| Owing to the complex and special living circumstance they reside, marine microorganisms usually coexist in the marine environment, and they gradually form a set of chemical defense system that is beneficial to the survival of themselves or the host. Most of the chemical defense substances they produce have biological activity, so more and more scientists considered that chemical defense mechanism is the main inducing factor for marine microorganisms to produce bioactive substances. However, there are few people studying about how chemical defense start by marine microorganisms and what is starting mechanism. Quorum sensing phenomenon has been gradually revealed with the development of microbiology, and some scholars proposed the hypothesis that chemical defense mechanism in microorganism, especially bacteria, is regulated by quorum sensing.Quorum sensing is a metabolic regulation and control process which enables the bacterial cells to sense changes in cell density and induce the expression of specific genes. This regulatory process is mediated through sensing the concentration changes of specific signal molecules being released either by bacteria themselves, or from external sources. Density-dependent is one of the most important characteristics of quorum sensing, that is, when the bacteria grow to a high cell density, the concentration of autoinducers (AI) they release could reach a critical threshold value. The bacteria could then sense the autoinducers and appear some specific physiological characters, including bioluminescence, the production of antibiotics, virulence genes expression, biofilm formation and so on. In this thesis, we have to solve the problem whether the production of chemical defense substances is regulated by quorum sensing in marine bacteria.Based on natural product chemistry and chemical ecology approach, a sponge-associated bacterium Pseudoalteromonas sp. NJ6-3-1 with remarkable biological activities and with the phenomenon of quorum sensing was chosen as case study, and the production of antibacterial substances was taken as a chemical defense response in this dissertation. On the one hand, the chemical defense substances produced by bacteria NJ6-3-1 was identified, and the production of antibacterial substances of bacteria NJ6-3-1 with density-dependence was confirmed, namely the production of antibacterial substances of bacteria NJ6-3-1 was regulated by quorum sensing. These results demonstrated sponge-associated bacteria NJ6-3-1 have chemical defense regulated by quorum sensing mechanism. On the other hand, quorum sensing signal molecules N-acyl homoserine lactones (AHLs) and diketopiperazines (DKPs), which are commonly considered to be quorum sensing signal molecules, were proved to exist in sponge-associated bacteria NJ6-3-1, and the autoinducer that regulated the metabolite of antibacterial substance in bacteria NJ6-3-1 was explored. Finally, the luxR gene fragments of quorum sensing transcriptional regulatory protein LuxR was obtained from whole genome of bacteria NJ6-3-1, thus we preliminary demonstrated that LuxI/LuxR quorum sensing regulatory system exist in bacteria NJ6-3-1 from the molecule level. This study will enrich the related theory of chemical defense induction mechanism of marine bacteria, deepen the understanding of bacteria regulation functions of quorum sensing, and provide a new way for the development of new medicinal natural products. This dissertation consists of six chapters as follow:Chapter one consists of two parts. In the first part, a brief introduction of sponge-associated microorganisms is given, including their types, secondary metabolites, and chemical defense mechanisms. In the second part, a brief introduction of bacterial quorum sensing system is summarized, and the topics discussed include the history of quorum sensing, classification of quorum sensing system and signal molecules, the formation and regulation mechanism of quorum sensing, the detection method of signal molecule AHLs, and the phenomenon of quorum sensing in marine bacteria. The research objective and experimental plan of this dissertation are also presented.In chapter two, the screening and identification of sponge-associated bacteria with antibacterial activity are discussed. At first, sponge-associated bacteria were isolated by dilution-plate method from marine organism sponge of South China Sea and East China Sea, and the antibacterial activity were screened for four pathogens as test strains by an agar diffusion method. At the same time, taxonomy was studied based on phylogenetic analysis of 16S rDNA sequences. The results showed that 19 strains (20 %) among total 95 isolated marine bacteria have antimicrobial activity. And the most active sponge-associated bacteria were identified to be the genus Bacillus.In chapter three, the antibacterial substances of sponge-associated bacteria NJ6-3-1 with cell density