogen E. coli O157:H7 around the disks soaked in culture liquids of active Rif mutants indicate a high antibacterial activity of the mutants compared to the parental strain (in the center). For further study, the mutants numerated on the plate were designated as follows: 19 - Rif1, 31 - Rif2, 33 - Rif 3, 14 - Rif 4, and 21 - Rif5.

Growth behavior and hydrogen peroxide accumulation by Rif mutants

To study the growth behavior of L. delbrueckii Rif mutants, LAPTg medium was inoculated 1:10 with overnight Rif cultures and incubated at 37 °C under anaerobic and aerobic (agitation 200 rpm) conditions. Biomass (OD₆₀₀) and H₂O₂ accumulation was assessed every 30 min (Figures 2 and 3).

The specific growth rate (0.8 ± 0.1 h^-1) of the cultures under aerobic and anaerobic conditions, does not differ significantly from one to another, but aerated cultures enter the stationary phase significantly earlier than anaerobic ones (Fig. 2). Aerobic cultures release hydrogen peroxide into the environment, which leads to premature growth inhibition, which occurs at different times of growth depending on the rate of H₂O₂accumulation. After autoinhibition, the populations of Rif mutants, as well as the parental strain remain constant for the entire time. As compared to the parental strain, all Rif mutants, except of Rif-3, eventually accumulated approximately the same amount of H₂O₂, but by a 20-50% lower biomass, as a result of an earlier transition to the stationary phase. When the level of H₂O₂reaches to approximately 120 mg/l, the growth, and thus the synthesis of H₂O₂ gradually cease. Due to auto-inhibition, the maximum level of H₂O₂ accumulation in all cultures was approximately the same (p ≥ 0.05) (Fig. 3). But in mid logarithmic phase the comparison of the H₂O₂ concentration revealed significant (p < 0.05) differences between the mutants as well as the parental strain; the Rif mutants produced 30–50% more hydrogen peroxide than the wild strain.

The Rif-3 mutant accumulates the most biomass and H₂O₂(P<0,05). In an anaerobically growing culture, neither accumulation of hydrogen peroxide, nor auto-inhibition was observed.

The effect of growth phase and aeration on H₂O₂ tolerance of L. delbrueckii

In contrast to the H₂O₂ MIC obtained earlier for non-aerated L. delbrueckii, the extracellular concentration of H₂O₂ causing auto-inhibition of aerobically growing cultures was significantly lower. Hence, the relative H₂O₂MICs of the cultures depending on the growth phase and aeration was studied (Fig. 4).

The MIC of hydrogen peroxide for anaerobically growing L. delbrueckii in stationary and exponentially phases differ from each other. Cells in log phase were about twice more sensitive to hydrogen peroxide than cells in the stationary phase. In turn, significantly less amount of H₂O₂ was needed to arrest the growth (auto-inhibition) of aerobically growing cultures.

Hydrogen peroxide production by Rif mutants in LAPTg at 5 °C

Since lactobacilli are widely used for food preservation at refrigeration temperature, the behavior of L. delbrueckii mutants at 5°C was studied. In the most of studies the production of hydrogen peroxide by LAB cells was examined in phosphate buffer (Amézquita and Brashears 2002, Ruby et al. 2009). But the formation of hydrogen peroxide must also be studied in complex media, since food, especially raw meat, is rich in nutrients.

Harvested L. delbrueckii cells were inoculated into fresh LAPTg at ~10⁸ CFU/ml and incubated at 5°C. The rate of accumulation of hydrogen peroxide, determined per every other day for five days, is shown in Figure 5.

The accumulation of hydrogen peroxide by wild and mutant strains gradually increased and reached its maximum on the fourth day (Fig. 5) and remained stable for at least ten days of cold incubation (data are not presented in the figure). During the entire period of cold exposure of the cultures, despite the active production of H₂O₂, no significant changes in the pH and in the live cell population was observed. With the same biomass without the growth Rif mutants have produced significantly more hydrogen peroxide in LAPTg than the wild strain (p <0.05). Despite the high amount of extracellular H₂O₂ accumulated in the cold, it does not lead to death of host. Interestingly in cold condition the Rif3 mutant lost its superiority over other mutants and became closer to the parental strain.

