Isotachophoretic quantification of total viable bacteria on meat and surfaces.

BACKGROUND: The quantification of microbes, particularly live bacteria, is of utmost importance in assessing the quality of meat products. In the context of meat processing facilities, prompt identification and removal of contaminated carcasses or surfaces is crucial to ensuring the continuous production of safe meat for human consumption. The plate count method and other traditional detection methods are not only labour-intensive but also time-consuming taking 24-48 h. RESULTS: In this report, we present a novel isotachophoretic quantification method utilizing two nucleic acid stains, SYTO9 and propionic iodide, for the detection of total viable bacteria. The study employed E. coli M23 bacteria as a model organism, with an analysis time of only 30 min. The method demonstrated a limit of detection (LOD) of 184 CFU mL-1 and 14 cells mL-1 for total viable count and total cell count, respectively. Furthermore, this new approach is capable of detecting the microbial quality standard limits for food contacting surfaces (10 CFU cm-2) and meat (1.99 × 104 CFU cm-2) by swabbing an area of 10 × 10 cm2. SIGNIFICANCE: In contrast to the culture-based methods usually employed in food processing facilities, this isotachophoretic technique enables easy and rapid detection (<30 min) of microorganisms, facilitating crucial decision-making essential for maintaining product quality and safety.

Performance evaluation of commercially available swabs for environmental monitoring: Uptake and release efficiency.

Safety and the quality of products rely on proper cleanliness procedures and good manufacturing practices in the production environment. The use of swabs for the collection of samples from surfaces has been a common practice in industries, medicine and forensic studies. To accommodate these different purposes, many varieties of swabs have been introduced into the market, and it is important to assess the performance of these swabs before incorporating into an environmental monitoring procedure. The overall effectiveness of a swab is determined by two factors: the number of bacteria that a swab can uptake from a surface and the number of picked-up bacteria the swab can elute into a releasing buffer. This study evaluated the uptake efficiency and release efficiency of four different commercially available swabs: CleanFoam (Texwipes, USA), FLOQSwabs (Copan diagnostic Inc., USA), Hydraflock swabs (Puritan medical products, USA), and Cotton swabs. Cotton swabs showed the highest uptake efficiency (96.5 ± 1.9%), whereas CleanFoam swabs (57.9 ± 20.3%) showed the least. Both flocked (FLOQSwabs and Hydraflock) swabs showed over 80% uptake efficiency. Releasing efficiency of swabs was tested with eight different releasing buffers. Cotton swabs displayed the lowest release efficiency with most of the tested releasing buffers. When employed with Tris HEPES, Tris MOPS, Tris TAPS, FLOQSwabs, and Hydraflock swabs exhibited releasing efficiency of over 75%. The overall efficiency of the swabs was determined using TAPS as the releasing buffer and the values obtained were 80.4 ± 9.8%, 54.7 ± 16.9%, 35.0 ± 12.7% and 25.2 ± 6.9% for Hydraflock swabs, FLOQSwabs, Cotton swabs and Cleanfoam swabs, respectively.

Isotachophoresis for rapid transformation of Escherichia coli.

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2-3 × 105 ) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 µm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (-16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.

Modelling growth and histamine formation of Klebsiella aerogenes TI24 isolated from Indonesian pindang.

Indonesian salted-boiled fish (pindang) is a popular traditional food in Indonesia, which is made from Scombroid fish such as tuna and mackerel. As with other traditionally prepared fish products, pindang has important economic and social values, especially for those living in the coastal areas of Indonesia. However, pindang is a major cause of histamine fish poisoning (HFP) for consumers. Klebsiella aerogenes T124, a relatively high histamine-producing isolate from pindang, was used to describe lag time (λ), growth rate (µmax), maximum population density (Nmax), and histamine production in histidine broth and artificially contaminated Grey mackerel. Broth was adjusted to 1.5, 6, 10 and 20% w/v NaCl; mackerel was treated with 6% w/w NaCl, a level common to Indonesian industry practice, or not treated with additional NaCl. Samples were incubated at 10, 15, 20 and 30 °C. In broth, µmax and Nmax were significantly affected by temperature and NaCl, respectively, with λ influenced by both parameters. In control fish, µmax was significantly affected by temperature and NaCl, except at 10 and 15 °C; for 6% NaCl treatment, growth was only observed at 20 and 30 °C. Under similar incubation conditions for broth and fish, histamine formation was markedly affected by NaCl concentration. In broth, -5.1 to -6.6 log µg of histamine was produced per CFU, versus -4.6 to -6.6 log µg per CFU in fish. This study demonstrated that mackerel treated with 6% NaCl and stored at 10-15 °C prevents growth of K. aerogenes strain TI24 and formation of toxic levels of histamine.

