Psychrotrophic Strains Research Articles

Proteolytic and Biofilm-forming Capacity of Bacteria Isolated from a UHT Milk Processing Plant

The destabilization of milk during processing, caused by bacteria, represents a significant challenge for the dairy industry. This study aimed to determine the spoilage potential of 31 psychrotrophic, mesophilic, or thermophilic strains isolated from the UHT-milk production chain. This encompassed the biofilm-forming ability on polystyrene and stainless-steel surfaces, along with the strains' caseinolytic activity and heat resistance. The effect of 5 mM MgCl2 on the biofilm-forming capacity was also determined. The results indicated that the spoilage potential is highly strain-dependent. On polystyrene Pseudomonas brenneri C10.3.4, Staphylococcus pasteuri RE1.9, and B. licheniformis SDP.15 were the strongest biofilm-formers of each group, whereas on stainless-steel, Pseudomonas lactis D1.11, Lacticaseibacillus paracasei P2.20 and Bacillus sonorensis C4.13 exhibited the highest capacity. Staphylococcus pasteuri RE1.9 and Staphylococcus epidermidis E3.6 biofilm cells showed the highest heat resistance. Moreover, biofilm-born cells of the spore-forming B. licheniformis SDP.15 and B. sonorensis C4.13 were significantly more heat-resistant than their free-cell equivalents. Furthermore, 22 of the assessed strains showed proteolytic activity, although only the proteases produced by P. lactis E6.10 maintained their enzymatic activity after being treated at 92°C. These findings may contribute to developing strategies to enhance the dairy industry's long-term economic sustainability. Keywords: UHT milk; spoilage potential; caseinolytic activity; biofilm-forming ability; thermal resistance

Evaluation of growth behavior of lactic acid bacteria isolated from kimchi via kinetic modeling

Lactic acid bacteria (LAB) are critical in kimchi fermentation. This study determined the effect of temperature on the growth of 11 predominant LAB (five psychrotrophic and six mesophilic strains) isolated from kimchi and analyzed their growth behavior using a kinetic model to understand changes in LAB communities during kimchi fermentation. The modified Gompertz model was more suitable for the growth of kimchi LAB rather than the logistic model in terms of RMSE, AICc, R2, adjusted R2, Af and Bf. Latilactobacillus sakei KCKM 0001 and Leuconostoc mesenteroides KCKM 0008 rapidly grew at all temperatures (5, 10, 15, 20, 25, 30, and 37 °C) among the strains analyzed for growth characteristics using the modified Gompertz model. Additionally, the growth of psychrotrophic strains was more active than that of the mesophilic strains, excluding L. sakei KCKM 0001 and Leu. mesenteroides KCKM 0008 at kimchi fermentation temperatures of 5–10 °C. The differences observed in the lag phase and maximum specific growth rate at each temperature showed a trend consistent with previously reported dominant LAB at kimchi fermentation temperatures. The predictive modeling results showed that the changes in LAB communities during kimchi fermentation could be explained by their growth behavior at different temperatures.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Effect of Thermostable Enzymes Produced by Psychrotrophic Bacteria in Raw Milk on the Quality of Ultra-High Temperature Sterilized Milk.

Ultra-high temperature sterilized milk (UHT) is a popular dairy product known for its long shelf life and convenience. However, protein gel aging and fat quality defects like creaming and flavor deterioration may arise during storage. These problems are primarily caused by thermostable enzymes produced by psychrotrophic bacteria. In this study, four representative psychrotrophic bacteria strains which can produce thermostable enzymes were selected to contaminate UHT milk artificially. After 11, 11, 13, and 17 weeks of storage, the milk samples, which were contaminated with Pseudomonas fluorescens, Chryseobacterium carnipullorum, Lactococcus raffinolactis and Acinetobacter guillouiae, respectively, demonstrated notable whey separation. The investigation included analyzing the protein and fat content in the upper and bottom layers of the milk, as well as examining the particle size, Zeta potential, and pH in four sample groups, indicating that the stability of UHT milk decreases over time. Moreover, the spoiled milk samples exhibited a bitter taste, with the dominant odor being attributed to ketones and acids. The metabolomics analysis revealed that three key metabolic pathways, namely ABC transporters, butanoate metabolism, and alanine, aspartate, and glutamate metabolism, were found to be involved in the production of thermostable enzymes by psychrotrophic bacteria. These enzymes greatly impact the taste and nutrient content of UHT milk. This finding provides a theoretical basis for further investigation into the mechanism of spoilage.

