Bacterial strains of the phylum Planctomycetes occur ubiquitously, but are often found on surfaces of aquatic phototrophs, e.g. alga. Despite slower growth, planctomycetes are not outcompeted by faster-growing bacteria in biofilms on such surfaces; however, strategies allowing them to compensate for slower growth have not yet been investigated. Here, we identified stieleriacines, a class of N-acylated tyrosines produced by the novel planctomycete Stieleria maiorica Mal15T, and analysed their effects on growth of the producing strain and bacterial species likely co-occurring with strain Mal15T. Stieleriacines reduced the lag phase of Mal15T and either stimulated or inhibited biofilm formation of two bacterial competitors, indicating that Mal15T employs stieleriacines to specifically alter microbial biofilm composition. The genetic organisation of the putative stieleriacine biosynthetic cluster in strain Mal15T points towards a functional link of stieleriacine biosynthesis to exopolysaccharide-associated protein sorting and biofilm formation. Kallscheuer, Jeske, Sandargo et al. demonstrate that stieleriacines, a class of N-acylated tyrosine derivatives, can alter species abundance in marine biofilms. This study suggests that slow-growing planctomycetes employ stieleriacines to counteract the growth advantage of their competitors.

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The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

Planctomycetes is a fascinating phylum of mostly aquatic bacteria, not only due to the environmental importance in global carbon and nitrogen cycles, but also because of a unique cell biology. Their lifestyle and metabolic capabilities are not well explored, which motivated us to study the role of Planctomycetes in biofilms on marine biotic surfaces. Here, we describe the novel strain Pan54T which was isolated from algae in a hydrothermal area close to the volcanic island Panarea in the Tyrrhenian Sea, north of Sicily in Italy. The strain grew best at pH 9.0 and 26 °C and showed typical characteristics of planctomycetal bacteria, e.g. division by polar budding, formation of aggregates and presence of stalks and crateriform structures. Phylogenetically, the strain belongs to the genus Rubinisphaera. Our analysis suggests that Pan54T represents a novel species of this genus, for which we propose the name Rubinisphaera italica sp. nov. We suggest Pan54T (= DSM 29369 = LMG 29789) as the type strain of the novel species.

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Rubinisphaera italica sp. nov. isolated from a hydrothermal area in the Tyrrhenian Sea close to the volcanic island Panarea

Traditionally recognized as environmental bacteria, Planctomycetes have just been linked recently to human pathology as opportunistic pathogens, arousing a great interest for clinical microbiologists. However, the lack of appropriate culture media limits our future investigations as no Planctomycetes have ever been isolated from patients' specimens despite several attempts. Several Planctomycetes have no cultivable members and are only recognized by 16S rRNA gene sequence detection and analysis. The cultured representatives are slow-growing fastidious bacteria and mostly difficult to culture on synthetic media. Accordingly, the provision of environmental and nutritional conditions like those existing in the natural habitat where yet uncultured/refractory bacteria can be detected might be an option for their potential isolation. Hence, we systematically reviewed the various natural habitats of Planctomycetes, to review their nutritional requirements, the physicochemical characteristics of their natural ecological niches, current methods of cultivation of the Planctomycetes and gaps, from a perspective of collecting data in order to optimize conditions and the protocols of cultivation of these fastidious bacteria. Planctomycetes are widespread in freshwater, seawater, and terrestrial environments, essentially associated to particles or organisms like macroalgae, marine sponges, and lichens, depending on the species and metabolizable polysaccharides by their sulfatases. Most Planctomycetes grow in nutrient-poor oligotrophic environments with pH ranging from 3.4 to 11, but a few strains can also grow in quite nutrient rich media like M600/M14. Also, a seasonality variation of abundance is observed, and bloom occurs in summer-early autumn, correlating with the strong growth of algae in the marine environments. Most Planctomycetes are mesophilic, but with a few Planctomycetes being thermophilic (50°C to 60°C). Commonly added nutrients are N-acetyl-glucosamine, yeast-extracts, peptone, and some oligo and macro-elements. A biphasic host-associated extract (macroalgae, sponge extract) conjugated with a diluted basal medium should provide favorable results for the success of isolation in pure culture.

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Planctomycetes as Host-Associated Bacteria: A Perspective That Holds Promise for Their Future Isolations, by Mimicking Their Native Environmental Niches in Clinical Microbiology Laboratories.

Seagrass meadows are ubiquitous, fragile and endangered marine habitats, which serve as fish breeding grounds, stabilize ocean floor substrates, retain nutrients and serve as important carbon sinks, counteracting climate change. In the Mediterranean Sea, seagrass meadows are mostly formed by the slow-growing endemic plant Posidonia oceanica (Neptune grass), which is endangered by global warming and recreational motorboating. Despite its importance, surprisingly little is known about the leaf surface microbiome of P. oceanica. Using amplicon sequencing, we here show that species belonging to the phylum Planctomycetes can dominate the biofilms of young and aged P. oceanica leaves. Application of selective cultivation techniques allowed for the isolation of two novel planctomycetal strains belonging to two yet uncharacterized genera.

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The Microbiome of Posidonia oceanica Seagrass Leaves Can Be Dominated by Planctomycetes.

