Soil substrate culturing approaches recover diverse members of Actinomycetota from desert soils of Herring Island, East Antarctica.

Antimicrobial resistance is an escalating health crisis requiring urgent action. Most antimicrobials are natural products (NPs) sourced from Actinomycetota, particularly the Streptomyces. Underexplored and extreme environments are predicted to harbour novel microorganisms with the capacity to synthesise unique metabolites. Herring Island is a barren and rocky cold desert in East Antarctica, remote from anthropogenic impact. We aimed to recover rare and cold-adapted NP-producing bacteria, by employing two culturing methods which mimic the natural environment: direct soil culturing and the soil substrate membrane system. First, we analysed 16S rRNA gene amplicon sequencing data from 18 Herring Island soils and selected the soil sample with the highest Actinomycetota relative abundance (78%) for culturing experiments. We isolated 166 strains across three phyla, including novel and rare strains, with 94% of strains belonging to the Actinomycetota. These strains encompassed thirty-five 'species' groups, 18 of which were composed of Streptomyces strains. We screened representative strains for genes which encode polyketide synthases and non-ribosomal peptide synthetases, indicating that 69% have the capacity to synthesise polyketide and non-ribosomal peptide NPs. Fourteen Streptomyces strains displayed antimicrobial activity against selected bacterial and yeast pathogens using an in situ assay. Our results confirm that the cold-adapted bacteria of the harsh East Antarctic deserts are worthy targets in the search for bioactive compounds.

Bosea psychrotolerans sp. nov., a psychrotrophic alphaproteobacterium isolated from Lake Michigan water.

Three strains of a Gram-stain negative bacterium were isolated from Lake Michigan water. 16S rRNA gene sequence analysis revealed that strain 1131 had sequence similarities to Bosea vaviloviae LMG 28367T, Bosea lathyri LMG 26379T, Bosea lupini LMG 26383T, Bosea eneae CCUG 43111T, Bosea vestrisii CCUG 43114T and Boseamassiliensis CCUG 43117T of 99.8, 99.1, 98.4, 98.4, 98.4 and 98.2 %, respectively. The average nucleotide identity value between strain 1131T and Bosea vaviloviae Vaf-18T was 93.4 % and the DNA relatedness was 38 %. The primary cellular fatty acids of strain 1131T were C16 : 1ω7c and C18 : 1ω7c. The primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major compound in the quinone system was ubiquinone Q-10 and in the polyamine pattern sym-homospermidine was predominant. Additional phenotypic characteristics included growth at 5-35 °C, pH values of pH 5.5-8.0, a salt tolerance range of 0.0-1.2 % (w/v), and production of an unknown water soluble brown pigment. After phenotypic, chemotaxonomic and genomic analyses, this isolate was identified as a novel species for which the name Bosea psychrotolerans is proposed. The type strain is 1131T (NRRL B-65405=LMG 30034).

Complete Genome Sequence and Comparative Analysis of Synechococcus sp. CS-601 (SynAce01), a Cold-Adapted Cyanobacterium from an Oligotrophic Antarctic Habitat.

Marine picocyanobacteria belonging to Synechococcus are major contributors to the global carbon cycle, however the genomic information of its cold-adapted members has been lacking to date. To fill this void the genome of a cold-adapted planktonic cyanobacterium Synechococcus sp. CS-601 (SynAce01) has been sequenced. The genome of the strain contains a single chromosome of approximately 2.75 MBp and GC content of 63.92%. Gene prediction yielded 2984 protein coding sequences and 44 tRNA genes. The genome contained evidence of horizontal gene transfer events during its evolution. CS-601 appears as a transport generalist with some specific adaptation to an oligotrophic marine environment. It has a broad repertoire of transporters of both inorganic and organic nutrients to survive in inhospitable environments. The cold adaptation of the strain exhibited characteristics of a psychrotroph rather than psychrophile. Its salt adaptation strategy is likely to rely on the uptake and synthesis of osmolytes, like glycerol or glycine betaine. Overall, the genome reveals two distinct patterns of adaptation to the inhospitable environment of Antarctica. Adaptation to an oligotrophic marine environment is likely due to an abundance of genes, probably acquired horizontally, that are associated with increased transport of nutrients, osmolytes, and light harvesting. On the other hand, adaptations to low temperatures are likely due to prolonged evolutionary changes.

Evaluation of conventional and response surface level optimisation of n-dodecane (n-C12) mineralisation by psychrotolerant strains isolated from pristine soil at Southern Victoria Island, Antarctica.

