The Environment Adapted Lifestyles Of Deep-sea Sediment Bacterium Pseudoalteromonas Sp.SM9913 And The Involved Physiological And Genetic Mechanisms

In the complex ocean ecosystem, heterotrophic bacteria are the major decomposer of dissolved organic matter and particle organic matter and play an enssential role in the matter cycling and energy flux in the ocean. The sediment in the seafloor is full of particle organic matter which is deposited from the seawater in the upper ocean. Bacteriobenthos, the major decomposer of the particle organic matter in the sediment, play a key role in the matter cycling and energy flux in the sediment. However, studies on the lifestyles of bacteriobenthos are rare. Pseudoalteromonas sp. SM9913 (Ps. sp. SM9913), isolated from the surficial sediment of the Okinawa Trough, is a typical deep-sea sediment heterotrophic bacterium. The extracellular proteases, exopolysaccharide and genome of Ps. sp. SM9913 have been studied by researchers in our laboratory. Based on these early studies, we studied the deep-sea sediment adaptaed lifestyles of Ps. sp. SM9913 here. Inspired by studies on the exopolysaccharide and genome of Pseudoalteromonas sp. SM9913 (Ps. sp. SM9913), we studied its lifestyles. Based on our results of physiological, genetic and transcrptomic studies, we primarily revealed the lifestyles of Ps. sp. SM9913 and the mechanisms involved in. And finally we propsed a model that describing the possible lifestyles of Ps. sp. SM9913.(1) The particle-associated lifestyle of benthonic bacterium Ps. sp. SM9913Based on the results of study on the exopolysaccharide, we speculated that Ps. sp. SM9913 might have particle-associated lifestyle in deep-sea sediment. We found that Ps. sp. SM9913 and Pseudoalteromonas haloplanktis TAC125 (Ps. haloplanktis TAC125) had similar abilities of attachment to clay particles, indicating a particle-associated life of Ps. sp. SM9913. The particle attachment ability of exopolysaccharide synthesis defective strain ΔepsT was remarkably lower than that of wild strain, complementation of gene epsT in ΔepsT could restore its ablility of attachment to clay particles. These results indicated that exopolysaccharide was important in the particle-associated lifestyle of Ps. sp. SM9913.(2) The characterization of the swimming and swarming motilities of benthonic bacterium Ps. sp. SM9913The genome of Ps. sp. SM9913 was sequenced and analyzed by Dr. Qin Qilong previously. In the genome of Ps. sp. SM9913, a gene cluster which has 37 genes was supposed to be the lateral flagella gene cluster and responsible for the synthesis of lateral flagella. Two gene clusters which have 34 and 22 genes respectively were supposed to be responsible for the synthsis of polar flagella. To confirm the results of genomic analysis, we tested the motility of Ps. sp. SM9913 and result showed that it had both swimming and swarming motilites. The edge of the swarming colony was observed with an inverted microscope. Cells of Ps. sp. SM9913 were observed to be randomly arranged and organized in a monolayer in the leading edge of the swarming colony. Cells in the monolayer moved individually along the long axes of themselves, whereas cells outside the monolayer could not move. The swarming pattern of Ps. sp. SM9913 was different from that of Escherichia coli and Bacillus subtilis. Swimming and swarming cells of Ps. sp. SM9913 were observed with an atomic force microscope, and results showed that each swimming cell had a truncated polar flagellum and several lateral flagella were found in each swarming cell. These results confirmed the results of genome analysis and for the first time the swimming and swarming motilities of an oceanic bacterium was well characterized.(3) The genetic bases for the swimming and swarming motilities of benthonic bacterium Ps. sp. SM9913 To confirm the genetic bases for the swimming and swarming motilities of Ps. sp. SM9913, we disrupted these two flagella gene clusters with gene knockout technique. The mutant ΔLF was obtained by deleting genes PSM_A0914 and PSM_A0915 in the lateral flagella gene cluster and ΔLF lost its swarming motility. The complementation of genes PSM_A0914 and PSM_A0915 in ΔLF could rescue the swarming motility of ΔLF. The mutant APF was obtained by deleting genes PSM_A2283, PSM_A2282 and PSM_A2281 in the polar flagella gene cluster. The swimming motility of APF was defective and the complementation of genes PSM_A2283, PSM_A2282 and PSM_A2281 in APF could rescue the swimming motility of ΔPF to the level comparable to that of the wild Ps. sp. SM9913. The mutant APFΔLF was obtained by deleting genes PSM_A0914 and PSM_A0915 in the mutant ΔPF. APFALF lost both swimming and swarming motilities. The deleted genes PSM_A0914, PSM_A0915, PSM_A2283, PSM_A2282 and PSM_A2281 were complemented in ΔPFΔLF and result showed that swimming and swarming motilities of ΔPFΔLF were both rescued. These results confirmed that the lateral flagella gene cluster was the genetic basis of swarming motility and the polar flagella gene cluster was the genetic basis of swimming motility.(4) The comparative analysis of transcriptomes between swarming and swimming cells of benthonic bacterium Ps. sp. SM9913To further understand the the regulation of swarming and swimming motilities in Ps. sp. SM9913, we sequenced the transcriptomes of swarming and swimming cells and comparatively analyzed them. The analysis showed that 946 genes were up-regulated in swarming cells and only 76 genes were down-regulated in swarming cells. All genes belonging to the lateral flagella gene cluster were remarkably up-regulated in swarming cells. We also found three genes adjacent to the lateral flagella gene cluster were similarly up-regulated and the alignment of these three genes showed that they belonged to the lateral flagella gene cluster too.We validated the results of RNA sequencing using real time fluorescent quantitative PCR. And results showed that four selected genes in the lateral flagella gene cluster were all significantly up-regulated and three selected genes in the polar flagella gene cluster were all slightly down-regulated in swarming cells. These results indicated that the results of RNA sequencing were reliable.Comparison of transcriptomes between swarming and swimming cells showed that 18 of 21 genes encoding methyl-accepting chemotaxis proteins were up-regulated in swarming cells. The most up-regulated gene was PSM_A2360 and the homolog of protein PSM_A2360 in Escherichia coli was shown to participate in the signal transduction of swarming motility which suggested that similar signal transduction might exsist in Ps. sp. SM9913.In the swarming cells of Ps. sp. SM9913, several genes encoding proteins which can synthesize or degrade cyclic dimeric guanosine monophosphate (c-di-GMP) and intereact with c-di-GMP were up-regulated. Gene PSM_A0906 in the lateral flagella gene cluster encoding a protein which had only one PliZ domain and the PliZ domain had been demonstrated to be able to bind c-di-GMP. These results suggested that c-di-GMP might participate in the the regulation of motility of Ps. sp. SM9913.Three two components system encoded by 6 paired genes were found to be up-regulated in swarming cells, and these 6 proteins happened to have no orthologs in its close relative Ps. haloplanktis TAC125. Among them, the two components system composed of PSM_A0531 and PSM_A0532 might directly regulate the transcription regulation of the lateral flagella gene cluster. The two components system composed of PSM_A2131 and PSM_A2132 might participate in the regulation of motility by adjusting the concentration of c-di-GMP within cells. The two components system composed of PSM_B0075 and PSM_ B0076 might participate in the regulation of motility indirectly by quorum sensing.The results of transcriptomes analysis also showed that six siderophore receptor genes and five genes encoding proteins in siderophores transport system were up regulated. Correspondingly, genes of iron storage proteins and iron-containing proteins were down regulated. The concentration of iron ion in the medium we used was measured to be 4.64± 0.07μM. We found that additional FeCl3 with final concentrations ranging from 1μM to 10 μM repressed the swarming of Ps. sp. SM9913. And the addition of FeCl3 at a concentration≤ 10μM in the medium had no inhibitory effect on the growth of Ps. sp. SM9913. These results indicated that the iron limitation in environment might induce the swarming of Ps. sp. SM9913.The comparative analysis of transcriptomes between swarming and swimming cells complemented the study of genomics and genetics and primarily revealed the physiological regulation mechanism between swarming and swimming motilities of Ps. sp. SM9913 which was the basis of further study on the regulation mechanism of motilities.(5) The role of exopolysaccharide in the swarming of Ps. sp. SM9913The slime layer is important to the swarming of some bacteria and exopolysaccharide is one of the major components of slime layer. We studied the role of exopolysaccharide in the swarming of Ps. sp. SM9913 using the mutant AepsT which had defect in the synthesis of exopolysaccharide. Swarming assay showed that the swarming motility of AepsT was weaker than that of wild Ps. sp. SM9913, which suggested that the lack of exopolysaccharide impaired the swarming of Ps. sp. SM9913.The expansions of the swarming colonies of wild Ps. sp. SM9913 and AepsT were studied quantitatively. Results showed that the colony expansion rate of wild Ps. sp. SM9913 increased from nearly zero to the maximum and then decreased back to nearly zero, while the colony expansion rate of AepsT was the maximum at the start and dereased to nearly zero as time went on. The swarming of Ps. sp. SM9913 had a lag phase and was population dependent while the swarming of AepsT did not have a lag phase and was population independent.The addition of surfactin on swarming plates could rescue the defect of AepsT, but the tendency of the swarming colony expansion was significantly different from that of the wild Ps. sp. SM9913. The addition of exopolysaccharide secreted by Ps. sp. SM9913 could partially rescue the defect of AepsT.These results suggested that exopolysaccharide participated in the regulation of swarming motility of Ps. sp. SM9913, which could help us understand the role of exopolysaccharide in the lifestyles of Ps. sp. SM9913.(6) Simulation study on the motile lifestyles of benthonic bacterium Ps. sp. SM9913By simulating the deep-sea sediment environment, the motilities of Ps. sp. SM9913 and its mutants in clay were measured and compared with that of planktonic bacterium Ps. haloplanktis TAC125. We compared the motilities of Ps. sp. SM9913 and Ps. haloplanktis TAC125 in clay. And results showed that Ps. sp. SM9913 had good motility in clay while the motility of Ps. haloplanktis TAC125 in clay was not observed. The motilities of flagella mutants ΔPF, ΔLF and ΔPFΔLF in clay were remarkably lower than that of the wild Ps. sp. SM9913 and motility of ΔPFΔLF was the lowest. These results suggested that Ps. sp. SM9913 might have swimming and swarming motilities in the deep-sea sediment environment, and polar and lateral flagella powered the movement in sediment. Futher genomic analysis found that having two flagella systems is prevalent among bacteria isolated from deep-sea sediment.(7) The proposal of a model describing the lifestyles of benthonic bacterium Ps. sp. SM9913A model describing the lifestyles of Ps. sp. SM9913 was proposed based on our results. In deep-sea sediment, Ps. sp. SM9913 could cluster around the substrate hot spots by swimming motility, and attach to the hot spots or surrounding clay particles with the help of exopolysaccharide. In the particle-associated lifestyle, some essential mutrients may become depleted due to the growth and propagation of bacterial cells, so Ps. sp. SM9913 may synthesis lateral flagella and disperse from the sessile lifestyle by swarming. The dispersed cells may shift to swimming lifestyle under appropriate conditions and restart a particle-associated lifestyle. For the first time a model describing the possible lifestyles of a deep-sea sediment bacterium in its in-situ habitat was proposed and provided an intuitive reference for further study.