Interspecific interactions of heterotrophic bacteria during chitin degradation

Abstract

In their natural habitats, bacteria live in multi-species microbial communities and are, thus, constantly interacting with bacteria of other phylogenetic groups. In order to prevail in these interspecific interactions, such as the competition for nutrients, bacteria have developed numerous strategies. During the degradation of polymers such interspecific interactions are likely to occur, because degradation starts as an extracellular process. In one possible interaction scenario, investor bacteria, which invest energy in the production of extracellular enzymes, face the danger of being exploited by opportunistic bacteria that compete for degradation products. To investigate such a scenario and to characterize the strategies employed by the bacteria involved, we established two co-culture model-systems consisting of bacteria that co-exist in aquatic environments and with the polymer chitin as carbon, nitrogen, and energy source. Aeromonas hydrophila strain AH-1N, which releases extracellular chitinases, was employed as investor bacterium in both co-cultures.In the first co-culture, chitin embedded in agarose served as substrate. Flavobacterium sp. strain 4D9, which cannot degrade embedded chitin due to its cell-associated chitinases, was employed as opportunistic bacterium. The strategies applied by strain 4D9 in order to acquire nutrients included active integration into the biofilm formed by strain AH-1N on the chitin beads and interception of the chitin monomer N-acetylglucosamine (GlcNAc), leading to overgrowth of strain AH-1N by strain 4D9 in the biofilm.In the second co-culture, suspended chitin served as substrate. Pseudomonas aeruginosa strain PAO1, which is unable to degrade chitin, was employed as opportunistic bacterium. In the first phase of the co-culture, strain PAO1 grew with ammonium, acetate, and possibly GlcNAc and other compounds, which were released by strain AH-1N. In the second phase, strain PAO1 produced quorum sensing (QS)-controlled secondary metabolites, among them the redox active pigment pyocyanin. Pyocyanin inhibited the enzyme aconitase of strain AH-1N through the production of reactive oxygen species causing a block of the citric acid cycle. This led to a massive acetate release by strain AH-1N, which supported substantial growth of strain PAO1. Strain AH-1N was finally inactivated by pyocyanin and presumably other secondary metabolites. Further investigation of this parasitic growth strategy of strain PAO1 revealed that, while catabolite repression of GlcNAc metabolism by acetate did not play a role, the action of isocitrate lyase was a key metabolic requirement for the transition into the second phase. Besides its role in acetate utilization, this enzyme was crucial for the utilization of GlcNAc. The ability to synthesize amino acids was a metabolic requirement of strain PAO1 as well. The overexpression of the QS effector protein PqsE regulating pyocyanin production could not restore formation of pyocyanin in auxotrophic mutants. P. aeruginosa possesses three QS systems. For the QS response of strain PAO1 in the co-culture, both the rhl and the 2-alkyl-4(1H)-quinolone system were crucial, whereas the las system was dispensable. Lack of the rhl signal synthase could be complemented by cross-talk with signals of strain AH-1N. By applying transposon mutagenesis and screening for mutants of strain PAO1 with defects in QS-regulated processes, we could identify several genes that were involved in the regulation of pyocyanin production. Among them were members of the gene cluster PA1415-PA1421, mutations of which led to a decreased production of pyocyanin and accelerated growth with the polyamine spermin.By employing our co-culture model systems to study interspecific interactions, we could identify strategies of bacteria that are likely to be important in their natural habitats. With regard to P. aeruginosa, our model system offers the possibility to study QS under conditions that are more ecologically relevant than in single culture.