Effect of peroxyacetic acid treatment and bruising on the bacterial community and shelf-life of baby spinach.

The importance of leaf integrity, i.e. the effects of bruising (mechanical damage), and sanitisation with peroxyacetic acid (PAA) on bacterial communities of ready-to-eat baby spinach remains unclear. Two shelf-life studies were conducted at 4 °C to investigate the effect of bruising and sanitisation on the growth of spoilage microorganisms. In the first experiment, both bruising treatments (100% and 40% of leaves) halved shelf life to 12 d, whereas intact leaves had a shelf-life of 23 d. Bruising had no influence on bacterial diversity during shelf-life, though some differences in the relative abundance of minor genera were observed. Pseudomonas and Pantoea were the most dominant bacterial genera, regardless of bruising treatment. High throughput amplicon sequencing also identified other spoilage bacteria including Chryseobacterium, Stenotrophomonas, Bacillus, Sphingobacterium, Erwinia and Flavobacterium. In the second experiment, washing of intact baby spinach with a sanitiser (80 mg/L: PAA) reduced microbial load as determined by aerobic plate count but did not immediately affect the presence/relative abundance of most of the genera of spoilage bacteria observed. During shelf-life, the bacterial diversity of sanitised leaves was significantly lower than on water-washed leaves. Although sanitisation resulted in a higher initial log reduction in microbial load compared to control (portable tap water), sanitisation did not extend the shelf life of baby spinach (23 d). Sanitised spinach had reduced bacterial diversity however, by the end of shelf life, both sanitised and water-washed spinach was dominated by Pseudomonas and Pantoea spoilage bacteria. This study demonstrated for the first time that the shorter shelf life of bruised leaves was related to faster microbial growth rather than changes in bacterial diversity or community composition.

Effect of glucose, pH and lactic acid on Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens within a commercial heat-shrunk vacuum-package film.

Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens are common spoilage organisms found within the microbiome of refrigerated vacuum-packaged (VP) beef. Extending and predicting VP beef shelf-life requires knowledge about how spoilage bacteria growth is influenced by environmental extrinsic and intrinsic factors. Multifactorial effects of pH, lactic acid (LA) and glucose on growth kinetics were quantified for C. maltaromaticum, B. thermosphacta and S. liquefaciens within a heat shrink-wrapped VP commercial film containing a simulated beef medium. LA, pH, and undissociated lactic acid (UDLA) significantly affected bacterial growth rate (p < 0.001), whereas 5.55 mM glucose produced a marginal effect. At 1.12 mM UDLA, growth rate and maximum population density decreased 20.9 and 3.5%, 56 and 7%, and 11 and 2% for C. maltaromaticum, B. thermosphacta, and S. liquefaciens, respectively.

qPCR quantification of Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Serratia liquefaciens growth kinetics in mixed culture.

Quantifying growth kinetics of specific spoilage microorganisms in mixed culture is required to describe the evolution of food microbiomes. A qPCR method was developed to selectively amplify individual meat spoilage bacteria, Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens, within a broth medium designed to simulate the composition of beef. An optimized method of DNA extraction was produced for standard curve construction. Method specificity was determined by individual single peaks in melt curves. Reaction efficiency for standard curves of C. maltaromaticum, B. thermosphacta and S. liquefaciens was high (R2 = 0.98-0.99), and linear quantification was achieved over a 5 log CFU/ml range. Coefficient of variation was calculated considering both threshold cycle (Ct) and bacterial concentration; the value did not exceed 14% for inter- or intra-runs for either method. Comparison of growth kinetic parameters derived from plate count and qPCR showed no significant variation (P > .05) for growth rate (GR) and maximum population density (MPD); lag phase duration (LPD) was not included in this comparison due to high innate variability. Log quantification of each isolate was validated in a mixed-culture experiment for all three species with qPCR and plate count differing less than 0.3 log CFU/ml (average 0.10 log CFU/ml, R2 = 0.98).

Characterisation of the Brochothrix thermosphacta sortase A enzyme.

