The community structure in the plant-associated microbiome depends collectively on host–microbe, microbe–microbe and host–microbe–microbe interactions. The ensemble of interactions between the host and microbial consortia may lead to outcomes that are not easily predicted from pairwise interactions. Plant–microbe–microbe interactions are important to plant health but could depend on both host and microbe strain variation. Here we study interactions between groups of naturally co-existing commensal and pathogenic Pseudomonas strains in the Arabidopsis thaliana phyllosphere. We find that commensal Pseudomonas prompt a host response that leads to selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of protection depends on plant genotype, supporting that these effects are host-mediated. Strain-specific effects are also demonstrated by one individual Pseudomonas isolate eluding the plant protection provided by commensals. Our work highlights how within-species genetic differences in both hosts and microbes can affect host–microbe–microbe dynamics.
The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.
Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked
fructooligosaccharides (IFOS) from sucrose. Previous studies have
shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here,
mutational analyses of four conserved calcium-binding site I (Ca-I)
residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for
these residues not only reduced the stability and activity of LrInu,
but also modulated the pattern of the IFOS produced. Circular dichroism
spectroscopy and molecular dynamics simulation indicated that these
mutations had limited impact on the overall conformation of the enzyme.
One of Ca-I residues most critical for controlling LrInu-mediated
polymerization of IFOS, Asp418, was also subjected to mutagenesis,
generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity
of these mutants demonstrated that the IFOS chain length could be
controlled by a single mutation at the Ca-I site.
The microbial population size, or load, in a host is a fundamental metric of colonization by commensals or infection by pathogens. Sequence-based measurement of DNA amount contributed by host and microbes in a sample provides a simple way of measuring microbial load, and it also has the ability to estimate microbiome diversity. Unfortunately, it is also very costly, especially when host DNA greatly outweighs microbial DNA. We introduce a robust two-step PCR strategy to quantify absolute abundance of the host-associated microbiome and describe its composition in a single, cost-effective amplicon library. We demonstrate the accuracy and flexibility of this method across multiple amplicons and hosts, and further present a simple technique that can be used, prior to sequencing, to optimize the host representation in a batch of libraries without a loss of information.
Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.
Sphingomonas
is one of the most abundant bacterial genera in the phyllosphere of wild
Arabidopsis thaliana
, but relative to
Pseudomonas
, the ecology of
Sphingomonas
and its interaction with plants is poorly described. We analyzed the genomic features of over 400
Sphingomonas
isolates collected from local
A. thaliana
populations, which revealed much higher intergenomic diversity than for the considerably more uniform
Pseudomonas
isolates found in the same host populations. Variation in
Sphingomonas
plasmid complements and additional genomic features suggest high adaptability of this genus, and the widespread presence of protein secretion systems hints at frequent biotic interactions. While some of the isolates showed plant-protective phenotypes in lab tests, this was a rare trait. To begin to understand the extent of strain sharing across alternate hosts, we employed amplicon sequencing and a bulk-culturing metagenomics approach on both
A. thaliana
and neighboring plants. Our data reveal that both
Sphingomonas
and
Pseudomonas
thrive on other diverse plant hosts, but that
Sphingomonas
is a poor competitor in dying or dead leaves.
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