| Members of the Pseudoalteromonas genus are typical marine bacteria. So far, all of the reported species of the Pseudoalteromonas genus are isolated from the marine environment, including deep sea and polar zones. Many studies suggest that Pseudoalteromonas strains have developed multiple adaptation mechanisms for the extreme environments, which make these strains widely distribute in various marine environment. Pseudoalteromonas strains are important models for the investigation of the mechanisms by which marine microorganisms are adapted to the marine environments. A further study of the environmental adaptation mechanisms of Pseudoalteromonas will be helpful to our understanding of how microorganisms are adapted to and evolve in the marine environments. Many Pseudoalteromonas strains can secrete bioactive substances and extracellular enzymes and thus may have potential applications in industry. In the thesis, Pseudoalteromonas strains from Arctic sea ice were studied to gain insights into their environment adaptation mechanism. Firstly, a filamentous phage was isolated from Arctic sea ice Pseudoalteromonas and its effects on the growth, stress tolerance and motility of the host were studied and a filamentous phage-mediated mechanism for the survival of Pseudoalteromonas in sea ice was proposed. Next, a conjugal transfer system with high efficiency was constructed in Ps. sp. SM9913and based on this conjugal transfer system, a gene knockout system was constructed in Ps. sp. SM9913. Lastly, a conjugal transfer system for Arctic sea ice Pseudoalteromonas sp. SM20429was constructed and a cold-adapted expression system was developed using Ps. sp. SM20429as the host. With this expression system, a cold-adapted metalloprotease that cannot be maturely expressed in Escherichia coli expression system was successfully expressed and purified using the cold-adapted expression system.Ⅰ. Environmental adaptation mechanisms of Pseudoalteromonas mediated by filamentous phage in sea icePseudoalteromonas is one of the predominant culturable bacterial groups within the Arctic sea ice ecosystem. Many studies show that the host mortality and adjusting of microbial community structure in polar areas are regulated by lytic viruses. However, the ecological role of filamentous phages that cannot split the host in sea ice is still unknown. In the dissertation, sequence analysis of plasmid pSM327, a plasmid previously isolated from Arctic sea ice strain Pseudoalteromonas sp. BSi20327, indicated that, pSM327may be the replicative form (RF) of a filamentous phage. In the following study, a filamentous phage was isolated from Ps. sp BSi20327culture. The basic characteristatics of the filamentous phage and their distribution in the Arctic sea-ice Pseudoalteromonas were studied. Furthermore, the ecological role of this filamentous phage was revealed.(1) Discovery of a filamentous phage from Arctic sea ice Pseudoalteromonas and its basic characteristics and distributionA filamentous phage named f327was extracted from the culture of Ps. sp. BSi20327and observed by electron microscope. f327exhibits a filament-like structure, with14nm in width and approximately1.5μm in length, agreeing well with the morphological characteristics shared by filamentous phages. The genomic type of O27is single-stranded DNA (ssDNA) and its genome contains6114nucleotides. Genome of f327is composed of9open reading frames (ORFs). Sequence analysis reveals that each pSM327ORF shows the greatest homology to the ORFs at the corresponding positions in the well-characterized filamentous phages. The genes of f327can be classified into4functional modules, replication, structure, assembly and regulation. The distribution of this filamentous phage in Arctic sea ice Pseudoalteromonas strains was investigated by PCR amplification. The results indicated that36%of the Pseudoalteromonas strains (19/53) contain f327or f327-like genes. Phylogenetic tree based on the rpoD gene and DNA-DNA hybridization analysis indicated that all these phage-containing strains belong to the same species of Pseudoalteromonas.(2) Ecological role of the filamentous phage and the molecular mechanism of f327affecting the hostTo investigate the role of f327in the sea ice ecosystem, an RF327-cured derivative of Ps. sp. BSi20327was obtained and named Ps. sp. BSi20327A. No filamentous phage ssDNA was detected in Ps. sp. BSi20327A and comparative transcriptomic analysis showed that the transcription level of RF327in Ps. sp. BSi20327A is3%-7%of those in Ps. sp. BSi20327. Comparison of Ps. sp. BSi20327and Ps. sp. BSi20327A showed that f327can reduce the host growth rate, lower the cell density of the host community, impair the host tolerance against NaCl and H2O2and increase the motility of the host.To determine the mechanism underlying how f327affects its host BSi20327, the whole transcriptomes of BSi20327and BSi20327A grown in different concentrations of NaCl were sequenced and compared. The results showed that, at3%NaCl, only genes encoded by phage f327and related to phage shock, flagellar assembly and bacterial chemotaxis were up-regulated in Ps. sp. BSi20327. As the processes of phage and flagella assembly are energy-intensive, the reduction in the growth rate of Ps. sp. BSi20327in3%NaCl may be mainly due to these two energy-intensive processes. At8%NaCl, in addition to those