Acinetobacter oleivorans IRS14 alleviates cold stress in wheat by regulating physiological and biochemical factors.

Climate change is responsible for extremely cold winters causing a significant loss in crop yield and productivity due to chilling stress. This study aims to investigate the potential of psychrotrophic plant growth-promoting rhizobacteria (PGPR) strain to promote wheat growth under cold stress and explore the adaptive responses of wheat. Wheat seeds and seedlings were inoculated with the psychrotrophic strain IRS14 and the plants were cultivated for five weeks at 6±2°C. The genetic, biochemical, physiological, and molecular analysis of the bacterium and plant was done to evaluate the effect of the PGPR strain in alleviating chilling stress. IRS14 possesses antioxidant activity and produced multiple phytohormones, which enhanced seed germination (∼50%) and plant growth (∼50%) during chilling stress. Here, we reported that the application of IRS14 helps to regulate the biochemical and metabolic pathways in wheat plants. It alleviates chilling stress and increases plant growth rate and biomass. Strain IRS14 in wheat effectively increased chlorophyll content, antioxidants, carotenoid, proline, and endogenous phytohormones compared with untreated wheat.

Biosynthesis, Characterization and Antibacterial Properties of CuO Nanoparticles of Psychrotrophic Bacteria

Copper oxide nanoparticles have many applications in industry and medicine. Nevertheless, few studies have been conducted in relation to the biological production of these nanoparticles. In the present study, the ability of isolated Psychrotrophic (cold-resistant) prokaryotes from the Zagros highlands located in Lorestan province of Iran and some isolated Streptomyces strains from the Oman Sea has been evaluated in the synthesis of copper oxide nanoparticles. The maximum lethal concentration of copper salts was determined for the strains and the strains with the highest level of resistance were selected. The biosynthesis of copper oxide nanoparticles was done extracellularly by inoculating Psychrotrophic and Streptomyces strains in liquid TSB culture medium containing 0.01 M copper sulfate salt and keeping the culture medium in a shaker incubator at 20 °C at 150 rpm. The production of copper oxide nanoparticles was evaluated by changing the color of the reaction solution from light blue to dark green. From the 44 cold-resistant strains and the investigated Streptomyces strains, all strains were able to tolerate concentrations higher than 5 mM. Of these, the strain OSNP13 belonging to the genus Microbacterium liquefaciens (X77444) has been able to synthesize the highest amount of copper oxide nanoparticles. The optical absorption of the solution containing synthesized nanoparticles was determined in the range of 250-350 nm by UV-vis spectrophotometer, which had a specific peak at 288 nm. And the copper oxide nanoparticles have an average size of 63.21 nm based on DLS analysis. The crystallographic characteristics of copper oxide nanoparticles were also determined by using XRD analysis, which showed that the prepared nanoparticles had a hexagonal crystal structure with a size of 31.29 nanometers. Then, the antibacterial activity of produced nanoparticles was evaluated. The MIC values ​​of copper oxide nanoparticles for E. coli and S. aureus bacteria were calculated as 125 and 250 micrograms per milliliter, respectively. The produced copper oxide nanoparticles have shown a good antimicrobial properties and can be suitable candidates for use as antimicrobial agents.

Atrazine decontamination by a newly screened psychrotroph Paenarthrobacter sp. KN0901 in an aquatic system: Metabolic pathway, kinetics, and hydroponics experiment

Atrazine residues running off the fields and entering water resources are a major threat to food security and the ecosystem. In this study, a psychrotrophic functional strain named KN0901 to remove atrazine residues was screened. KN0901 could degrade 30 mg·L−1 atrazine in 4 days at 15ºC with 105 CFU·mL−1 incubation. The phylogenetic results showed KN0901 belonged to Paenarthrobacter sp. PCR results showed that the functional genes consist of trzN, atzB, and atzC, suggesting atrazine was transformed to cyanuric acid by KN0901. KN0901 could degrade atrazine without adding exogenous carbon and nitrogen sources. What’s more, KN0901 could tolerate extreme low temperature (5ºC) and high atrazine concentration (100 mg·L−1). When growth and degradation curves were compared, the results indicated the length of lag time showed significant correlation to atrazine degradation rate. The hydroponic experiments showed that the toxicity of atrazine was significantly reduced with KN0901 treatment. The study provided an effective, economic, and eco-friendly bioremediation measure to address atrazine contamination.

