- Currently, little is known about the biological and molecular responses of Xcc to low temperatures.. - Results: Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. - At low temperature Xcc formed branching structured motility. - Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. - Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress.. - Conclusions: Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.. - Keywords: Xanthomonas, Low temperature stress, Motility, Biofilm formation, Fatty acids, Metabolism. - Low temperature is one of the most prevalent abiotic stresses. - signaling pathway and salicylic acid (SA) also participate in responding to low temperature stress [10–12].. - Impact of low temperature in the regulation of bacterial physiology has been reported. - coli adapts to low temperature environment by increasing the ratio of straight-chain unsaturated fatty acids (SCUFA) to straight-chain saturated fatty acids (SCFAs) [15]. - In general, bacteria adapt to low temperature environment by regulating several cellular fac- tors such as fatty acid desaturases [17], cold shock proteins (CSPs) [18] and transcriptional regulators . - Although, plant response to cold stress has been extensively stud- ied [5–8], but limited information is available about the impact of low temperature on plant pathogen, Xantho- monas. - To gain insight into the molecular mechanisms of Xcc in response to low temperature, RNA-seq tech- nology was employed along with physiological experi- ments to examine the spectrum and impact of low temperature on gene expression profiles and physio- logical changes in Xcc.. - Negative effects of low temperature on Xcc growth Temperature is a crucial environmental factor that de- termines the growth of pathogens [26]. - Xcc exhibited slower growth at low temperature and different lag phases at different temperatures (Fig. - Colony forming units (CFU) of Xcc strain in different growth phases at 15 °C and 28 °C were measured by dilution plate count method, which revealed significant effect of low temperature on Xcc growth (Fig. - Effects of low temperature on swarming motility and biofilm formation of Xcc. - Comparative analysis of Xcc motility and biofilm. - formation at 28 °C and 15 °C revealed that biofilm forma- tion was increased at low temperature (Fig. - At low temperature bacteria densely gathered to form closely packed biofilm layer (Fig.. - Colonization phase occurred at the temperatures higher than 28 °C whereas significantly reduced swarming mobility of Xcc was noted at low temperature (Fig. - Edge morphology of Xcc colonies at different temperatures was studied under inverted microscopes (Fig. - Formation of an uneven and indefinite boundary at low temperature was also observed under the microscope (Fig. - This implies a unique way of Xcc to adapt in low temperature environment.. - Low temperature modulates UFAs of Xcc. - 1 Reduced Xcc strain growth at low temperatures. - b Colony forming units (CFU) of Xcc strains during different growth phases at 15 °C and 28 °C. - Little is known about the FA composition of Xcc at low temperatures. - Growth at low temperature resulted in the decrease of iso-C 15:0 per- centage and increased percentage of anteiso to iso fatty acids ratio (Fig. - These results appeared consistent with the changes in membrane phospholipids for adapt- ing to low temperature environment [36].. - b Several stages of Xcc biofilm formation on the interstitial surfaces between glass slides and nutrient-agar medium at different temperatures. - c Image of Xcc biofilm formation stage. - Low temperature regulates the expression of genes involved in several functional categories. - In order to investigate the effect of low temperature on Xcc, RNA-Seq of Xcc was carried out at different tem- peratures. - The Q20 value of Xcc grown at 28 °C and 15 °C remained as 96.69 and 96.97%, respectively whereas the genome of Xcc was used as reference (NC_. - 5) and results indicated acceptable quality of Xcc RNA sequencing.. - To further explore the genes in response to low temperature, gene expressions were compared before and after low temperature treatments at a genome-wide level.. - a Swarming motility of Xcc wild- type strain on rich YEB medium plates at 28 °C and 15 °C after 3 days. - b The characteristic image of Xcc colonies edge morphology were captured by inverted microscope at 28 °C and 15 °C. - c Microscopic images of Xcc edge expressing green fluorescent protein. - Response of Xcc genes involved in carbon and nitrogen metabolism at low temperature. - Due to the effect of low temperature on Xcc growth (Fig. - Results of Xcc carbon metabolism at. - low temperature revealed that 90.7% genes, mainly in- volved in carbon and central carbon metabolism were down-regulated (Additional file 3: Table S3). - Five genes involved in the glycolysis pathway and pyruvic acid metabolism were up-regulated indicating that low temperature does not in- hibit their activities (Additional file 4: Table S4). - These re- sults demonstrated that low temperature might block other pathways to limit energy for cell growth and metab- olism. - Overall, results suggest that low temperature dis- rupts carbon and nitrogen metabolism in Xcc.. - Low temperature alters genes expression of flagellar and type IV pilus systems in Xcc. - As expected, low temperature affected flagella assembly however var- ied effects of temperature on Xcc flagella assembly genes were observed (Additional file 6: Table S6). - Low Table 1 Compositions of Xcc fatty acid phospholipids at. - Fatty acid. - 4 Differences in the compositions of Xcc fatty acid phospholipids at different temperatures. - UFA, unsaturated fatty acid. - BCFA, branched-chain fatty