BACKGROUND: Biodegradation of hydrocarbons in Antarctic soil has been reported to be achieved through the utilisation of indigenous cold-adapted microorganisms. Although numerous bacteria isolated from hydrocarbon-contaminated sites in Antarctica were able to demonstrate promising outcomes in utilising hydrocarbon components as their energy source, reports on the utilisation of hydrocarbons by strains isolated from pristine Antarctic soil are scarce. In the present work, two psychrotolerant strains isolated from Antarctic pristine soil with the competency to utilise diesel fuel as the sole carbon source were identified and optimised through conventional and response surface method. RESULTS: Two potent hydrocarbon-degraders (ADL15 and ADL36) were identified via partial 16S rRNA gene sequence analysis, and revealed to be closely related to the genus Pseudomonas and Rhodococcus sp., respectively. Factors affecting diesel degradation such as temperature, hydrocarbon concentration, pH and salt tolerance were studied. Although strain ADL36 was able to withstand a higher concentration of diesel than strain ADL15, both strains showed similar optimal condition for the cell's growth at pH 7.0 and 1.0% (w/v) NaCl at the conventional 'one-factor-at-a-time' level. Both strains were observed to be psychrotrophs with optimal temperatures of 20 °C. Qualitative and quantitative analysis were performed with a gas chromatograph equipped with a flame ionisation detector to measure the reduction of n-alkane components in diesel. In the pre-screening medium, strain ADL36 showed 83.75% of n-dodecane mineralisation while the reduction of n-dodecane by strain ADL15 was merely at 22.39%. The optimised condition for n-dodecane mineralisation predicted through response surface methodology enhanced the reduction of n-dodecane to 99.89 and 38.32% for strain ADL36 and strain ADL15, respectively. CONCLUSIONS: Strain ADL36 proves to be a better candidate for bioaugmentation operations on sites contaminated with aliphatic hydrocarbons especially in the Antarctic and other cold regions. The results obtained throughout strongly supports the use of RSM for medium optimisation.

Paludicola psychrotolerans gen. nov., sp. nov., a novel psychrotolerant chitinolytic anaerobe of the family Ruminococcaceae.

A psychrotolerant chitinolytic bacterium, designated NC1253T, was isolated from Zoige wetland on the Qinghai-Tibetan Plateau. This strain was a Gram-stain-positive, spore-forming and rod-shaped anaerobe. NC1253T grew at 4-35 °C, at pH 6.0-8.5 and could grow on chitin as the only carbon resource. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain NC1253T represented a novel bacterial genus within the family Ruminococcaceae. Strain NC1253T has less than 91.0 % similarity with other type strains, such as Harryflintia acetispora V20-281aT (90.9 %), Clostridium methylpentosum DSM 5476T (90.8 %), Anaerotruncus colihominis DSM 17241T (89.8 %), Eubacterium siraeum DSM 15702T (89.6 %), and Acetanaerobacterium elongatum Z7T (89.6 %). The major components of the cellular fatty acids were iso-C14 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 35.4 mol%. Phenotypic, chemotaxonomic and phylogenetic characteristics allowed strain NC1253T to be clearly distinguished from genera in the family Ruminococcaceae. On the basis of polyphasic taxonomic data, the isolate is considered to represent a novel genus and novel species in the family Ruminococcaceae, for which the name Paludicola psychrotolerans gen. nov., sp. nov., is proposed. The type species is NC1253T (DSM 104738T=KCTC 15582T).

A novel ∼34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: production, purification, and characterization.

An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²âº but decreased by Mg²âº, Mn²âº, Zn²âº, Fe²âº, and Ca²âº. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.

Antimicrobial efficacy of lauric arginate against Campylobacter jejuni and spoilage organisms on chicken breast fillets.