Gram-positive bacteria utilise class A sortases to coat the surface of their cells with a diversity of proteins that facilitate interactions with their environment and play fundamental roles in cell physiology and virulence. A putative sortase A gene was identified in the genome of the poorly studied meat spoilage bacterium Brochothrix thermosphacta. To understand how this bacterium mediates interactions with its environment, an N-terminal truncated, His-tagged variant of this protein (His6-BtSrtA) was expressed and purified. Catalytic activity of recombinant His6-BtSrtA was investigated, including sorting motif recognition of target proteins and bioconjugation activity. Further, the B. thermosphacta genome was examined for the presence of sortase A (SrtA) protein substrates. His6-BtSrtA readily formed intermediate complexes with LPXTG-tagged proteins. Although the reaction was inefficient, nucleophilic attack of the resultant thioacyl intermediates by tri-glycine was observed. Genome examination identified 11 potential SrtA substrates, two of which contained protein domains associated with adherence of pathogens to host extracellular matrix proteins and cells, suggesting the B. thermosphacta SrtA may be indirectly involved in its attachment to meat surfaces. Thus, further work in this area could provide crucial insight into molecular mechanisms involved in the colonisation of meat by B. thermosphacta.

Genomic and metabolic characterization of spoilage-associated Pseudomonas species.

Pseudomonas are common spoilage agents of aerobically stored fresh foods. Their ability to cause spoilage is species- and may be strain-specific. To improve our understanding of the meat and milk spoilage agents Pseudomonas fragi and Pseudomonas lundensis, we sequenced the genomes of 12 P. fragi and seven P. lundensis isolates. These genomes provided a dataset for genomic analyses. Key volatile organic compounds (VOCs) produced or metabolised by the isolates were determined during their growth on a beef paste and where possible, metabolic activity was associated with gene repertoire. Genome analyses showed that the isolates included in this work may belong to more than two Pseudomonas species with possible spoilage potential. Pan-genome analyses demonstrated a high degree of diversity among the P. fragi and genetic flexibility and diversity may be traits of both species. Growth of the P. lundensis isolates was characterised by the production of large amounts of 1-undecene, 5-methyl-2-hexanone and methyl-2-butenoic acid. P. fragi isolates produced extensive amounts of methyl and ethyl acetate and the production of methyl esters predominated over ethyl esters. Some of the P. fragi produced extremely low levels of VOCs, highlighting the importance of strain-specific studies in food matrices. Furthermore, although usually not considered to be denitrifiers, all isolates generated molecular nitrogen, indicating that at least some steps of this pathway are intact.

Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi.

Pseudomonas fragi is a meat and milk spoilage bacterium with high iron requirements; however, mechanisms of iron acquisition remain largely unknown. The aim of this work was to investigate siderophore production as an iron acquisition system for P. fragi. A vibrioferrin siderophore gene cluster was identified in 13 P. fragi, and experiments were conducted with a representative strain of this group (F1801). Chromeazurol S assays showed that P. fragi F1801 produced siderophores under iron starvation at optimum growth and refrigeration temperature. Conversely, supplementation of low iron media with 50 µM FeCl3 repressed transcription of the vibrioferrin genes and siderophore production. Disruption of the siderophore receptor (pvuA) caused polar effects on downstream vibrioferrin genes, resulting in impaired siderophore production of the ΔpvuA mutant. Growth of this mutant was compared to growth of a control strain (Δlip) with wild-type vibrioferrin genes in low iron media supplemented with iron chelators 2,2΄-bipyridyl or apo-transferrin. While 25 µM 2,2΄-bipyridyl caused impaired growth of ΔpvuA, growth of the mutant was completely inhibited by 2.5 µM apo-transferrin, but could be restored by FeCl3 addition. In summary, this work identifies a vibrioferrin-mediated iron acquisition system of P. fragi, which is required for growth of this bacterium under iron starvation.

Isotachophoretic Fluorescence in Situ Hybridization of Intact Bacterial Cells.

A counter-pressure-assisted capillary isotachophoresis method in combination with a sieving matrix and ionic spacer was used to perform in-line fluorescence in situ hybridization (FISH) of bacterial cells. A high concentration of sieving matrix (1.8% w/v HEC) was introduced at one end of the capillary, and the bacterial cells were suspended in the spacer electrolyte for injection. Using a 2 min injection with 18 psi counter-pressure, 50% of the cells injected into the capillary were hybridized with the fluorescently labeled oligonucleotide, and the excess unhybridized probe was separated from the hybridized cell-probe complexes in a two-stage ITP method. With an LOD (6.0 × 104 cells/mL) comparable with the CE analysis of a sample processed using an off-line FISH protocol, the total analysis time was reduced from 2.5 h to 30 min. Provided the appropriate probe is selected, this approach can be used for specific detection of bacterial cells in aqueous samples.