acid. - temperature treatment resulted in up-regulation of four genes and down-regulation of two genes suggesting that low temperature may disrupt flagella assembly of Xcc.. - Microscopic analysis of twitching assay at low temperature depicted that multi- cellular organization at the edges of subsurface twitching zones of Xcc cells has blurred and irregular boundary lines (Fig. - Taken together, results suggest that low temperature may disrupt flagella assembly and up- regulate type IV pilus genes expression leading to differen- tial motility in Xcc.. - predominately down-regulated at low temperature Xcc treatment. - As low temperature influenced UFAs, DEGs related to fatty acid biosynthesis, phospholipid synthe- sis and lipid A synthesis were analyzed (Fig. - Thereby, changing the membrane phospholipid component of Xcc to adapt in low temperature environment.. - Pathogenesis-associated genes in Xcc are negatively regulated by low temperature. - 6).Therefore, we analyzed the effect of low temperature on pathogenesis-associated genes expression in Xcc. - Six pathogenesis related DEGs were influenced by low temperature treatment of whichone was up-regulated and 5 were down-regulated (Additional file 9: Table S9). - Results showed that 78.3% of genes related to these systems were down-regulated at low temperature (Additional file 10: Table S10). - Low temperature is a common environmental factor and cold shock is known to restrict bacterial growth [43]. - During the study we ob- served significantly effected Xcc growth rate at low temperature (Fig. - that low temperature down-regulated expression of genes involved in carbon and nitrogen metabolism but had little effect on genes related to glycolysis pathway and pyruvic acid metabolism (Additional file 3: Table S3, Additional file 4: Table S4). - 6a) implying that ribosomal proteins might have special functions at low temperature like other bacteria [44]. - Similar to Vibrio cholerae, Xcc formed more and tighter biofilms at low temperature (Fig. - At low temperature motility was significantly influenced and Xcc formed an uneven and indefinite col- ony boundary (Fig. - Transcriptome analysis revealed diverse effects of low temperature on Xcc flagella assembly genes (Additional file 6: Table S6).. - Results suggested that low temperature could be disrup- tive to flagellar system and subsequently disorganizes flagellar assembly. - Interestingly, low temperature stimu- lated the expression of genes related to type IV pilus systems (Additional file 6: Table S6). - Microscopic analysis of twitching assays depicted that subsurface twitching zones of Xcc cells have blurred and irregular boundary lines at low temperatures (Fig. - This is an uncharac- terized aspect of Xcc and underlying mechanism is the focus of our studies. - Moreover, type IV pilus participates in biofilm formation [48, 49] and that might be a potential pathway of Xcc to increase biofilm formation at low temperatures.. - Membrane fluidity is essential for the survival of bacteria at low temperature [30] and bacteria have evolved various. - This study revealed that low temperature stimulates the biosyn- thesis of unsaturated fatty acids by increasing the percent- age of n-C 16:1 cis-9 (Fig. - It is an effective way to adjust the membrane fluidity for adapting at low temperature. - How- ever, Xcc mechanism of altering the ratio of anteiso to iso fatty acids at low temperature is unknown. - This might be a potential mechanism to alter the ratio of branched-chain fatty acids at low temperature through the selection of substrates. - 8) revealed that 88.9% genes were down- regulated (Additional file 8: Table S8) implying that more energy is used for basal metabolism of bacteria at low temperature.. - However, we found that a large portion of genes related to Xcc pathogenesis were down-regulated at low temperature (Additional file 8: Table S8), including CRP-like protein. - a Microanalysis of multi-cellular organization at the edges of twitching zones of Xcc cells at 28 °C. - b Microanalysis of the multi-cellular organization at the edges of twitching zones of Xcc cells at 15 °C. - syrin- gae, we found that low temperature influenced Xcc secretion system that might reduce the injection of ef- fector proteins or toxins into host cells (Additional file 10:. - Meanwhile, we speculate that low temperature may initiate additional uncharacterized mechanisms to control Xcc virulence.. - In short, physiological characteristics of Xcc at low temperature were examined and genome wide transcrip- tional analysis presented specific response of Xcc at low temperature. - Results suggested that many biological pro- cesses participate to respond at low temperature includ- ing carbon, nitrogen and fatty acid metabolism. - Data of this study provide insights into molecular mech- anisms of Xcc to adapt at low temperature and present. - 8 Membrane FA composition biosynthesis in response to low temperature in Xcc. - FadL, long-chain fatty acid transport protein. - For microscopic analysis of biofilm formation assays, overnight cultures of Xcc strains expressing GFP in YEB medium were collected by centrifugation, washed with fresh medium and adjusted to OD 600 = 1.0. - Analysis of fatty acid composition. - List of genes related to fatty acid metabolism in Xcc regulated by temperature.. - Critical role of anteiso-C 15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. - Role of sigma(B) in adaptation of Listeria monocytogenes to growth at low temperature. - FabH selectivity for anteiso branched-chain fatty acid precursors in low-temperature adaptation in Listeria monocytogenes. - Phaseolicola NPS3121 at low temperature:
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