Antagonism of Rif 4 mutant against E. coli O157:H7 at 5°C

The antagonistic effect of most active Rif-3 mutant against the pathogen E. coli O157:H7 was determined at 5°C co-cultivation at ratios of 10:1, 100:1 and 1000:1 respectively. Data of E. coli O157:H7 survival determined on MacConkey agar are shown in Figure 6.

As can be seen from Figure 6, there is a direct relationship between the number of lactobacilli and the death of E. coli O157:H7. At a ratio of 1000:1, Rif-4 practically eliminated the pathogen (p < 0.05) within 4 days of co-cultivation. At a ratio of 100:1, complete elimination occurred on the 6^th day, which is not far behind 1000:1. Whereas, at a ratio of 10:1, the E. coli population decreased only by 1.6 log in 6 days. The optimal ratio of lactic acid bacteria to pathogen is most likely in the range of 1000:1 to 100:1.

On the first day of co-cultivation, when the H₂O₂ concentration (see fig. 4) had not yet reached to the MIC of E. coli (~ 34 mg/ml), a significant decrease in E. coli viable cell count was observed depending of lactobacilli concentration (Fig5). According to our earlier data (Pashayan and Hovhannisyan 2021), death at sub-inhibitory concentrations of H₂O₂ can be caused by L. delbrueckii co-aggregation with the pathogen and direct release of H₂O₂ on it. Further experiments revealed 39 ± 1.5% level of co-aggregation between L. delbrueckii and E. coli.

In lactic acid bacteria, under aerobic conditions, nicotinamide adenine dinucleotide NAD molecules react with oxygen to form hydrogen peroxide, which is mainly produced by oxidases, including NADH oxidases (Nox), pyruvate oxidase (Pox) and lactate oxidase (Lox) (Condon 1987; Hertzberger et al. 2014). Transformation of endogenous superoxide anions (O₂⁻) can also make some contribution to the accumulation of H₂O₂ by the action of superoxide dismutase (SOD), which is present in most LAB (Britton et al. 1978). LAB are genetically devoid of catalase activity, since they cannot synthesize hemoporphyrins, therefore, they can get rid of the hydrogen peroxide only with the help of NADH peroxidase (Whittenbury 1964), thus any imbalance between NADH oxidase and NADH peroxidase triggered by rif mutations, can result in the changes of the H₂O₂ concentration. Rif mutations can affect H₂O₂ yield by modification of permeability or influx/efflux mechanisms. The mechanism of increased H₂O₂ production in the Rif3 mutant under optimal growth conditions evidently differs from other mutants. This is reminiscent of some Salmonella mutants that have been shown to accumulate high levels of hydrogen peroxide due to faster production than release of H₂O₂. (Thomas and Pera 1983). Thus, the changes caused by rif mutations in expression each of more than dozen gens organized in operons and regulons involved in regulation, formation and transportation of hydrogen peroxide in bacteria can cause enhancement of H₂O₂ yield.

The Rif mutants eventually accumulated approximately the same amount of H₂O₂, but with a 20–50% lower biomass than the parental strain, as a result of an earlier transition to the stationary phase. Marty-Teysset et al. (2000) showed that the earlier entry into stationary phase and subsequent lower biomass production of aerated cultures were due to the accumulation of the inhibitory concentrations of H₂O₂ in the growth medium by directly adding 0.75 mM exogenous H₂O₂ to L. bulgaricus during the exponential phase.

It has been shown that lactic acid bacteria exhibit an inducible oxidative stress response when exposed to sub-lethal concentrations of H₂O₂ (Condon 1983). This response protects them if they are subsequently exposed to lethal concentrations of H₂O₂. The effect appears to be related to other stress responses such as heat-shock and is similar, in some but not all aspects, to that of previously reported for enteric bacteria (Condon 1987). Imlay (2015) also demonstrated role of transcription factors that defend bacteria against reactive oxygen species. The long-term persistence of LAB populations after autoinhibition (Fig. 3) supports this opinion.