Application of electrolysed oxidising water as a sanitiser to extend the shelf-life of seafood products: a review.

Electrolysed oxidising water (E.O. water) is produced by electrolysis of sodium chloride to yield primarily chlorine based oxidising products. At neutral pH this results in hypochlorous acid in the un-protonated form which has the greatest oxidising potential and ability to penetrate microbial cell walls to disrupt the cell membranes. E.O. water has been shown to be an effective method to reduce microbial contamination on food processing surfaces. The efficacy of E.O. water against pathogenic bacteria such as Listeria monocytogenes, Escherichia coli and Vibrio parahaemolyticus has also been extensively confirmed in growth studies of bacteria in culture where the sanitising agent can have direct contact with the bacteria. However it can only lower, but not eliminate, bacteria on processed seafoods. More research is required to understand and optimise the impacts of E.O. pre-treatment sanitation processes on subsequent microbial growth, shelf life, sensory and safety outcomes for packaged seafood products.

Insight into the Genome of Brochothrix thermosphacta, a Problematic Meat Spoilage Bacterium.

Brochothrix thermosphacta is a dominant but poorly studied meat spoilage organism. It is a close relative of the foodborne pathogen Listeria monocytogenes, and Brochothrix constitutes the second genus in the Listeriaceae family. Here, the genomes of 12 B. thermosphacta strains were sequenced, assembled into draft genomes, characterized, and compared with the genomes of Brochothrix campestris and L. monocytogenes Phenotypic properties including biogenic amine production and antibiotic and heavy metal susceptibilities were tested. Comparative genomic analyses revealed a high degree of similarity among the B. thermosphacta strains, with bacteriophage genes constituting a significant proportion of the accessory genome. Genes for the production of the malodorous compounds acetate, acetoin, butanediol, and fatty acids were found, as were stress response regulatory genes, which likely play important roles in the spoilage process. Amino acid decarboxylases were not identified in the genomes, and phenotypic testing confirmed their absence. Orthologs of Listeria virulence proteins involved in virulence regulation, intracellular survival, and surface protein anchoring were found; however, key virulence genes were absent. Analysis of antibiotic susceptibility showed that strains were sensitive to the four tested antibiotics, except for one tetracycline-resistant isolate with plasmid-mediated tetracycline resistance genes. Strains tolerated higher levels of copper and cobalt than of cadmium although not at concentrations high enough to categorize the strains as being resistant. This study provides insight into the Brochothrix genome, links previous phenotypic data and data provided here to the gene inventory, and identifies genes that may contribute to the persistence of this organism in the food chain.IMPORTANCE Despite increasing knowledge and advances in food preservation techniques, microbial spoilage of foods causes substantial losses, with negative social and economic consequences. To better control the contamination and microbial spoilage of foods, fundamental knowledge of the biology of key spoilage bacteria is crucial. As a common meat spoilage organism, B. thermosphacta contributes substantially to spoilage-associated losses. Nonetheless, this organism and particularly its genome remain largely unstudied. This study contributes to improving our knowledge of the Brochothrix genus. Spoilage-relevant pathways and genes that may play a role in the survival of this organism in a food processing environment were identified, linking previous phenotypic data and data provided here to the gene inventory of Brochothrix and establishing parallels to and differences from the closely related foodborne pathogen L. monocytogenes.

Microbial and sensorial models for head-on and gutted (HOG) Atlantic Salmon (Salmo salar) stored from 0 to 15 °C.

Predictive models offer efficient means to manage the quality and safety of highly perishable seafood. Salmon is an increasingly popular seafood, and relies on well managed domestic and international supply chains to minimize growth of spoilage and pathogenic bacteria. While the literature describes predictive models for smoked and modified atmosphere packaged salmon, there are no reported models for spoilage bacteria and Listeria monocytogenes on head-on and gutted (HOG) aerobically-stored Atlantic salmon. Predictive models were developed for microbial and sensorial degradation of HOG Atlantic salmon stored at 0-15 °C until the end of shelf-life. Total Viable Count (TVC) and Pseudomonas spp. had similar growth rates at 0, 5 and 10 °C, but TVC rate was higher at 15 °C. L. monocytogenes growth rate at 0 °C was 0.004 log10 cfu/h, and showed a log-linear increase (R(2) = 0.99) to 0.079 log10 cfu/h at 15 °C. Sensory Quality Index (QI) scores were 2.4, 4.5, and 7.2 times greater at 5, 10 and 15 °C, respectively, compared to 0 °C. QI and TVC rates had a relatively strong relationship at 5 (R(2) = 0.87), 10 (R(2) = 0.80) and 15 °C (R(2) = 0.78), compared to 0 °C (R(2) = 0.50). These models are potential tools to manage the safety and quality of HOG Atlantic salmon in supply chains.