The objectives of this study were to determine the antimicrobial efficacy of lauric arginate (LAE) against Campylobacter jejuni (in broth and on chicken breast fillets) and spoilage microorganisms (on chicken breast fillets). In vitro antimicrobial activity of LAE was determined by treating C. jejuni (in pure culture) with 0 (control), 50, 100, and 200 mg/L of LAE solutions at 4°C for 2 h. Inoculated chicken samples with C. jejuni were treated with 0, 200, and 400 mg/kg of LAE, packaged, and stored at 4°C for 7 d for determining the efficacy of LAE against C. jejuni on meat. Noninoculated skinless chicken breast fillet samples were treated with 0, 200, and 400 mg/kg of LAE and were used for analysis of LAE treatments on growth of mesophilic and psychrotrophic organisms on d 0, 3, 9, and 14 during storage at 4°C. Lauric arginate was highly effective against C. jejuniin vitro with no detectable survivors. Lauric arginate significantly (P ≤ 0.05) reduced C. jejuni counts on chicken breast fillets with 200 and 400 mg/kg treatments. Lauric arginate at 400 mg/L gave a maximum reduction of ~1.5 log cfu/g of C. jejuni during 7 d of storage at 4°C without any change in pH of meat. Treating chicken breast fillets with 400 mg/kg of LAE caused 2.3 log cfu/g reduction of psychrotrophs (P ≤ 0.05) compared with the control on d 0 of storage. However, no difference existed (P ≥ 0.05) in the growth of psychrotrophs on chicken breast fillets after treatment with 200 and 400 mg/kg of LAE compared with the control after 3 d. The LAE treatments had no effect (P ≥ 0.05) on growth of mesophilic organisms. The results of the study indicated that LAE is effective in reducing C. jejuni on chicken breast fillets.

Cold-tolerant phosphate-solubilizing Pseudomonas strains promote wheat growth and yield by improving soil phosphorous (P) nutrition status.

It is well-known that phosphate-solubilizing bacteria (PSB) promote crop growth and yield. The information regarding characterization of PSB isolated from agroforestry systems and their impact on wheat crops under field conditions is rarely known. In the present study, we aim to develop psychrotroph-based P biofertilizers, and for that, four PSB strains (Pseudomonas sp. L3, Pseudomonas sp. P2, Streptomyces sp. T3, and Streptococcus sp. T4) previously isolated from three different agroforestry zones and already screened for wheat growth under pot trial conditions were evaluated on wheat crop under field conditions. Two field experiments were employed; set 1 includes PSB + recommended dose of fertilizers (RDF) and set 2 includes PSB - RDF. In both field experiments, the response of the PSB-treated wheat crop was significantly higher compared to the uninoculated control. In field set 1, an increase of 22% in grain yield (GY), 16% in biological yield (BY), and 10% in grain per spike (GPS) was observed in consortia (CNS, L3 + P2) treatment, followed by L3 and P2 treatments. Inoculation of PSB mitigates soil P deficiency as it positively influences soil alkaline phosphatase (AP) and soil acid phosphatase (AcP) activity which positively correlated with grain NPK %. The highest grain NPK % was reported in CNS-treated wheat with RDF (N-0.26%, P-0.18%, and K-1.66%) and without RDF (N-0.27, P-0.26, and K-1.46%), respectively. All parameters, including soil enzyme activities, plant agronomic data, and yield data were analyzed by principal component analysis (PCA), resulting in the selection of two PSB strains. The conditions for optimal P solubilization, in L3 (temperature-18.46, pH-5.2, and glucose concentration-0.8%) and P2 (temperature-17°C, pH-5.0, and glucose concentration-0.89%), were obtained through response surface methodology (RSM) modeling. The P solubilizing potential of selected strains at <20°C makes them a suitable candidate for the development of psychrotroph-based P biofertilizers. Low-temperature P solubilization of the PSB strains from agroforestry systems makes them potential biofertilizers for winter crops.

Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk.

Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk. The BRET-based biosensor is highly selective for bacterial protease activity compared with other proteases tested, notably including plasmin, which is abundant in milk. It incorporates a novel peptide linker that is selectively cleaved by P. fluorescens AprX proteases. The peptide linker is flanked by green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the linker by bacterial proteases from Pseudomonas fluorescens strain 65, leads to a 95% decrease in the BRET ratio. We applied an azocasein-based calibration method to the AprX biosensor using standard international enzyme activity units. In a 10-min assay, the detection limit for AprX protease activity in buffer was equivalent to 40 pg/mL (≈0.8 pM, 22 µU/mL) and 100 pg/mL (≈2pM, 54 µU/mL) in 50% (v/v) full fat milk. The EC50 values were 1.1 ± 0.3 ng/mL (87 µU/mL) and 6.8 ± 0.2 ng/mL (540 µU/mL), respectively. The biosensor was approximately 800x more sensitive than the established FITC-Casein method in a 2-h assay, the shortest feasible time for the latter method. The protease biosensor is sensitive and fast enough to be used in production settings. It is suitable for measuring bacterial protease activity in raw and processed milk, to inform efforts to mitigate the effects of heat-stable bacterial proteases and maximise the shelf-life of dairy products.

Isolation and identification of psychrotrophic lactic acid bacteria in godo, the traditional fermented soy food in Japan.