Influence of Isolation Temperature on Isolating Diverse Lactic Acid Bacteria from Kimchi and Cultural Characteristics of Psychrotrophs.

Kimchi is a traditional Korean fermented vegetable that is stored and fermented at low temperatures. However, kimchi lactic acid bacteria (LAB) are typically isolated under mesophilic conditions, which may be inappropriate for isolating the diverse LAB. Therefore, this study investigated the suitable conditions for isolating various LAB from kimchi. Here, LAB were isolated from four kimchi samples using MRS, PES, and LBS media and varying isolation temperatures (30, 20, 10, and 5°C). Then, MRS was selected as the suitable medium for LAB isolation. A comparison of culture-dependent and culture-independent approaches indicated that 5°C was not a suitable isolation temperature. Thus, the number and diversity of LAB were determined at 30, 20, and 10°C using 12 additional kimchi samples to elucidate the effect of isolation temperature. With the exception of two samples, most samples did not substantially differ in LAB number. However, Leuconostoc gelidum, Leuconostoc gasicomitatum, Leuconostoc inhae, Dellaglioa algida, Companilactobacillus kimchiensis, Leuconostoc miyukkimchii, Leuconostoc holzapfelii, and Leuconostoc carnosum were isolated only at 10 and 20°C. The growth curves of these isolates, except Leu. holzapfelii and Leu. carnosum, showed poor growth at 30°C. This confirmed their psychrotrophic characteristics. In Weissella koreensis, which was isolated at all isolation temperatures, there was a difference in the fatty acid composition of membranes between strains that could grow well at 30°C and those that could not. These findings can contribute to the isolation of more diverse psychrotrophic strains that were not well isolated under mesophilic temperatures.

Clostridium gelidum sp. nov., a psychrotrophic anaerobic bacterium isolated from rice field soil.

A cold-adapted or psychrotrophic anaerobic bacterial strain (C5S11T) was isolated from rice field soil in Japan using an enrichment culture incubated at 5 °C. C5S11T grew at 0 °C and optimum growth was observed at 10 °C. Cells of C5S11T were Gram-stain-positive, motile, spore-forming rods with peritrichous flagella. C5S11T was assigned to a large branch consisted of various mesophilic species of the genus Clostridium in the phylogenetic trees reconstructed using the 16S rRNA gene sequences. The most closely related species of the strain was Clostridium chromiireducens DSM 23318T (98.5 % sequence similarity). C5S11T fermented various carbohydrates, including polysaccharides (starch, inulin, pectin and xylan), and produced acetate, butyrate and H2 as major products. The major cellular fatty acids were C16 : 0, C16 : 1ω9c, C14 : 0 and C16 : 1ω7c. The diagnostic diamino acid of the cell wall peptidoglycan was meso-diaminopimelic acid. The genome size of C5S11T was 6.04 Mb and the genomic DNA G+C content was 29.0 mol%. Average nucleotide identity by blast (ANIb), avaerage amino acid identity (AAI) and digital DNA–DNA hybridization (dDDH) values between the genomes of C5S11T and C. chromiireducens DSM 23318T were 80.0, 75.2 and 26.4 %, respectively. C5S11T had a gene encoding cold shock protein (RNA chaperone) in the genome, homologues of which have been found in psychrophilic species of the genus Clostridium . On the basis of the differences in the phylogenetic, genomic and phenotypic characteristics of C5S11T from those of the closely related species, a novel species, Clostridium gelidum sp. nov., is proposed to accommodate the strain. The type strain is C5S11T (=NBRC 114689T = DSM 112608T).

Genome Functional Analysis of the Psychrotrophic Lignin-Degrading Bacterium Arthrobacter sp. C2 and the Role of DyP in Catalyzing Lignin Degradation.

In the cold regions of China, lignin-rich corn straw accumulates at high levels due to low temperatures. The application of psychrotrophic lignin-degrading bacteria should be an effective means of overcoming the low-temperature limit for lignin degradation and promoting the utilization of corn straw. However, this application is limited by the lack of suitable strains for decomposition of lignin; furthermore, the metabolic mechanism of psychrotrophic lignin-degrading bacteria is unclear. Here, the whole genome of the psychrotrophic lignin-degrading bacterium Arthrobacter sp. C2, isolated in our previous work, was sequenced. Comparative genomics revealed that C2 contained unique genes related to lignin degradation and low-temperature adaptability. DyP may participate in lignin degradation and may be a cold-adapted enzyme. Moreover, DyP was proven to catalyze lignin Cα-Cβ bond cleavage. Deletion and complementation of the DyP gene verified its ability to catalyze the first-step reaction of lignin degradation. Comparative transcriptomic analysis revealed that the transcriptional expression of the DyP gene was upregulated, and the genetic compensation mechanism allowed C2ΔDyP to degrade lignin, which provided novel insights into the survival strategy of the psychrotrophic mutant strain C2ΔdyP. This study improved our understanding of the metabolic mechanism of psychrotrophic lignin-degrading bacteria and provided potential application options for energy-saving production using cold-adapted lignin-degrading enzymes.