dependence is discussed. SectionΙandΠ, the antibacterial metabolites were isolated and purified from sponge-associated bacteria NJ6-3-1 with remarkable antibacterial activity. And combined with the chemical method and the biotechnology, two antibacterial substances in bacteria NJ6-3-1 was ultimately obtained by active tracking, the structure of two compounds was determined by many structure identification methods. The formula of the first antibacterial substance was C15H13BrO3, whose molecular weight was 320, and structure was named as 2-(1'-bromine-1',3'-butadiene)-4-(2'',4''-dienevaleric acid)-phenol. It has different inhibition effect to Bacillus subtilis, Staphylococcus aureus and Saccharomyces cerevisiae, minimum inhibitory concentration (MIC) in turn was 500μg/mL, 125μg/mL and 250μg/mL. The molecular weight of the second antibacterial substance was 520, whose interim formula was C23H24BrN2SO5. It has inhibition effect to Staphylococcus aureus, MIC was 500μg/mL. SectionШ, the content of antibacterial substances C23H24BrN2SO5 of bacteria NJ6-3-1 depends on cell density was determined by simultaneous detection of the cell density of bacteria NJ6-3-1 under different culture times and the content of antibacterial substance C23H24BrN2SO5 in metabolite crude extracts of bacteria NJ6-3-1. The metabolism of antibacterial substance C23H24BrN2SO5 of bacteria NJ6-3-1 with cell density-dependence was further proved by detecting antibacterial activity of metabolite crude extracts of bacteria NJ6-3-1 under different culture times, and cell density threshold value is OD630=0.4. In chapter four, the AHLs in the metabolites of sponge-associated bacteria NJ6-3-1 is discussed. SectionΙ, the detection method of quorum sensing signal molecule AHLs in bacteria was established, and the production of AHLs in sponge-associated bacteria NJ6-3-1 was detected combined with these methods. The results showed that signal molecule AHLs which can induce biosensor strain A. tumefaciens A136 to produce blue pigment exist in bacteria NJ6-3-1, and bacteria NJ6-3-1 contains three AHLs, N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-tetradecanoyl-L-homoserine lactone (C14-HSL) by Gas Chromatography-Mass Spectrometry (GC-MS) detection. It demonstrated that this method could be applied to screen AHLs-producing bacteria from large quantities of bacteria. SectionΠ, C6-HSL, C8-HSL and C14-HSL, as quorum sensing signal molecules and the metabolites of bacteria NJ6-3-1, was added respectively in the culture of bacteria NJ6-3-1 at low cell density, and the metabolite crude extracts of bacteria NJ6-3-1 were obtained after adding different inducers. Through analyzing antibacterial activity by bioautography and the content of antibacterial substance C23H24BrN2SO5 by High Performance Liquid Chromatography (HPLC), the results showed C8-HSL can induce low-density bacteria NJ6-3-1 to produce antibacterial activity, and can induce the metabolite content of antibacterial substance C23H24BrN2SO5 of low-density bacteria NJ6-3-1 to increase, thus we concluded that C8-HSL is very likely a quorum sensing autoinducer which are produced by bacteria NJ6-3-1 to regulate the metabolite of antibacterial substance.In chapter five, the DKPs in the metabolites of sponge-associated bacteria NJ6-3-1 is discussed. At first, cyclo-(ΔVal-L-Val), cyclo-(L-Phe-L-Val) and cyclo-(L-Pro-L-Leu), as quorum sensing signal molecule and the metabolites of bacteria NJ6-3-1, was added respectively in the culture of bacteria NJ6-3-1 at low cell density, and the metabolite crude extract of bacteria NJ6-3-1 were obtained after adding different inducers. Through detecting of antibacterial activity by bioautography, the results showed cyclo-(L-Phe-L-Val) can induce low-density bacteria NJ6-3-1 to produce antibacterial activity, so it is probably that cyclo-(L-Phe-L-Val) is a quorum sensing autoinducer which are produced by bacteria NJ6-3-1 to regulate the metabolite of antibacterial substance. Secondly, 100 L broth of bacteria NJ6-3-1 were fermented in order to get more DKPs compounds from the bacteria NJ6-3-1, and twelve DKPs were isolated and purified from the metabolites of bacteria NJ6-3-1. In addition, the structure of twelve DKPs was identified by means of physical and chemical properties and spectroscopy. Furthermore, one DKPs compound, namely cyclo-(Tyr-Phe) has not yet been reported to be found in the microbial.In chapter six, the molecular basis of quorum sensing in sponge-associated bacteria NJ6-3-1 is discussed. The luxR gene fragment was sequenced. The luxR gene fragment of quorum sensing transtritional regulatory protein was found to exist in bacteria NJ6-3-1 by comparison whole genome of bacteria NJ6-3-1 (Ningbo University), thus the LuxI/LuxR quorum sensing regulatory system was initially confirmed to exist in bacteria NJ6-3-1 from genetic aspect.
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