up-regulated at3%NaCl, genes encoding key enzymes in the TCA cycle are down-regulated in Ps. sp. BSi20327, which would result in a reduction in energy production. Genes related to transcription elongation factor and ribosome assembly are also down-regulated in Ps. sp. BSi20327, which likely decreases the rate of gene transcription and translation. Therefore, at8%NaCl, over-expression of f327in Ps. sp. BSi20327not only consumes energy but also affects energy production and gene transcription and translation of the host, which will severely reduce the host growth rate. Bacteria usually respond to hyperosmolarity by accumulating compatible solutes, such as some amino acids (glutamate and proline) and sugars, by absorbing or synthesizing them. Based on transcriptome analysis, genes involved in amino acid transport and glutamate and proline synthesis, are up-regulated in both Ps. sp. BSi20327and Ps. sp. BSi20327A in3-8%NaCl. However, these genes are up-regulated to a greater extent in Ps. sp. BSi20327compared to Ps. sp. BSi20327A, especially at8%NaCl. Because these genes are related to the accumulation of compatible solutes to cope with the hyperosmotic environment, this result suggests that8%NaCl is more stressful to Ps. sp. BSi20327than that to Ps. sp. BSi20327A. Therefore, Ps. sp. BSi20327needs to accumulate more compatible solutes to resist the hyperosmolarity. Due to existence of O27, genes related to flagella assembly and bacterial chemotaxis are up-regulated in Ps. sp. BSi20327, which enhance the motility of the host.At last, the model of how the filamentous phage regulates the host community to improve the survival of the host in Arctic sea ice was proposed. In winter, the bacteria in sea ice are confronted with high salinity and nutrient deficiency due to the low temperatures and long polar nights. The reduction in the growth rate and NaCl tolerance of Ps. sp. BSi20327caused by f327would diminish the community scale of Ps. sp. BSi20327and thereby reduce nutrient consumption; in the meantime, motility enhancement caused by f327can help Ps. sp. BSi20327access environments suitable for growth. These regulatory effects of phage f327on the host cells are helpful for the survival of the host community during the long nutrient-deficient polar night winter days. In summer, although the salinity in the sea ice decreases as temperature increases, the sea ice bacteria are faced with rich nutrients and a high concentration of H2O2due to the polar day and intense sunlight. Owing to the presence of f327, the H2O2tolerance of Ps. sp. BSi20327decreases, which would lead to a high mortality rate of Ps. sp. BSi20327due to the high concentration of H2O2. In addition, the presence of f327also reduces the cell density of the Ps. sp. BSi20327community. These mechanisms may help avoid the over-blooming of BSi20327during the months of round-the-clock sunlight and the nutrient-rich summer.II. Construction of the gene knockout system in Pseudoalteromonas sp. SM9913Pseudoalteromonas sp. SM9913, isolated from the deep-sea sediment at1855m, is a psychrophilic strain. The genome of Ps. sp. SM9913has been sequenced and some specific features of how Ps. sp. SM9913adapts to the deep-sea environment have been revealed, which are different from the Pseudoalteromonas living in other environments. However, due to the lack of a genetic system in Ps. sp. SM9913, it is difficult to verify the predicted features by experiments. Therefore, it is necessary to develop a genetic system for the reverse genetics in Ps. sp. SM9913to investigate the genetic mechanism of how the bacterium adapts to the deep-sea sedimentary environment. Although an electroporation genetic transformation system was constructed in Ps. sp. SM9913in our previous work, due to its very low transformation efficiency, the downstream genetic manipulation cannot be performed. In the dissertation, a conjugal transfer system with high efficiency was constructed. Based on this conjugal transfer system, a gene knockout system was constructed.(1) Construction of the conjugal transfer systemThe antibiotic sensitivity of Pseudoalteromonas sp. SM9913was characterized and the erythromycin resistance gene was chosen as the selection marker. In the previous work, pSM429(a plasmid from the sea-ice bacterium Pseudoalteromonas sp. BSi20429) was screened and its functional region was studied. The minimal region for replication of pSM429, the conjugative transfer initiation origin and erythromycin resistance gene were introduced into the bone vector pGEM-T easy vector, respectively. The resultant shuttle plasmid, pOriT-4EM, can replicate in both Pseudoalteromonas and Escherichia coli. Afterwards, using E. coli ET12567(pUZ8002) as the donor, pOriT-4EM was transferred into Ps. sp. SM9913by conjugation. A conjugal transfer system with high efficiency (1.8x10-3) was constructed after optimizing the mating conditions, which lays the foundation of genetic manipulation in this strain.(2) Construction of the gene knockout system in Ps. sp. SM9913The epsT gene which encodes the UDP-glucose lipid carrier transferase was selected as the target gene for gene inactivation by in-frame deletion. Two homologous fragments of about800bp were selected from the flanking sequences of epsT gene. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and levansucrase (sacB) gene was the counter-selection marker. The epsT gene was in-frame deleted with a two-step integration-segregation strategy after transferring the suicide vector pMT into Ps. sp. SM9913. The obtained△epsT mutant showed approximately73%decrease in the yield of exopolysaccharides. No selection marker was introduced in the chromosome, which makes it possible to knockout several genes in the same host. Thus, a system for gene knockout in Ps. sp. SM9913was successfully constructed, which is helpful for further investigating the molecular mechanism of Pseudoalteromonas adapted to the deep-sea environment.III. Construction of the cold-adapted heterologous expression system for PseudoalteromonasAlong with the discovery and study of cold adapted microbes, the cold-adapted enzymes secreted by them have drawn much attention due to their great values in fundamental research and potentials in application. However, expression of genes of cold-adapted enzymes in the mostly used mesophilic E. colt host often results in the enzymatically inactive inclusion bodies. The reason for the inactivity is misfolding of the polypeptide chains when they are expressed in a common E. coli strain at37℃. Although lowering the induction temperature may improve the expression of some cold-adapted proteins, the expression time has to be prolonged at low temperatures. Therefore, development of the expression system which uses cold-adapted microbe as the host will be helpful for both the fundamental research and industrial applications of the cold-adapted enzymes. In our previous work, an expression system was preliminarily developed using Ps. sp. SM20429(a plasmid-cured strain of Ps. sp. BSi20429) as the host, but proteins must be expressed at25-30℃in that system. In the dissertation, strong promoter of Ps. sp. BSi20429was screened by construction of the reporter plasmid, and expression vector was constructed. Three cold-adapted enzymes were successfully expressed in Ps. sp. BSi20429at low temperatures.(1) Construction of the conjugal transfer system and screening of the strong promoter at low temperatures by construction of reporter plasmidsThe erythromycin resistance gene in the shuttle vector pOriT-4EM was replaced by the chloromycetin resistance gene and the resultant plasmid was named as pOriT-4CM. Based on the Ps. sp. SM9913conjugal transfer system, the Ps. sp. SM20429transfer system was constructed using Ps. sp. SM20429as the recipient strain and an efficiency of4x10-3was obtained. A series of reporter plasmids were constructed using the promoter of the xylanase gene, cold shock protein gene (CspA) and heat shock protein gene (dnaK) from Ps. sp. BSi20429and the erythromycin resistance gene and the gene encoding the catalytic domain and the signal peptide of the cold-adapted cellulase Cel308from the Arctic sea ice strain Ps. sp. BSw20308as the reporter gene. The reporter plasmids were transferred into Ps. sp. SM20429by conjugation. The erythromycin resistance and cellulase activity were tested to compare the strength of the promoters. The results showed that the erythromycin resistance gene was expressed under the control of the promoter of the xylanase gene and CspA gene. The cellulase was also expressed under the control of these two promoters at5-15℃, but the promoter of the xylanase gene is stronger than the CspA promoter at10-15℃. Therefore, the promoter of the xylanase gene was chosen for construction of the protein expression system in SM20429at low temperatures. (2) Construction of a cold-adapted expression system and recombinant expression of cold-adapted enzymesPseudoalterin, a novel M23A subfamily protease secreted by Psendoalteromonas sp. CF6-2from deep-sea sediment, is a cold-adapted enzyme with low optimal temperature (25℃) and high thermolability. However, because it cannot mature by autoprocess, pseudoalterin is unable to be expressed as a mature form in E. coli, which severely impedes the further study of its structure and function.A DNA fragment containing the promoter of the xylanase gene, multiple cloning sites and6repeated CAC (for His tag) was synthesized and introduced into the bone vector pOriT-4CM to construct the expression vector, pEV. Pseudoalterin gene was cloned into pEV to construct the expression plasmid for pseudoalterin. After transferring the expression plasmid into Ps. sp. SM20429, pseudoalterin was expressed using spelt xylan as the inducer. By optimizing the inducer concentration and induction temperature, active pseudoalterin was produced and purified from the culture by Ni affinity chromatography. N-terminal sequencing indicated that the recombinant pseudoalterin has the same N-terminal sequence as the native enzyme purified from the culture of the wild strain. The yield of the recombinant pseudoalterin was16-20U/ml with the specific activity of252+13U/mg. To detect the applicability of the expression system, the enhanced green fluorescent protein (EGFP) and another two cold-adapted proteins from Pseudoalteromonas sp. SM9913(UDP-N-acetylglucosamine2-epimerase and UDP-N-acetyl-mannosamine dehydrogenase) were expressed and purified. The development of this expression system is helpful for the further study of pseudoalterin and can be used for the expression of other cold-adapted proteins that are intractable for the E. coli expression system. |
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