Counter-pressure-assisted ITP with electrokinetic injection under field-amplified conditions for bacterial analysis.

Counter-pressure was used to extend the duration of field-amplified sample injection in isotachophoresis (FASI-ITP) in order to improve the detection of bacterial cells. Using 0.51-µm negatively charged encapsulated fluorescent beads as a model, the counter-pressure, injection and separation voltages, and times were optimized. Using 6-min 8,963-Pa counter-pressure FASI-ITP injections at -12 kV followed by mobilization of the ITP band with continued injection at -6 kV, the limit of detection (LOD) for Escherichia coli was improved to 78 cells/mL, a factor of 4 when compared with FASI-ITP without counter-pressure.

Proteomic Insight into Functional Changes of Proteorhodopsin-Containing Bacterial Species Psychroflexus torquis under Different Illumination and Salinity Levels.

The extremely psychrophilic proteorhodopsin-containing bacterial species Psychroflexus torquis is considered to be a model sea-ice microorganism, which has adapted to an epiphytic lifestyle. So far, not much is known about proteorhodopsin-based phototrophy and associated life strategies of sea ice bacteria, although it has been previously shown that P. torquis can gain growth advantage from light using a proteorhodopsin proton pump, the activity of which is influenced by environmental salinity. The comprehensive quantitative proteomic study performed here indicated that P. torquis responds to changing salinity and illumination conditions. Proteins in the electron-transfer chain were down-regulated at a suboptimal salinity level, TonB-dependent transporters increased in abundance under supra-optimal salinity and decreased under suboptimal salinity. In addition, several anaplerotic CO2 fixation proteins and three putative light sensing proteins that contain PAS and GAF domains became more abundant under illumination. Furthermore, central metabolic pathways (TCA and glycolysis) were also induced by both salinity stress and illumination. The data suggest that P. torquis responded to changes in both light energy and salinity to modulate membrane and central metabolic proteins that are involved in energy production as well as nutrient uptake and gliding motility processes that would be especially advantageous during the polar summer ice algal bloom.

The use of microbial gene abundance in the development of fuel remediation guidelines in polar soils.

Terrestrial fuel spills in Antarctica commonly occur on ice-free land around research stations as the result of human activities. Successful spill clean-ups require appropriate targets that confirm contaminated sites are no longer likely to pose environmental risk following remediation. These targets are based on knowledge of the impacts of contaminants on the soil ecosystem and on the response of native biota to contamination. Our work examined the response of soil microbial communities to fuel contamination by measuring the abundance of genes involved in critical soil processes, and assessed the use of this approach as an indicator of soil health in the presence of weathered and fresh fuels. Uncontaminated and contaminated soils were collected from the site of remediation treatment of an aged diesel spill at Casey Station, East Antarctica in December 2012. Uncontaminated soil was spiked with fresh Special Antarctic Blend (SAB) diesel to determine the response of the genes to fresh fuel. Partly remediated soil containing weathered SAB diesel was diluted with uncontaminated soil to simulate a range of concentrations of weathered fuel and used to determine the response of the genes to aged fuel. Quantitative PCR (qPCR) was used to measure the abundance of rpoB, alkB, cat23, and nosZ in soils containing SAB diesel. Differences were observed between the abundance of genes in control soils versus soils containing weathered and fresh fuels. Typical dose-response curves were generated for genes in response to the presence of fresh fuel. In contrast, the response of these genes to the range of weathered fuel appeared to be due to dilution, rather than to the effect of the fuel on the microbial community. Changes in microbial genes in response to fresh contamination have potential as a sensitive measure of soil health and for assessments of the effect of fuel spills in polar soils. This will contribute to the development of remediation guidelines to assist in management decisions on when the impact of a fuel spill warrants remediation.

Phytoremediation of hydrocarbon contaminants in subantarctic soils: an effective management option.