Efficient conversion of hemicellulose into 2, 3-butanediol by engineered psychrotrophic Raoultella terrigena: mechanism and efficiency

Low-temperature biorefineries inhibit the multiplication of undesired microorganisms, improve product purity and reduce economic costs. Herein, to improve the 2,3-butanediol (2,3-BD) bioconversion efficiency from hemicellulose, a psychrotrophic hemicellulose-degrading strain Raoultella terrigena HC6 with high β-xylosidase activity 1520 U/mL was isolated and genetically modified. Xylan (hemicellulose replacement) was depolymerized into xylooligosaccharides (XOS) and xylose by HC6, which were further converted into 2,3-BD. Transcriptomic analysis revealed that β-xylosidase gene (xynB) and xylose isomerase gene (xylA), which are beneficial for increasing the carbon flux from xylan to 2,3-BD, were significantly upregulated 56.9-fold and 234-fold, respectively. A recombinant strain was constructed by overexpressing xynB in HC6, which obtained 0.389 g/g yield of 2,3-BD from hemicellulose extracted from corn straw at 15 °C. This study proposed a promised strategy for the bioconversion of agricultural waste into 2,3-BD at low temperatures and provides a basis for future efforts in the achievement of carbon neutrality.

Synergism With ε-Polylysine Hydrochloride and Cinnamon Essential Oil Against Dual-Species Biofilms of Listeria monocytogenes and Pseudomonas lundensis.

Various pathogenic and spoilage bacteria frequently coexist in meat processing environments and can form multispecies biofilms, causing significant health and economic issues. Despite the prevalence and coexistence, only less is known about possible interactions between Listeria monocytogenes (LM) and spoilers like Pseudomonas species, and their community-wide resistance against natural preservatives. This study evaluates the interactions between mono- or dual-species biofilms formed by LM and Pseudomonas lundensis (PL), as well as the sensitivity of these bacteria in dual-species biofilms to ε-polylysine hydrochloride (ε-PLH) alone or combined with cinnamon essential oil (CEO). The results showed that the biofilm cell density of P. lundensis in dual species was higher (p < 0.05) than LM, constituting about 85% of the total population. More biofilms and exopolysaccharide both in mono- or dual species of the two psychrotrophic strains were greatly produced at 15°C than at 30°C. The biomass, biovolume, and thickness of dual-species biofilms were significantly lower than single PL biofilm when tested using crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy, indicating the competitive interactions between them prevail. Additionally, ε-PLH significantly reduced the biofilm development as mono- and dual species in a concentration-dependent manner, especially single LM biofilm, which was consistent with the decrease in autoinducer-2 (AI-2) activity. LM as dual-species biofilms exhibited lower sensitivity to ε-PLH than its mono-biofilm probably due to protective effect conferred by PL. ε-PLH in combination with CEO, at the maximum sublethal concentrations (MSCs), showed enhanced inhibitory activity against dual-species biofilm formation, as evidenced by thin spare spatial structures and reduced AI-2 activity. In addition, the preformed dual biofilms were dramatically eradicated following treatment with ε-PLH combined with CEO at higher than minimum inhibitory concentration in comparison with either of the compounds used alone, indicating the synergistic antibiofilm of the two preservatives. This study reveals the competitive interactions between the two strains in dual-species biofilms, in which the dominant PL significantly contributed toward the tolerance of LM to ε-PLH, and the use of combined preservatives shows it is an effective strategy to control the multispecies biofilms in meat processing.

