Limited diffusive fluxes of substrate facilitate coexistence of two competing bacterial strains

Soils are known to support a great bacterial diversity down to the millimeter scale, but the mechanisms by which such a large diversity is sustained are largely unknown. A feature of unsaturated soils is that water usually forms thin, poorly-connected films, which limit solute diffusive fluxes. It has been proposed, but never unambiguously experimentally tested, that a low substrate diffusive flux would impact bacterial diversity, by promoting the coexistence between slow-growing bacteria and their potentially faster-growing competitors. We used a simple experimental system, based on a Petri dish and a perforated Teflon® membrane to control diffusive fluxes of substrate (benzoate) whilst permitting direct observation of bacterial colonies. The system was inoculated with prescribed strains of Pseudomonas, whose growth was quantified by microscopic monitoring of the fluorescent proteins they produced. We observed that substrate diffusion limitation reduced the growth rate of the otherwise fast-growing Pseudomonas putida KT2440 strain. This strain out-competed Pseudomonas fluorescens F113 in liquid culture, but its competitive advantage was less marked on solid media, and even disappeared under conditions of low substrate diffusion. Low diffusive fluxes of substrate, characteristic of many unsaturated media (e.g. soils, food products), can thus promote bacterial coexistence in a competitive situation between two strains. This mechanism might therefore contribute to maintaining the noncompetitive diversity pattern observed in unsaturated soil

Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We compare the transfer frequency of a mobilizable plasmid to that of a mobilizing and conjugal plasmid measured for a model strain and for the assayed community. With Pseudomonas putida carrying the gfp-tagged mobilizable RSF1010 plasmid as donor strain, we conducted solid surface mating experiments with either a P. putida strain carrying the mobilizing plasmid RP4 or a model bacterial community that was extracted from the inner walls of a domestic shower conduit. Additionally, we estimated the permissiveness of the same community for RP4 using P. putida as donor strain. The permissiveness of the model community for RP4 (at 1.16x10-4 transconjugants per recipient (T/R)) was similar to that previously measured for soil microbial communities. RSF1010 was mobilized by the model community at a frequency of 1.16x10-5 T/R, only one order of magnitude lower than its permissiveness to RP4. This mobilization frequency is unexpectedly high considering that (i) mobilization requires the presence of mobilizing conjugal plasmids within the permissive fraction of the recipients; (ii) in pure culture experiments with P. putida retromobilization of RSF1010 through RP4 only took place in approximately half of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities

Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR

Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantitative PCR methods to enumerate ammonia‐oxidizing bacteria (AOB): one targeting the phylogenetic gene 16S rRNA and the other, the functional gene amoA. Cloning‐sequencing with both primer sets on DNA from two waterworks revealed contrasting images of AOB diversity. The amoA‐based approach preferentially recovered sequences belonging to Nitrosomonas Cluster 7 over Cluster 6A ones, while the 16S rRNA one yielded more diverse sequences belonging to three AOB clusters, but also a few non‐AOB sequences, suggesting broader, but partly unspecific, primer coverage. This was confirmed by an in silico coverage analysis against sequences of AOB (both isolates and high‐quality environmental sequences). The difference in primer coverage significantly impacted the estimation of AOB abundance at the waterworks with high Cluster 6A prevalence, with estimates up to 50‐fold smaller for amoA than for 16S rRNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets