Two of the Mechanims Used by Bacteria to Modify the Environment: Quorum Sensing and ACC Deaminase

Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. QS has been shown to be involved in many aspects of bacterial life, including virulence, bioluminescence, symbiosis, antibiotic production, swarming and swimming motility, biofilm formation, conjugation and growth inhibition. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, in the first part of this thesis, isolation and characterization of new QS systems from metagenomic libraries constructed using DNA from activated sludge and soil were described. Using an Agrobacterium biosensor strain, three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homolog proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxIQS6-1 directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxIQS10-1 directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxIQS10-2 directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxIQS6-1. Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. Its ability to transfer and integrate foreign DNA into plant genome also makes it a useful tool for plant genetic engineering. Ethylene, the gaseous plant hormone, has been reported to be important for both crown gall development and A. tumefaciens mediated transformation efficiency to plants. ACC deaminase, an enzyme that can break down ACC, the direct precursor of ethylene biosynthesis in plants, is a mechanism used by some plant growth promoting bacteria (PGPB) to promote plant growth by reducing stress ethylene levels. In the second part of this thesis, the effect of ACC deaminase on A. tumefaciens induced crown gall development and on A. tumefaciens mediated transformation efficiency was studied. By either introduction of an ACC deaminase encoding gene into the virulent strain A. tumefaciens C58 or co-inoculation of A. tumefaciens C58 with an ACC deaminase containing PGPB P. putida UW4, using different plant systems including tomato plants and castor bean plants, it was found that the presence of an ACC deaminase significantly inhibited crown gall development. It was also found that introduction of an acdS gene into the disarmed A. tumefaciens strain GV3101::pMP90 reduced the ethylene levels evolved by plants during infection and cocultivation process and increased the transformation efficiency of commercialized canola cultivars. The A. tumefaciens D3 strain was reported to contain an ACC deaminase encoding gene (acdS). In this study it was determined that this strain is an avirulent strain and shows plant growth promoting activity. When co-inoculated with A. tumefaciens C58 on castor bean stems, both the wild type and the acdS knockout mutant showed biocontrol activity and were able to significantly inhibit crown gall formation, with the wild type strain showing slightly better tumor inhibition effects. The mutation of acdS and its regulatory gene lrpL in A. tumefaciens D3 was also found to affect QS signal production of this strain, which indicates a cross talk between the two sets of genes

Genetic and Functional Diversity of Pseudomonas aeruginosa Lipopolysaccharide

Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium–host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed of three domains, namely, lipid A, core oligosaccharide, and the distal O antigen (O-Ag). Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band). Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa. The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa. The underlying factors contributing to this diversity will be thoroughly discussed and presented in the context of its contributions to host–pathogen interactions and the control/prevention of infection

Co-evolution with Staphylococcus aureus leads to lipopolysaccharide alterations in Pseudomonas aeruginosa

Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureus prevails in childhood, whereas Pseudomonas aeruginosa progressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosa adapts to S. aureus by evolving P. aeruginosa in the presence and absence of S. aureus. P. aeruginosa populations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wsp mutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureus and conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wsp and LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosa LPS mutations are a direct consequence of S. aureus imposed selection in vitro

Sorry Pope, It Hasn't Worked

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Additional file 11: Figure S3. of Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa

Homologues of P.aeruginosa pilV2 sequence and pilV adhesins of plasmid R64 of Salmonella enterica serovar Typhimurium LT2 (Multiple Sequence Alignment, Clustal Omega software, EMBL-EBI). (TIF 2355 kb

Additional file 6: Figure S2. of Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa

Role of pslB gene in PAPI-1 transfer. Results were shown as mean ± SD for three independent replicates. Statistical significance was analyzed by One-way ANOVA compared to the positive control (ns: no significant difference; and *** p < 0.001). (TIF 217 kb