Quorum Interference In Bacteria And Its Effect On Physiological Function

Many bacterial species use small molecules named autoinducer (AI) as signals to coordinate their activities. This type of communication has been termed as "quorum sensing". Lots of relevant researches have been going on, but most bacteria still have little characterized genetic backgrounds of this system. In this study, we developed a rapid method (quorum interference) about evaluate functions of quorum-sensing by introducing two plasmids into the target strains, examining changes of AI accumulation level and influence on physiological functions. One will overexpress the Agrobacteriu quorum-sensing regulator TraR to competitively binding AHL of target strains, the other will inactivate AHL by AHL-degrading enzyme AttM.The symbiotic relationships of the nitrogenfixing rhizobia and their legume hosts are the results of an intricate signaling network between the host and symbionts. The lux-type quorum sensing has been linked to various phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important to the establishment of a successful symbiosis. However, there still have no detailed studies on quorum-sensing regulatory systems in Mesorhizobium genus, a moderately growing rhizobium. On the other hand, the gastrointestinal tract is the site of the largest and most complex environment which harbors large amount of Enterobacteriaceae, many of them are opportunistic pathogen. For bacterial pathogens, quorum-sensing has been proposed to aid the disease process by controlling many behaviors such as biofilm formation, virulence factors production and infection. Although researches on quorum-sensing of bacterial pathogens are popular now, in the Enterobacteriaceae, however, only a few AHL-based quorum-sensing systems have been reported. So we went on works on these strains, including eight type strains of the genus Mesorhizobium and 53 strains belonging to the Enterobacteriaceae which were isolated from blood samples of patients with high fever admitted to the Sir Run Run Shaw Hospital in Hanzhou, China.Bioassay and TLC detections showed AHL activities in genus Mesorhizobium when cultured in TY medium, and the AHL profiles were quite different. M. tianshanense CCBAU 3306, M. chaoconense LMG19008 and M. mediterraneum USDA3392 produced at least five different AHL molecules while M. loti NZP 2213 and M. ciceri USDA3378 showed low level in this condition.TraR, the Agrobacteriu quorum-sensing regulator, was overpressed in M. huakuii and tested to determine whether TraR interferes with the M. huakuii! s endogenous quorum systems. The results indicate that TraR proteins can recognize the AHL signals of M. huakuii, which are distinctly different from the TraR cognate AHL 3-oxo-C8-HSL, and actively regulate traI expression in M. huakuii. However, the unknown M. huakuii quorum-sensing regulators failed to activate the A. tumefaciens quorum-sensing target gene traI, this may result from either low level of regulatory gene expression or barrier of the regulators to recognize the traI promoter region. The two systems are interchangeable to a certain extent. The same results were showed in M.tianshanense. Competition is common in quorum-sensing systems, regulators can combine various AI signals in different efficiency. In this system, TraR proteins could trap intracellularly the AI produced by Mesorhizobium synthase(s) but failed to active the expression of target gene. Sequestering AI by overproduced TraR proteins will disrupt regulation of Mesorhizobium quorum-sensing, because there have not enough quorum signals for its own quorum-sensing regulators, especially in those strains of low basic AI production level.AttM, an AHL-lactonase from A. tumefaciens, can inactivate AHL signals by hydrolyzing the lactone ring to produce acyl homoserine. A plasmid that constitutively expresses attM was introduced into M. huakuii, but failed to reduce its AI activity, while the same construct significantly reduced AI production in A. tumefaciens and M. tianshanense. It suggested that autoinducers of M. huakuii were not popular AHL signals. Reference other results, it is possible that autoinducers of M. tianshanense are AHLs, but M. huakuii's are cyclic dipeptides which are infrequent in Rhizobium.TraR overexpression also blocked some physiological phenomena in M. huakuii, including biofilm formation and root-hair deformation inducement, but had only a little influence on root hair attachment and nodulation formation. The possible reason is the overexpression of TraR just inhibit the target gene expression of M. huakuii temporarily. The rest AI which were not trapped by TraR protein will accumulate to the proper concentration finally alone with the increasing of cell density, and all delayed functions will be recoverd then. The other study on the M. tianshanense mutant of AI degradation by AttM showed a 65% reduction in the efficiency of root hair adherence, suggesting that quorum-sensing positively regulates this phenomenon. These data provide strong evidence that quorum sensing plays a critical role in the Mesorhizobium symbiotic process.In the study of Enterobacteriaceae, 53 strains were cultured under different nutrition and oxygen concentration growth conditions. At last, supernatants from nine strains displayed various AHL activities under at least one of the growth conditions, most of them have not been reported before. Among these, C. freundii SRRSH27, E. cloacae SRRSH59 and S. marcescens SRRSH68 produced significant amounts of AHL under all four tested conditions. E. aerogenes SRRSH2, K. pneumoniae SRRSH17, K. pneumoniae SRRSH20 and K. oxytoca SRRSH73 produced high AHL activities only when grown microaerophilically in LB medium. S. marcescens SRRSH67 produced more AHL in rich media, whilst strain S. marcescens SRRSH69 preferred aerobic growth conditions for AHL production. TLC analyses showed most of the strains produced more than one type of AHLs, whilst retaining production of the same AHLs, albeit at different levels, under different growth conditions. One exception was seen in S. marcescens SRRSH68, where it produced an extra AHL when grown in LB aerobically. The results indicated that the expression of AHL synthases in these strains may have been affected by the growth conditions.Usually, it is hard to study so many strains without more genetic backgrounds of quorum sensing by introducing plasmids or screening AI synthase at one time. In this study, we tried a simple method to evaluate the basic information of their quorum-sensing systems. DH5a(pYC146) contains the plasmid which can overexpress AttM. AI activity of Cell-free supernatants was assayed after mixing cultures of pathogen and DH5a(pYC146) overnight to classify its AI type roughly. The activity all reduced in different degree so that we consider AHLs type autoinducer were contained in them. The worse degrade effect may result from multiple AI structure that AttM can not work on (such as cyclic dipeptides), or some long-chain AHLs which affect specificity of enzyme to substrate, but this method is worth referencing.During the degrading course, the biofilm structure of S. marcescens SRRSH45 was disrupted at the same time, it seemed that quorum-sensing can positive regulate its biofilm formation. Furthermore, it proved that this testing method is effective.Another AI-deficient mutant S. marcescens SRRSH67-1 was more motile than the wild-type on 0.4% agar plates, suggesting quorum sensing may negatively regulate motility in this clinical isolate.