Godo is a traditional fermented soy food made in Aomori prefecture, Japan. It is mainly made of soybeans, rice koji, and salt. Since godo ripens during the long and severe winter in northeast Japan, it is assumed that lactic acid bacteria inhabiting godo have cold tolerance. We aimed to investigate the presence or absence of psychrotrophic lactic acid bacteria in godo. The viable counts of estimated lactic acid bacteria ranged from 106 to 108 cfu/g. In addition, aerobic and anaerobic microorganisms were detected in four godo products though the microbial population differed from sample to sample. Twenty-two bacterial strains were able to be isolated from godo, and all of the isolated strains were Gram-positive and catalase-negative. Some of the isolates grew well at 10°C. The carbohydrate fermentation profile of the selected three strains was determined by API50 CHL analysis. These strains were identified as Leuconostoc mesenteroides, and Latilactobacillus sakei by 16S rRNA gene sequence analysis. Leuconostoc mesenteroides strains HIT231 and HIT252, and Latilactobacillus sakei strain HIT273 could grow at 5°C in MRS broth, but their optimum growth temperature was 20°C-30°C. These results suggest that psychrotrophic lactic acid bacteria presumed to be derived from rice koji are present in godo, which is one of the factors in the low temperature ripening of godo in winter.

Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature.

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.

Psychrotrophic plant beneficial bacteria from the glacial ecosystem of Sikkim Himalaya: Genomic evidence for the cold adaptation and plant growth promotion.

Commercial biofertilizers tend to be ineffective in cold mountainous regions due to reduced metabolic activity of the microbial inoculants under low temperatures. Cold-adapted glacier bacteria with plant growth-promoting (PGP) properties may prove significant in developing cold-active biofertilizers for improving mountain agriculture. With this perspective, the cultivable bacterial diversity was documented from the East Rathong glacier ecosystem lying above 3900 masl of Sikkim Himalaya. A total of 120 bacterial isolates affiliated to Gammaproteobacteria (53.33%), Bacteroidetes (16.66%), Actinobacteria (15.83%), Betaproteobacteria (6.66%), Alphaproteobacteria (4.16%), and Firmicutes (3.33%) were recovered. Fifty-two isolates showed many in vitro PGP activities of phosphate solubilization (9-100 µg/mL), siderophore production (0.3-100 psu) and phytohormone indole acetic acid production (0.3-139 µg/mL) at 10 °C. Plant-based bioassays revealed an enhancement of shoot length by 21%, 22%, and 13% in ERGS5:01, ERMR1:04, and ERMR1:05, and root length by 14%, 17%, 11%, and 22% in ERGS4:06, ERGS5:01, ERMR1:04, and ERMR1:05 treated seeds respectively. An increased shoot dry weight of 4-29% in ERMR1:05 and ERMR1:04, and root dry weight of 42-98% was found in all the treatments. Genome analysis of four bacteria from diverse genera predicted many genes involved in the bacterial PGP activity. Comparative genome study highlighted the presence of PGP-associated unique genes for glucose dehydrogenase, siderophore receptor, tryptophan synthase, phosphate metabolism (phoH, P, Q, R, U), nitrate and nitrite reductase, TonB-dependent receptor, spermidine/putrescine ABC transporter etc. in the representative bacteria. The expression levels of seven cold stress-responsive genes in the cold-adapted bacterium ERGS4:06 using real-time quantitative PCR (RT-qPCR) showed an upregulation of all these genes by 6-17% at 10 °C, and by 3-33% during cold-shock, which indicates the cold adaptation strategy of the bacterium. Overall, this study signifies the psychrotrophic bacterial diversity from an extreme glacier environment as a potential tool for improving plant growth under cold environmental stress.

Screening of microorganisms producing cold-active oxidoreductases to be applied in enantioselective alcohol oxidation. An Antarctic survey.

Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph.

Cold Temperature Growth of Clinical and Food Isolates of Bacillus cereus ‡.

A collection of 27 Bacillus cereus food and clinical isolates were screened for the ability to grow at cold temperatures. Growth was examined using fluid or solid nutrient media or milk incubated at 10, 7, or 5°C. Fourteen isolates were capable of visible colony formation on brain heart infusion (BHI) agar by day 7 at 10°C; two isolates formed visible colonies by day 10 at 7°C on Trypticase soy agar. Nineteen of the isolates could grow in BHI at 7°C if previously adapted to 7°C over a five-week period. Both food and clinical isolates demonstrated a cold adaptation response. This should be considered when modeling B. cereus growth in foods or in assessing shelf life or safety relative to B. cereus .