Assessing the inhibitory activity of culture supernatants against foodborne pathogens of two psychrotrophic bacteria isolated from river trout

There is a need for new natural products with antimicrobial activity to treat multidrug resistant bacteria that can cause human illness. Some of them are foodborne pathogens. Two different Gram-negative psychrotrophic strains were isolated from healthy trout river samples (Salmotrutta). Based on phenotypic characterization, proteomics, genotyping and phylogenetic analyses of 16 rRNA gene, strains TCPS12 and TCPS13 were identified as Shewanellabaltica and Pseudomonasfragi, respectively. Both of them produced an exopolysaccharide that showed antimicrobial activity against four foodborne pathogens. P. fragi supernatant (AS13) showed higher antimicrobial activity than S. baltica supernatant (AS12) against all tested pathogens. The stability of the antimicrobial activity of AS13 was assessed against Enterococcus faecalis ATCC 29212 under different conditions. This solution was stable when exposed for 30 min to temperatures ranging from 40 to 100 °C. In addition, it retained its activity within a pH range of 2–8 during 2 h of incubation, showing higher activity at pH 6. Serine proteases and α-amylase inactivated significantly the antimicrobial activity of AS13, suggesting that the active molecule could most likely be a glycoprotein. These products are interesting for their possible application as biopreservatives in the food industry.

Molecular Detection of Bacillus cereus in Milk by Polymerase Chain Reaction

It is a valuable and good food source with the ingredients it contains in milk, but if hygienic conditions are not ensured during milking, storage and processing, diseases related to milk and dairy products are likely to occur. More than 90% of these diseases are of bacterial origin. B. cereus, found in the Bacillaceae family, is a significant source of contamination in milk and dairy products due to the presence of psychrotrophic strains. In recent years, it has been reported that milk and milk products contaminated with B. cereus have adverse effects on human health, and toxic substances are formed during the storage of contaminated milk and milk products. &#x0D; This study, it is aimed to molecularly identify the B. cereus bacteria species that cause adverse effects such as food poisoning and loss of quality in milk. In this study, the DNA of Bacillus cereus, one of the microorganisms found in raw milk, was isolated and toxic gene regions were determined at the molecular level by PCR method using specific primers to the gene regions Bacillus cereus. Six primers specific for nheA, nheB, nheC, hblA, hblC, hblD and 16S rRNA gene regions were used for molecular detection of B. cereus in milk samples. At the end of the study, Bacillus cereus bacteria were detected in 8 of the 13 milk samples. It has been revealed that B. cereus can be detected quickly and reliably in milk and dairy products by molecular species identification using specific primers.

Impact of microbial inoculants combined with humic acid on the fate of estrogens during pig manure composting under low-temperature conditions

To investigate the efficiency of psychrotrophic cellulose-degrading fungal strains (PCDFSs) and estrogen-degrading bacteria (EDBs) combined with humic acid (HA) on estrone (E1) and 17-β-estradiol (E2) degradation, five compost groups (T, HA, EDB, PCDFS, and CK) were prepared and composted for 32 days at 11–14°C. The results indicated that inoculation increased the temperature to 62.2°C and promoted E1 degradation to the lowest level of 100.1 ng/kg, while E2 was undetected from day 16. Metagenomic analysis revealed that inoculation altered the microbial community structure by increasing the abundance of cellulose-degrading fungi, especially Meyerozyma (16.7%) (among PCDFSs), and of estrogen-degrading bacteria, particularly Microbacterium (13.4%) (involved in EDBs). Moreover, inoculation increased the levels (>0.500%) of Gene Ontology (GO) associated with estrogen degradation, like 3-β-hydroxy-delta 5-steroid dehydrogenase and monooxygenase. Redundancy analysis demonstrated that temperature and Microbacterium were positively correlated with estrogen degradation. Structural equation model indicated that temperature and estrogen-degrading bacterial genera exhibited positive, significant (p < 0.001) and direct impacts on estrogen degradation. This is the first study to suggest that applying microbial inoculants and HA could accelerate estrogen degradation during composting in cold regions. The research outcomes offer a practical reference for managing compost safety, thereby decreasing its potential environmental and human health impacts.

Production of extracellular lipase from psychrotrophic bacterium Oceanisphaera sp. RSAP17 isolated from arctic soil.