Planctomycetes are part of the PVC superphylum together with Verrucomicrobia, Chlamydiae and others. They are budding bacteria with very distinctive characteristics, such as a remarkable morphology and cell biology. Planctomycetes can be found in almost all habitats, and seem to have a preference for marine biotic and abiotic surfaces, on which they frequently occur in biofilm-forming communities. To extend the number of axenic cultures of planctomycetal strains, we isolated Pan97T from a biofilm in a volcanic site close to the Italian island Panarea in the Thyrrhenian Sea. The physiology, genome and morphology of the novel strain were characterised revealing typical planctomycetal characteristics, such as, division by polar budding and presence of crateriform structures. The strain shows pear-shaped cells of 1.5 ± 0.3 µm × 0.8 ± 0.2 µm and forms white- to cream-coloured colonies on solid medium. Strain Pan97T is mesophilic and neutrophilic, since growth was observed  at a pH range of 5.5–9.5 with optimal growth at pH 7.0 and at a temperature range of 15–40 °C with a maximal growth rate at 36 °C. Pan97T has a genome size of 6,496,182 bp with a G + C content of 56.2%. 5264 protein-coding genes were identified, of which 2141 genes (41%) encode hypothetical proteins. Based on the phylogenetic analysis, we suggest that Pan97T (DSM 101992T = LMG 29460T) represents a novel species of a novel genus within the family Planctomycetaceae, for which we propose the name Bremerella gen. nov., with strain Pan97T classified as Bremerella volcania sp. nov. Based on our analysis, we also propose the reclassification of Blastopirellula cremea Lee et al. 2013 as Bremerella cremea comb. nov., as this species is considered to be the type species of the novel genus Bremerella.

Description of the novel planctomycetal genus Bremerella, containing Bremerella volcania sp. nov., isolated from an active volcanic site, and reclassification of Blastopirellula cremea as Bremerella cremea comb. nov.

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Erratum: Fuerstia Marisgermanicae Gen. Nov., Sp. Nov., an Unusual Member of the Phylum Planctomycetes from the German Wadden Sea (Front. Microbiol. (2016) 7 (2079), 10.3389/fmicb.2016.02079)

Abstract As of today, the majority of environmental microorganisms remain uncultured and is therefore referred to as ‘microbial dark matter’ (MDM). Hence, genomic insights into these organisms are limited to cultivation-independent approaches such as single-cell- and metagenomics. However, without access to cultured representatives for verifying correct taxon-assignments, MDM genomes may cause potentially misleading conclusions based on misclassified or contaminant contigs, thereby obfuscating our view on the uncultured microbial majority. Moreover, gradual database contaminations by past genome submissions can cause error propagations which affect present as well as future comparative genome analyses. Consequently, strict contamination detection and filtering need to be applied, especially in the case of uncultured MDM genomes. Current genome reporting standards, however, emphasize completeness over purity and the de facto gold standard genome assessment tool, checkM, discriminates against uncultured taxa and fragmented genomes. To tackle these issues, we present a novel contig classification, screening, and filtering workflow and corresponding open-source python implementation called MDMcleaner, which was tested and compared to other tools on mock and real datasets. MDMcleaner revealed substantial contaminations overlooked by current screening approaches and sensitively detects misattributed contigs in both novel genomes and the underlying reference databases, thereby greatly improving our view on ‘microbial dark matter’.

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How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria, and provide novel insights into the in situ host profile of these plasmids. This article is protected by copyright. All rights reserved.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1462-2920.16262

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.

https://www.mdpi.com/1422-0067/24/5/4270/pdf?version=1678255119

Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics

The environmental prevalence of antimicrobial resistance (AMR) has come into focus under the One Health concept. Wastewater treatment systems are among the significant sources of AMR in the environment. In such systems, it is uncertain to which extent antimicrobials present at sub-inhibitory concentrations constitute a selective pressure for bacterial maintenance and acquisition of antibiotic resistance (AR) genes. Here, we mapped AMR to inhibitors of folate biosynthesis in an aerated and a non-aerated horizontal subsurface flow treatment wetland receiving the same pre-treated municipal wastewater. General water characteristics and the concentrations of folate inhibitors were determined to define the ambient conditions over the longitudinal axis of the two treatment wetlands. Profiling of AMR as well as class 1 integrons, a mobile carrier of AR genes against folate inhibitors and other antimicrobials, was carried out by cultivation-dependent and -independent methods. The wetlands achieved mean reductions of AR gene copy numbers in the effluents of at least 2 log, with the aerated system performing better. The folate inhibitors had no noticeable effect on the prevalence of respective AR genes. However, there was a transient increase of AR gene copy numbers and AR gene cassette composition in class 1 integrons in the aerated wetland. The comparison of all data from both wetlands suggests that higher levels of cellular stress in the aerated system promoted the mobility of AR genes via enhancing the activity of the DNA recombinase of the class 1 integron. The findings highlight that environmental conditions that modulate the activity of this mobile genetic element can be more important for the fate of associated AR genes in treatment wetlands than the ambient concentration of the respective antimicrobial agents. By extrapolation, the results suggest that cellular stress also contributes to the mobility of AR gene in other wastewater treatment systems.

A-K Kaster

Papers

Erratum: Fuerstia Marisgermanicae Gen. Nov., Sp. Nov., an Unusual Member of the Phylum Planctomycetes from the German Wadden Sea (Front. Microbiol. (2016) 7 (2079), 10.3389/fmicb.2016.02079)

How clear is our current view on microbial dark matter? (Re-)assessing public MAG & SAG datasets with MDMcleaner

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics

Cellular stress affects the fate of microbial resistance to folate inhibitors in treatment wetlands.