Mechanisms of subzero growth in the cryophile Planococcus halocryophilus determined through proteomic analysis.

The eurypsychrophilic bacterium Planococcus halocryophilus is capable of growth down to -15°C, making it ideal for studying adaptations to subzero growth. To increase our understanding of the mechanisms and pathways important for subzero growth, we performed proteomics on P. halocryophilus grown at 23°C, 23°C with 12% w/v NaCl and -10°C with 12% w/v NaCl. Many proteins with increased abundances at -10°C versus 23°C also increased at 23C-salt versus 23°C, indicating a closely tied relationship between salt and cold stress adaptation. Processes which displayed the largest changes in protein abundance were peptidoglycan and fatty acid (FA) synthesis, translation processes, methylglyoxal metabolism, DNA repair and recombination, and protein and nucleotide turnover. We identified intriguing targets for further research at -10°C, including PlsX and KASII (FA metabolism), DD-transpeptidase and MurB (peptidoglycan synthesis), glyoxalase family proteins (reactive electrophile response) and ribosome modifying enzymes (translation turnover). PemK/MazF may have a crucial role in translational reprogramming under cold conditions. At -10°C P. halocryophilus induces stress responses, uses resources efficiently, and carefully controls its growth and metabolism to maximize subzero survival. The present study identifies several mechanisms involved in subzero growth and enhances our understanding of cold adaptation.

Differential Production of Pigments by Halophilic Bacteria Under the Effect of Salt and Evaluation of Their Antioxidant Activity.

Microorganisms that survive in the high salt environment have been shown to be a potential source for metabolites with pharmaceutical importance. In the present study, we have investigated the effect of 5 and 10% (w/v) NaCl on growth, biochemical changes, and metabolite production in seven moderately halophilic bacteria isolated from the salterns/mangrove area of South India. Metabolite production by Bacillus VITPS3 increased by 3.18-fold in the presence of 10% (w/v) NaCl concentration. Total phenolic and flavonoid content increased in Bacillus VITPS5 (11.3-fold) and Planococcus maritimus VITP21 (5.99-fold) whereas ß-carotene content was less at higher NaCl concentrations. VITP21 and VITPS5, in response to NaCl, produced metabolites with higher (6.72- and 4.91-fold) DPPH and ABTS radical scavenging activity. UV/visible spectrophotometry of the extracts confirmed the presence of flavonoids, phenolics, and related compounds. 1H-NMR spectra indicated substantial changes in the metabolite production in response to salt concentration. Principal component analysis (PCA) revealed that VITP21 extracts exhibited the highest antioxidant activity compared with other extracts. The present study presents the first report on the comparative analysis of pigment production by moderate halophilic bacteria, in response to the effect of salt and their relation to radical scavenging property.

Enhanced Microbial Survivability in Subzero Brines.

It is well known that dissolved salts can significantly lower the freezing point of water and thus extend habitability to subzero conditions. However, most investigations thus far have focused on sodium chloride as a solute. In this study, we report on the survivability of the bacterial strain Planococcus halocryophilus in sodium, magnesium, and calcium chloride or perchlorate solutions at temperatures ranging from +25°C to -30°C. In addition, we determined the survival rates of P. halocryophilus when subjected to multiple freeze/thaw cycles. We found that cells suspended in chloride-containing samples have markedly increased survival rates compared with those in perchlorate-containing samples. In both cases, the survival rates increase with lower temperatures; however, this effect is more pronounced in chloride-containing samples. Furthermore, we found that higher salt concentrations increase survival rates when cells are subjected to freeze/thaw cycles. Our findings have important implications not only for the habitability of cold environments on Earth but also for extraterrestrial environments such as that of Mars, where cold brines might exist in the subsurface and perhaps even appear temporarily at the surface such as at recurring slope lineae.

Accelerating the sludge disintegration potential of a novel bacterial strain Planococcus jake 01 by CaCl2 induced deflocculation.

The present study investigates the impacts of phase separated disintegration through CaCl2 (calcium chloride) mediated biosurfactant producing bacterial pretreatment. In the initial phase of the study, the flocs were disintegrated (deflocculation) with 0.06g/gSS of CaCl2. In the subsequent phase, the sludge biomass was disintegrated (cell disintegration) through potent biosurfactant producing new novel bacteria, Planococcus jake 01. The pretreatment showed that suspended solids reduction and chemical oxygen demand solubilization for deflocculated - bacterially pretreated sludge was found to be 17.14% and 14.14% which were comparatively higher than flocculated sludge (treated with bacteria alone). The biogas yield potential of deflocculated - bacterially pretreated, flocculated, and control sludges were observed to be 0.322(L/gVS), 0.225(L/gVS) and 0.145(L/gVS) respectively. To our knowledge, this is the first study to present the thorough knowledge of biogas production potential through a novel phase separated biosurfactant bacterial pretreatment.

Planococcus maritimus VITP21 synthesizes (2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-ß-d-fructofuranose under osmotic stress: a novel protein stabilizing sugar osmolyte.

A halotolerant bacterium, Planococcus maritimus VITP21 isolated from a saltern region in Kumta along the Arabian Sea Coast of India was found to have increased cellular levels of sugars (up to 2.3-fold) under osmotic stress when grown in minimal medium with glucose as the sole carbon and energy source supplemented with 10% w/v NaCl. The major sugar osmolyte which increased with the concentration of NaCl in the growth medium was purified and characterized using various nuclear magnetic resonance spectroscopy techniques. The sugar was found to be similar to sucrose but with the C-2 hydroxyl group of the glucose ring substituted with acetamido group, which is not previously reported for its natural synthesis by any other organism. This novel sugar, (2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-ß-d-fructofuranose, exhibited stabilizing effect on a model protein α-amylase by increasing the apparent midpoint transition, onset temperature of denaturation, and free energy of thermal unfolding.

Bacterial growth at -15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1.

Planococcus halocryophilus strain Or1, isolated from high Arctic permafrost, grows and divides at -15 °C, the lowest temperature demonstrated to date, and is metabolically active at -25 °C in frozen permafrost microcosms. To understand how P. halocryophilus Or1 remains active under the subzero and osmotically dynamic conditions that characterize its native permafrost habitat, we investigated the genome, cell physiology and transcriptomes of growth at -15 °C and 18% NaCl compared with optimal (25 °C) temperatures. Subzero growth coincides with unusual cell envelope features of encrustations surrounding cells, while the cytoplasmic membrane is significantly remodeled favouring a higher ratio of saturated to branched fatty acids. Analyses of the 3.4 Mbp genome revealed that a suite of cold and osmotic-specific adaptive mechanisms are present as well as an amino acid distribution favouring increased flexibility of proteins. Genomic redundancy within 17% of the genome could enable P. halocryophilus Or1 to exploit isozyme exchange to maintain growth under stress, including multiple copies of osmolyte uptake genes (Opu and Pro genes). Isozyme exchange was observed between the transcriptome data sets, with selective upregulation of multi-copy genes involved in cell division, fatty acid synthesis, solute binding, oxidative stress response and transcriptional regulation. The combination of protein flexibility, resource efficiency, genomic plasticity and synergistic adaptation likely compensate against osmotic and cold stresses. These results suggest that non-spore forming P. halocryophilus Or1 is specifically suited for active growth in its Arctic permafrost habitat (ambient temp. ∼-16 °C), indicating that such cryoenvironments harbor a more active microbial ecosystem than previously thought.