Cold-active extracellular lipases produced by different psychrotrophs are important for various industrial applications. We have isolated a Gram-negative, rod-shaped, aerobe, non-pigment producing psychrotrophic bacterial strain RSAP17 (MTCC 12991, MCC 4275) from the unexplored Arctic soil sample of NyAlesund, Svalbard, Norway (78° 55″ N, 11° 54″ E). The detailed morphological, biochemical, and molecular characteristics were investigated to characterize the isolate RSAP17. Analyses of the 16S rDNA sequence of strain RSAP17 (Accession no. MK391379) shows the closest match with Oceanisphaera marina YM319T (99.45%) and Oceanisphaera sediminis TW92 JCM 17329T (97.40%). The isolate is capable of producing extracellular lipase but not amylase, cellulase or urease. The optimal parameters for lipase production have been found in tributyrin based (10mL/L) agar media supplemented with 3% (w/v) NaCl after 2-3days of incubation at 20-22°C temperature and pH 9 at shaking condition. We have purified the extracellular lipase from the RSAP17 grown culture supernatant through 75% ammonium sulfate precipitation followed by dialysis and DEAE cellulose column chromatography. The invitro lipolytic activity of the purified lipase enzymes has been done through zymogram analysis. The molecular weight found for the lipase is 103.8 kD. The optimal activity of the purified lipase has been found at 25°C and pH 9. MALDI-TOF-MS study of the purified lipase showed the highest match with the sequence of prolipoprotein diacylglyceryl transferase with 44% sequence coverage. Further study on large-scale production, substrate utilization and enzymatic kinetics of this lipase could unravel its possibility in future biotechnological applications.

The effect of pH on the growth rate of Bacillus cereus sensu lato: Quantifying strain variability and modelling the combined effects of temperature and pH

In this study, the effect of pH, alone or in combination with temperature, on the maximum growth rate (μmax) of B. cereus sensu lato was investigated. In phase 1, the effect of pH at 30 °C was studied for 16 mesophilic strains and 2 psychrotrophic strains of Bacillus cereus sensu lato. The μmax vs. pH relationship was found to show a similar pattern for all the strains. Several pH models from literature were evaluated and the best performing ‘growth rate vs. pH’ model selected. A stochastic model was then developed to predict the maximum specific growth rate of mesophilic B. cereus at 30 °C as a function of pH, the intra-species variability being incorporated via considering the model parameters (e.g. pHmin) randomly distributed. The predicted maximum specific growth rates were acceptably close to independent published data. In phase 2, the combined effects of temperature and pH were studied. Growth rates were also generated at 15, 20 and 40 °C for a selection of strains and the pH model was fitted at each temperature. Interestingly, the results showed that the estimates for the pHmin parameter for mesophilic strains were lower at 20–30 °C than near the optimum temperature (40 °C), suggesting that experiments for the determination of this parameter should be conducted at lower-than-optimum temperatures. New equations were proposed for the relationship between temperature and the minimum pH-values, which were also consistent with the experimental growth boundaries. The parameters defining this equation quantify the minimum temperature for growth observed experimentally, the temperature of maximum enzyme stability and the maximum temperature for growth. Deviations from the Gamma hypothesis (multiplicative effects of environmental factors on the maximum specific growth rate) were observed near the growth limits, especially at 40 °C. To improve model performance, two approaches, one based on a minimum pH-term (doi: https://doi.org/10.3389/fmicb.2019.01510) and one based on an interaction term (doi: http://dx.doi.org/10.1016/S0168-1605(01)00640-7) were evaluated.

Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6–7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species.

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

CspB and CspC are induced upon cold shock in Bacillus cereus strain D2.

Bacillus cereus D2, a psychrotrophic strain, plays an essential role in the restoration of heavy metal-contaminated soils, especially at low temperatures. However, the cold shock response mechanisms of this strain are unclear. In this study, the cold shock response of B.cereus D2 was characterized; as per the Arrhenius curve, 10°C was chosen as the cold shock temperature. Six cold shock-like proteins were found and temporarily named cold shock protein (Csp)1-6; the respective genes were cloned and identified. Quantitative real-time PCR results showed that csp1, csp2, csp3, and csp6 were overexpressed under cold shock conditions. Interestingly, after cloning the respective encoding genes into the pET-28a (+) vector and their subsequent transformation into E.coli BL21 (DE3), the strains expressing Csp2 and Csp6 grew faster at 10°C, showing a large number of bacteria. These results suggest that Csp2 and Csp6 are the major cold shock proteins in B.cereus D2. Of note, the comparison of amino acid sequences and structures showed that Csp2 and Csp6 belong to the CspB and CspC families, respectively. Additionally, we show that the number of hydrophobic residues is not a determining feature of major Csps, while, on the other hand, the formation of an α-helix in the context of a leucine residue is the most dominant difference between major and other Bacillus and E.coli Csps.