We have shown that the H₂O₂ tolerance (MIC) of anaerobically growing L. delbrueckii depends on the growth phase. It has been found that exponentially growing L. delbrueckii cells are significantly more sensitive to H₂O₂ than their non-growing counterparts. Similar results were obtained by Tibessard et al. (2001) for S. thermophilus. During aerobic growth the H₂O₂ concentration causing auto-inhibition was about two times lower than exogenous H₂O₂ MIC determined for anaerobically growing L. delbrueckii. The main difference between the aerated and non-aerated cultures is the existence of intracellular H₂O₂ in aerobically growing cells, which may contribute to cell killing. The steady-state intracellular concentration of H₂O₂ depends on dissipating rates by scavenging enzymes and by efflux from the cell (Seaver and Imlay 2001). Uhl and Dukan (2016) studied the contribution of H₂O₂ internal and external concentrations in the bacterial inhibition and suggested the concepts of “maximal internal H₂O₂ concentration” and “cumulative H₂O₂ concentration” (i.e., the total amount of H₂O₂). They concluded that the cumulative H₂O₂ concentration, rather than maximal internal H₂O₂ concentration, is involved in the H₂O₂-mediated death of bacterial cells.

In our experiments L. delbrueckii strains produced large amounts of hydrogen peroxide in complete media at both 37°C and at 5°C temperatures. Some of authors avoided the use of complete media for peroxide biosynthesis, ostensibly referring to the presence of active catalase in the yeast and/or meat extracts included in their composition (Jaroni and Brashears 2000). However, it is unlikely that such a large tetramer enzyme of four polypeptide chains, each over 500 amino acids long (Kirkman and Gaetani 1984) will be able to recover and show any activity after denaturation caused by high-temperature sterilization.

Despite the potential application of peroxide production for food preservation, the mechanism of its production at refrigeration temperature remains poorly understood. It has been suggested that lactate oxidase (Lox) is involved in the formation of peroxide by L. delbrueckii subsp. lactis (Villegas and Gilliland 1998).

The bactericidal effect of H₂O₂ on E. coli O157:H7 is explained by its strong oxidative effect on bacterial cells and the destruction of the main cellular macromolecules (Imlay 2002). The lethality of pathogen caused by H₂O₂ has bimodal pattern (Imlay and Linn 1986). Mode I killing occurs through DNA damage from hydroxyl radicals (OH⁻) produced via the Fenton reaction (Imlay et al. 1988). Mode II killing occurs at higher concentrations of H₂O_2, but at a slower rate, which probably involves inactivation of housekeeping enzymes by oxidation of active site thiols (Imlay 2002). In addition to the indicated mechanisms, we propose another H₂O₂ impact on E. coli, that was found in co-cultivation experiments with lactobacilli. This mechanism is based on the ability of microorganisms to co-aggregate and directly affect the target subject (Pashayan and Hovhannisyan 2021). At a co-aggregation level of about 40%, the probability of lactobacilli contact with E. coli at ratios above 100:1 is very high. This property is especially valuable in solid-state cultivation of lactobacilli application in ground beef, during refrigerated storage, where the rate of accumulation of H₂O₂ is not high enough.

LAB

Lactic acid bacteria

N-methyl-N′-nitro-N-nitrosoguanidine

MIC

Minimal inhibitory concentration

Optical density.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The strain Lactobacillus delbrueckii subsp. lactis MDC 9617 (GenBank accession no. HQ379117 and HQ379180) and Rif mutants received during this study are available in the Microbial Depository Center (MDC) of the Republic of Armenia.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Science Committee of Ministry of Education, Science, Culture and Sports of the Republic of Armenia in the frame of Basic Funding I32

Authors' contributions

AHB, MMP, GGG and LVD performed the experiments, analyzed all data and interpreted the results, prepared and wrote the manuscript. EHG designed the experiments, interpreted the results and revised the manuscript. HGH supervised the study, critically interpreted the results, provided discussion, revised and edited the manuscript. All authors read and approved the final manuscript.

Authors' information

¹SPC “Armbiotechnology” NAS RA, Yerevan 0056, Armenia

²Yerevan State Medical University after Heratsi, Yerevan 0025, Armenia

Acknowledgements

We would like to thank Arpine Sukiasyan for helping to improve the manuscript.

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Enhancement of Hydrogen Peroxide Production by RNA Polymerase rif Mutations of Lactobacillus delbrueckii subsp. lactis MDC 9617

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