Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis

Abstract

Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions.

Fig 1. Impact of wapH mutation on cold growth and survival.

Growth of the wild type, wapH and pSEVAwapH strains. A. Growth at 30°C. B. Growth at 8°C. C. Survival at low temperatures. Erlenmeyer were inoculated and incubated at 30°C until reached an OD_600nm of 0.5 and then incubated at 8°C. Samples were taken at 0, 16 and 42 h and CFU/ ml was determined. Survival was calculated as (CFU/ml _{T = 16h or 42h}/CFU/ml _{T = 0}) *100. Values represent mean ± SD of triplicate independent cultures.

Fig 2. Aggregation assays.

A. Aggregation assay at 30°C of the wild type (wt), wapH and complemented strain (pSEVAwapH). Values represent mean ± SD of 5 independent measurements. B. Aggregation assay with different strains expressing mCherry protein and mixed with an unmarked strain. Values represent media ± SD of 5 independent measurements. C. Microscopic visualization of mixed aggregates using cells grown at 30°C or from cold shock experiments. Strains expressing fluorescent proteins were mixed and settled for15 min. An aliquot from the bottom of the tube was taken and aggregates were observed in a confocal microscope using 1000X magnification. Representative images from triplicate independent experiments are shown.

Fig 3. Cell permeability and polymyxin sensitivity assay.

A. SDS sensitivity assay of the wild type (wt), wapH and complemented strain (pSEVAwapH). Cells were cultured at 30°C or 8°C and CFU/ml was determined in LB plates with and without SDS. B. Sensitivity to polymixin B of the wild type (wt), wapH and complemented strain (pSEVAwapH) was performed by using disk inhibition assay with cells cultured at 30°C or 8°C. Values represent media ± SD of triplicate independent measurements. * denotes significant differences (Mann Whitney Test).

Fig 4. Nanomechanical determinations using atomic force microscopy (AFM) in live and hydrated cells.

A Surface to Volume ratio (S/V) was determined using Gwyddion software. B. Cell elasticity determination at different culture conditions. Force-distance curves were obtained using MultiMode 8 with a Nanoscope V controller, Bruker in contact mode for at least 10 cells per condition in 10 different points along the major axis per triplicate. Adjustment to the Sneedon model was performed between 0 and 2 nN and the Young module was calculated. C. K_bacterium determinations as a measure of bacterial turgor pressure. Adjustment to the Hooke’s law was performed between 2 and 4 nN in the same curves Force-distance described above. Wild type (wt), wapH and complemented strain (pSEVAwapH).

Fig 5. Proposed model to explain the effect of low temperatures in P.extremaustralis envelope characteristics and the impact of the wapH in cold adaptation.