Accidental fuel spills on world heritage subantarctic Macquarie Island have caused considerable contamination. Due to the island's high latitude position, its climate, and its fragile ecosystem, traditional methods of remediation are unsuitable for on-site clean up. We investigated the tolerance of a subantarctic native tussock grass, Poa foliosa (Hook. f.), to Special Antarctic Blend (SAB) diesel fuel and its potential to reduce SAB fuel contamination via phytoremediation. Toxicity of SAB fuel to P. foliosa was assessed in an 8 month laboratory growth trial under growth conditions which simulated the island's environment. Single seedlings were planted into 1 L pots of soil spiked with SAB fuel at concentrations of 1000, 5 000, 10,000, 2000 and 40,000 mg/kg (plus control). Plants were harvested at 0, 2, 4 and 8 months and a range of plant productivity endpoints were measured (biomass production, plant morphology and photosynthetic efficiency). Poa foliosa was highly tolerant across all SAB fuel concentrations tested with respect to biomass, although higher concentrations of 20,000 and 40,000 mg SAB/kg soil caused slight reductions in leaf length, width and area. To assess the phytoremediation potential of P. foliosa (to 10 000 mg/kg), soil from the planted pots was compared with that from paired unplanted pots at each SAB fuel concentration. The effect of the plant on SAB fuel concentrations and the associated microbial communities found within the soil (total heterotrophs and hydrocarbon degraders) were compared between planted and unplanted treatments at the 0, 2, 4 and 8 month harvest periods. The presence of plants resulted in significantly less SAB fuel in soils at 2 months and a return to background concentration by 8 months. Microbes did not appear to be the sole driving force behind the observed hydrocarbon loss. This study provides evidence that phytoremediation using P. foliosa is a valuable remediation option for use at Macquarie Island, and may be applicable to the management of fuel spills in other cold climate regions.

Extensive gene acquisition in the extremely psychrophilic bacterial species Psychroflexus torquis and the link to sea-ice ecosystem specialism.

Sea ice is a highly dynamic and productive environment that includes a diverse array of psychrophilic prokaryotic and eukaryotic taxa distinct from the underlying water column. Because sea ice has only been extensive on Earth since the mid-Eocene, it has been hypothesized that bacteria highly adapted to inhabit sea ice have traits that have been acquired through horizontal gene transfer (HGT). Here we compared the genomes of the psychrophilic bacterium Psychroflexus torquis ATCC 700755(T), associated with both Antarctic and Arctic sea ice, and its closely related nonpsychrophilic sister species, P. gondwanensis ACAM 44(T). Results show that HGT has occurred much more extensively in P. torquis in comparison to P. gondwanensis. Genetic features that can be linked to the psychrophilic and sea ice-specific lifestyle of P. torquis include genes for exopolysaccharide (EPS) and polyunsaturated fatty acid (PUFA) biosynthesis, numerous specific modes of nutrient acquisition, and proteins putatively associated with ice-binding, light-sensing (bacteriophytochromes), and programmed cell death (metacaspases). Proteomic analysis showed that several genes associated with these traits are highly translated, especially those involved with EPS and PUFA production. Because most of the genes relating to the ability of P. torquis to dwell in sea-ice ecosystems occur on genomic islands that are absent in closely related P. gondwanensis, its adaptation to the sea-ice environment appears driven mainly by HGT. The genomic islands are rich in pseudogenes, insertional elements, and addiction modules, suggesting that gene acquisition is being followed by a process of genome reduction potentially indicative of evolving ecosystem specialism.

Light-stimulated growth of proteorhodopsin-bearing sea-ice psychrophile Psychroflexus torquis is salinity dependent.

Proteorhodopsins (PRs) are commonly found in marine prokaryotes and allow microbes to use light as an energy source. In recent studies, it was reported that PR stimulates growth and survival under nutrient-limited conditions. In this study, we tested the effect of nutrient and salinity stress on the extremely psychrophilic sea-ice bacterial species Psychroflexus torquis, which possesses PR. We demonstrated for the first time that light-stimulated growth occurs under conditions of salinity stress rather than nutrient limitation and that elevated salinity is related to increased growth yields, PR levels and associated proton-pumping activity. PR abundance in P. torquis also is post-transcriptionally regulated by both light and salinity and thus could represent an adaptation to its sea-ice habitat. Our findings extend the existing paradigm that light provides an energy source for marine prokaryotes under stress conditions other than nutrient limitation.