Inter-microbial competition for N and plant NO3− uptake rather than BNI determines soil net nitrification under intensively managed Brachiaria humidicola

Brachiaria humidicola (syn. Urochloa humidicola) has been acknowledged to control soil nitrification through release of nitrification inhibitors (NI), a phenomenon conceptualized as biological nitrification inhibition (BNI). Liming and N fertilization as features of agricultural intensification may suppress BNI performance, due to a decrease in NI exudation, increased NH3 availability and promotion of ammonia oxidizing bacteria (AOB) over archaea (AOA). A 2-year three-factorial pot trial was conducted to investigate the influence of soil pH and soil microbial background (ratio of archaea to bacteria) on BNI performance of B. humidicola. The study verified the capacity of B. humidicola to reduce net nitrification rates by 50 to 85% compared to the non-planted control, irrespective of soil pH and microbial background. The reduction of net nitrification, however, was largely dependent on microbial N immobilization and efficient plant N uptake. A reduction of gross nitrification could not be confirmed for the AOA dominated soil, but possibly contributed to reduced net nitrification rates in the AOB-dominated soil. However, this putative reduction of gross nitrification was attributed to plant-facilitated inter-microbial competition between bacterial heterotrophs and nitrifiers rather than BNI. It was concluded that BNI may play a dominant role in extensive B. humidicola pasture systems, while N immobilization and efficient plant N uptake may display the dominant factors controlling net nitrification rates under intensively managed B. humidicola.

Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Phage tail-like nanostructures affect microbial interactions between Streptomyces and fungi

Extracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with the fungi. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.

Interactions between microbial diversity and substrate chemistry determine the fate of carbon in soil

Microbial decomposition drives the transformation of plant-derived substrates into microbial products that form stable soil organic matter (SOM). Recent theories have posited that decomposition depends on an interaction between SOM chemistry with microbial diversity and resulting function (e.g., enzymatic capabilities, growth rates). Here, we explicitly test these theories by coupling quantitative stable isotope probing and metabolomics to track the fate of 13C enriched substrates that vary in chemical composition as they are assimilated by microbes and transformed into new metabolic products in soil. We found that differences in forest nutrient economies (e.g., nutrient cycling, microbial competition) led to arbuscular mycorrhizal (AM) soils harboring greater diversity of fungi and bacteria than ectomycorrhizal (ECM) soils. When incubated with 13C enriched substrates, substrate type drove shifts in which species were active decomposers and the abundance of metabolic products that were reduced or saturated in the highly diverse AM soils. The decomposition pathways were more static in the less diverse, ECM soil. Importantly, the majority of these shifts were driven by taxa only present in the AM soil suggesting a strong link between microbial identity and their ability to decompose and assimilate substrates. Collectively, these results highlight an important interaction between ecosystem-level processes and microbial diversity; whereby the identity and function of active decomposers impacts the composition of decomposition products that can form stable SOM.

Imaging Flow Cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members

In nature, bacteria are establishing differentiated communities referred to as biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.

Identification of Burkholderia and Penicillium isolates from kauri (Agathis australis) soils that inhibit the mycelial growth of Phytophthora agathidicida

Phytophthora agathidicida is a highly virulent pathogen of kauri (Agathis australis) and the causal agent of dieback disease in New Zealand’s kauri forests. This study aimed to identify microbial isolates isolated from kauri forest soils that inhibited the growth of P. agathidicida. Three different forms of in vitro bioassays were used to assess the inhibition of each isolate on the mycelial growth of P. agathidicida. Furthermore, head space (HS) solid-phase micro-extraction coupled with gas chromatography-mass spectrometry (SPME-GCMS) was performed to identify if the microbial isolates emitted volatile organic compounds (VOCs), which may be contributing to inhibition. This research identified several bacterial isolates belonging to the genus Burkholderia that inhibited the mycelial growth of P. agathidicida. Furthermore, several VOCs produced by these isolates were putatively identified, which may be responsible for the inhibition observed in the bioassays. Several isolates of Penicillium were identified that inhibit Phytophthora agathidicida, with the culture filtrate of one isolate being found to strongly inhibit P. agathidicida mycelial growth. These isolates of Burkholderia and Penicillium appear to exhibit multiple modes of antagonism against P. agathidicida, including microbial competition and the production of diffusible and volatile anti-microbial compounds. Although further research is needed to better define their mechanisms of inhibition, these findings have identified candidate microbial antagonists of P. agathidicida.

Phage Tail-like Nanostructures Affect Microbial Interactions between Streptomyces and Fungi

Extracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with S. pombe. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.

Prodigiosin inhibits bacterial growth and virulence factors as a potential physiological response to interspecies competition

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 μg/μL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 μg/μL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.