Sanin Musovic

Cultivation-Independent Examination of Horizontal Transfer and Host Range of an IncP-1 Plasmid among Gram-Positive and Gram-Negative Bacteria Indigenous to the Barley Rhizosphere

ABSTRACT The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 × 10−2 ± 3.84 × 10−2. This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.

Novel Assay To Assess Permissiveness of a Soil Microbial Community toward Receipt of Mobile Genetic Elements

ABSTRACT There is a wealth of evidence indicating that mobile genetic elements can spread in natural microbial communities. However, little is known regarding the fraction of the community that actually engages in this behavior. Here we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness. Conjugal transfer of a broad-host-range plasmid labeled with a zygotically inducible green fluorescent protein (RP4::gfp) from a donor strain (Pseudomonas putida) to a soil bacterial suspension was examined. The mixture of cells was incubated on membrane filters supported by different solid media. Plasmid transfer was scored by in situ visualization of green fluorescent transconjugant microcolonies, and host range was determined by traditional plating or microcolony isolation by using a micromanipulator. Among the conditions tested, the highest plasmid transfer incidence (approximately 1 transfer per 104 soil bacteria) was measured after 48 h of incubation on either a 10% soil extract or a 10-fold diluted R2A medium. Stereomicroscopy combined with image analysis allowed easy examination and enumeration of green fluorescent microcolonies. In all experiments, however, stereomicroscopy consistently underestimated the number of conjugation events (approximately 10-fold) in comparison to confocal laser scanning microscopy. The plasmid host range was broad and included bacteria belonging to the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria classes of proteobacteria. The isolation of transconjugant microcolonies by micromanipulation greatly extended the estimated plasmid host range among soil bacteria. The new approach can be applied to examine the permissiveness of various communities toward receipt of different mobile elements.

Long-term manure exposure increases soil bacterial community potential for plasmid uptake.

Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent and was dominated by β- and γ-Proteobacteria.

Ecological patterns, diversity and core taxa of microbial communities in groundwater-fed rapid gravity filters.

Here, we document microbial communities in rapid gravity filtration units, specifically serial rapid sand filters (RSFs), termed prefilters (PFs) and after- filters (AFs), fed with anoxic groundwaters low in organic carbon to prepare potable waters. A comprehensive 16S rRNA-based amplicon sequencing survey revealed a core RSF microbiome comprising few bacterial taxa (29–30 genera) dominated by Nitrospirae, Proteobacteria and Acidobacteria, with a strikingly high abundance (75–87±18%) across five examined waterworks in Denmark. Lineages within the Nitrospira genus consistently comprised the second most and most abundant fraction in PFs (27±23%) and AFs (45.2±23%), respectively, and were far more abundant than typical proteobacterial ammonium-oxidizing bacteria, suggesting a physiology beyond nitrite oxidation for Nitrospira. Within the core taxa, sequences closely related to types with ability to oxidize ammonium, nitrite, iron, manganese and methane as primary growth substrate were identified and dominated in both PFs (73.6±6%) and AFs (61.4±21%), suggesting their functional importance. Surprisingly, operational taxonomic unit richness correlated strongly and positively with sampling location in the drinking water treatment plant (from PFs to AFs), and a weaker negative correlation held for evenness. Significant spatial heterogeneity in microbial community composition was detected in both PFs and AFs, and was higher in the AFs. This is the first comprehensive documentation of microbial community diversity in RSFs treating oligotrophic groundwaters. We have identified patterns of local spatial heterogeneity and dispersal, documented surprising energy–diversity relationships, observed a large and diverse Nitrospira fraction and established a core RSF microbiome.

Internal Porosity of Mineral Coating Supports Microbial Activity in Rapid Sand Filters for Groundwater Treatment

ABSTRACT A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH4 + removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 μm) of the mineral coating, which had a thickness of up to 600 ± 51 μm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.

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.