Morphological and physiological adaptations of psychrophilic Pseudarthrobacter psychrotolerans YJ56 under temperature stress

Both culture-independent and culture-dependent analyses using Nanopore-based 16S rRNA sequencing showed that short-term exposure of Antarctic soils to low temperature increased biomass with lower bacterial diversity and maintained high numbers of the phylum Proteobacteria, Firmicute, and Actinobacteria including Pseudarthrobacter species. The psychrophilic Pseudarthrobacter psychrotolerans YJ56 had superior growth at 13 °C, but could not grow at 30 °C, compared to other bacteria isolated from the same Antarctic soil. Unlike a single rod-shaped cell at 13 °C, strain YJ56 at 25 °C was morphologically shifted into a filamentous bacterium with several branches. Comparative genomics of strain YJ56 with other genera in the phylum Actinobacteria indicate remarkable copy numbers of rimJ genes that are possibly involved in dual functions, acetylation of ribosomal proteins, and stabilization of ribosomes by direct binding. Our proteomic data suggested that Actinobacteria cells experienced physiological stresses at 25 °C, showing the upregulation of chaperone proteins, GroEL and catalase, KatE. Level of proteins involved in the assembly of 50S ribosomal proteins and L29 in 50S ribosomal proteins increased at 13 °C, which suggested distinct roles of many ribosomal proteins under different conditions. Taken together, our data highlights the cellular filamentation and protein homeostasis of a psychrophilic YJ56 strain in coping with high-temperature stress.


Figure S1 .
Figure S1.Culture-independent bacterial communities in Antarctic soil using Oxford Nanopore sequencing tools.The same amounts of soil samples (1 g) were inoculated in an R2A liquid at 13 °C and 30 ℃, respectively.Incubated soil samples were harvested at specific periods (0, 7, 14, and 28 days) to estimate biomass (DNA) and cell density.a, c The bacterial biomass was estimated using DNA quantification and the bacterial community was analyzed using the CLC Genomics Workbench ver.21 (Qiagen, Germany) software.b, d Cell density was measured using the CFU assay.*N.D: not detected.

Figure S2 .
Figure S2.Culture-independent bacterial communities in Antarctic soil using T-RFLP analysis.a and b T-RFLP analysis for assessing culture-independent bacterial communities in Antarctic soil.The T-RFLP analysis was performed by using the restriction enzymes MspI and Rasl.The soil samples for the analysis were incubated in an R2A liquid medium at 13 °C and harvested at specific periods (0, 7, 14, and 28 days).a The peak of strain YJ56 was shown in 160 to 163 bps of fragment lengths distinguished by a red line.b The peak of strain YJ56 was shown in 454 to 457 bps of fragment lengths distinguished by a red line.The peaks identified by species were indicated by using arrows.

Figure S3 .
Figure S3.Growth test of psychrophilic bacteria isolated from Antarctic soil.A total of the 60 strains were inoculated in R2A medium and grown at 13 °C for 5 days.The OD600 values of each strain were measured by the spectrometer (24-hour unit intervals).Mesophilic Escherichia coli ATCC 25922 was used as a negative control.The red dotted line indicates OD600 of 3.0.

Figure S4 .
Figure S4.Phase-contrast microscopic observation of strain YJ56 cultured grown on an R2A liquid medium at 13℃ (a and b) and 25℃ (c and d) until the exponential phase (OD600: 1.0) using a confocal laser scanning microscope (Carl Zeiss, Germany) with 5 μm of the scale bar.Red and yellow arrows indicate cell lengths and widths of strain YJ56, respectively.

Figure S5 .
Figure S5.Comparative genomic analysis of P. psychrotolerans YJ56 and other Pseudarthrobacter species.a.The "A" in the yellow box represents the site with relatively abundant rimJ gene copies among specific contained genomic sequences in strain YJ56.b.Venn diagram represents the distribution of shared and unique gene clusters by OrthoVenn2 analysis.

Figure S6. 2 -
Figure S6.2-DE image analysis of proteins expressed from strain YJ56 cultured at 13 ℃ or 25 °C.The images were analyzed with Image Master Platinum 5.0 image analysis program.Green and red spots indicate paired (n: 238) and non-paired spots (n: 265), respectively.

Figure S7. 2 -
Figure S7.2-DE analysis for identifying different protein expressions in strain YJ56 cultured at 13 ℃ or 25 °C.Proteins with increased abundance in strain YJ56 cultured at a 13 ℃ or b 25 °C.The total number of selected spots was five and ten at 13 ℃ and 25 °C, respectively.Proteins only identified in strain YJ56 cultured at c 13℃ or d 25 °C.The total number of selected spots was four and nine for each temperature condition (13 ℃ and 25 °C), respectively.Selected proteins were indicated with yellow, white, and green triangles.Red circles are a standard point of arrangement for gel images.

Table S2 .
General genome features of P. psychrotolerans YJ56 and five species closest to Pseudarthrobacter based on OrthoANI analysis.

Table S3 .
Unique proteins in P. psychrotolerans YJ56 were determined using BLASTP on the NCBI database.Proteins are categorized by related function.Hypothetical proteins were not listed.

Table S4 .
Insertion sequence analysis of the "A" site with abundant rimJ copies in genomic sequence of strain YJ56 (114,355 to 121,867 bp).

Table S5 .
Identification of proteins expressed from strain YJ56 cultured at two different temperatures.

N-terminal sequence Theoretical Experimental Sequence coverage (%) Mr (KDa) pI Mr (KDa) pI Proteins with increased abundance at 13 °C compared to 25 °C
Changes in protein levels are reported as the ratio between the normalized protein spot volume from cells grown at 13 °C and 25°C (V13 °C /V25 °C or V25 °C /V13 °C , %), for proteins present at a higher level.

Table S6 .
Comparison of the cellular fatty acid compositions (%) of strain YJ56 cultured in an R2A liquid medium at 13 ℃ or 25 °C.The data represents percentages of the total fatty acids.Highlighted percentages in bold indicate major fatty acid components (>5.0%).-,Not detected.

Table S7 .
Statistics of Nanopore sequencing (MinION) data in Antarctic soil.

Table S8 .
Primers were used in this study.

Table S9 .
Proteins with an increased abundance in strain YJ56 cultured at 13 °C compared with 25 °C.The quantity of protein in each spot was normalized to the total valid spot intensity.Through comparing each protein gel image in strain YJ56 under different temperature conditions, significantly changed spots were selected based on a rate increased/decreased 1.8-fold or by complete appearance or disappearance.The final number of selected proteins for LC/MS analysis was 5 (bold) among 21 identified spots.

Table S10 .
Proteins with an increased abundance in strain YJ56 cultured at 25 °C compared with 13 °C.The quantity of protein in each spot was normalized to the total valid spot intensity.Through comparing each protein gel image in strain YJ56 under different temperature conditions, significantly changed spots were selected based on a rate increased/decreased 1.8-fold or by complete appearance or disappearance.The final number of selected proteins for LC/MS analysis was 13 (bold) among 37 identified spots.

Table S11 .
Proteins only identified in strain YJ56 cultured at 13 °C.Strain YJ56 expressed 94 proteins at 13 °C.The number of selected proteins for LC/MS analysis was 4 spots (bold).

Table S12 .
Proteins only identified in strain YJ56 cultured at 25 °C.Strain YJ56 expressed 171 proteins at 25 °C.The number of selected proteins for LC/MS analysis was 9 spots (bold).