Rhodopirellula Baltica Research Articles

Genomic and functional insights of a mucin foraging Rhodopirellula halodulae sp. nov.

Nine novel strains were obtained from various algal and seagrass samples. The analysis of the 16S rRNA gene-based phylogenetic tree revealed monophyletic placement of all novel strains within the Rhodopirellula genus. The type strain was identified as JC737T, which shared 99.1 % 16S rRNA gene sequence identity with Rhodopirellula baltica SH1T, while strain JC740 was designated as an additional strain. The genome sizes of strains JC737T and JC740 were 6.6 and 6.7 Mb, respectively, and the G + C content was 56.2 %. The strains cladded distinctly in the phylogenomic tree, and the ANI and dDDH values of the strain JC737T were 75.8–76.1 % and 20.8–21.3 %, respectively, in comparison to other Rhodopirellula members. The strain demonstrated a versatile degradation capability, exhibiting a diverse array of complex polysaccharides, including mucin which had not been previously identified within the members of the phylum Planctomycetota. The phylogenomic, pan-genomic, morphological, physiological, and genomic characterization of the strain lead to the proposal to describe the strain as Rhodopirellula halodulae sp. nov.

Antimicrobial Resistance Profile of Planctomycetota Isolated from Oyster Shell Biofilm: Ecological Relevance within the One Health Concept

Background: Planctomycetota isolation in pure culture is still challenging with most of the reported data coming from molecular-based methods. Here, we intended to isolate Planctomycetota from the filter-feeder Pacific oyster Magallana gigas, extending the search to a not yet explored natural reservoir and to characterize their antimicrobial resistance phenotype. Methods: Oyster samples from different supermarkets and from a farm producer were subject to isolation in selective medium. Inoculation was performed from the shell biofilm and after an enrichment of the edible content. Results: Planctomycetota isolates (n = 65) were only obtained from the shell biofilm with four different species identified: Rhodopirellula baltica (n = 62), Rhodopirellula rubra (n = 1), Rhodopirellula heiligendammensis (n = 1) and Gimesia chilikensis (n = 1). This study reports the first association of Planctomycetota members with oysters and the first description of R. heiligendammensis in Portugal. Moreover, R. rubra, originally identified in Portugal, was isolated from oysters of French origin. Antibiotic susceptibility testing, conducted in strains belonging to two species never assayed before revealed multidrug resistance phenotypes with bacteria showing resistance to several classes of clinically relevant antibiotics (e.g., β-lactams and aminoglycosides). Conclusion: The ecological role and impact of Planctomycetota on oyster holobiont and, ultimately, in public health, under the One Health concept, is discussed.

A Bacterial Myeloperoxidase with Antimicrobial Properties.

The four mammalian peroxidases (myeloperoxidase, eosinophilperoxidase, lactoperoxidase, and thyroid peroxidase) are widely studied in the literature. They catalyze the formation of antimicrobial compounds and participate in innate immunity. Owing to their properties, they are used in many biomedical, biotechnological, and agro-food applications. We decided to look for an enzyme that is easiest to produce and much more stable at 37 °C than mammalian peroxidases. To address this question, a peroxidase from Rhodopirellula baltica, identified by bioinformatics tools, was fully characterized in this study. In particular, a production and purification protocol including the study of heme reconstitution was developed. Several activity tests were also performed to validate the hypothesis that this peroxidase is a new homolog of mammalian myeloperoxidase. It has the same substrate specificities as the human one and accepts I-, SCN-, Br-, and Cl- as (pseudo-) halides. It also exhibits other auxiliary activities such as catalase and classical peroxidase activities, and it is very stable at 37 °C. Finally, this bacterial myeloperoxidase can kill the Escherichia coli strain ATCC25922, which is usually used to perform antibiograms.

Homology Modeling and Probable Active Site Cavity Prediction of Uncharacterized Arsenate Reductase in Bacterial spp.

The arsC gene-encoded arsenate reductase is a vital catalytic enzyme for remediation of environmental arsenic (As). Microorganisms containing the arsC gene can convert pentavalent arsenate (As[V]) to trivalent arsenite (As[III]) to be either retained in the bacterial cell or released into the air. The molecular mechanism governing this process is unknown. Here we present an in silico model of the enzyme to describe their probable active site cavities using SCFBio servers. We retrieved the amino acid sequence of bacterial arsenate reductase enzymes in FASTA format from the NCBI database. Enzyme structure was predicted using the I-TASSER server and visualized using PyMOL tools. The ProSA and the PROCHECK servers were used to evaluate the overall significance of the predicted model. Accordingly, arsenate reductase from Streptococcus pyogenes, Oligotropha carboxidovorans OM5, Rhodopirellula baltica SH 1, and Serratia ureilytica had the highest quality scores with statistical significance. The plausible cavities of the active site were identified in our examined arsenate reductase enzymes which were abundant in glutamate and lysine residues with 6 to 16 amino acids. This in silico experiment may contribute greatly to the remediation of arsenic pollution through the utilization of microbial species.

Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis

D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min−1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.

Draft Genome Sequence of Rhodopirellula baltica Strain BR-MGV, a Planctomycete Isolated from Brazilian Mangrove Soil.

Members of the phylum Planctomycetes, which are capable of surviving in a wide range of environments, are some of the least-explored bacteria. Here, we report the near-complete draft genome sequence and annotation of the planctomycete Rhodopirellula baltica BR-MGV, which was isolated from the soil of a contaminated Brazilian mangrove.

Planctomycetes attached to algal surfaces: Insight into their genomes

Planctomycetes are bacteria with complex molecular and cellular biology. They have large genomes, some over 7Mb, and complex life cycles that include motile cells and sessile cells. Some live on the complex biofilm of macroalgae. Factors governing their life in this environment were investigated at the genomic level. We analyzed the genomes of three planctomycetes isolated from algal surfaces. The genomes were 6.6Mbp to 8.1Mbp large. Genes for outer-membrane proteins, peptidoglycan and lipopolysaccharide biosynthesis were present. Rubripirellula obstinata LF1T, Roseimaritima ulvae UC8T and Mariniblastus fucicola FC18T shared with Rhodopirellula baltica and R. rubra SWK7 unique proteins related to metal binding systems, phosphate metabolism, chemotaxis, and stress response. These functions may contribute to their ecological success in such a complex environment. Exceptionally huge proteins (6000 to 10,000 amino-acids) with extracellular, periplasmic or membrane-associated locations were found which may be involved in biofilm formation or cell adhesion.

Determining the bacterial cell biology of Planctomycetes

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.

Developing Techniques for the Utilization of Planctomycetes As Producers of Bioactive Molecules.

Planctomycetes are conspicuous, ubiquitous, environmentally important bacteria. They can attach to various surfaces in aquatic habitats and form biofilms. Their unique FtsZ-independent budding cell division mechanism is associated with slow growth and doubling times from 6 h up to 1 month. Despite this putative disadvantage in the struggle to colonize surfaces, Planctomycetes are frequently associated with aquatic phototrophic organisms such as diatoms, cyanobacteria or kelp, whereby Planctomycetes can account for up to 50% of the biofilm-forming bacterial population. Consequently, Planctomycetes were postulated to play an important role in carbon utilization, for example as scavengers after phototrophic blooms. However, given their observed slow growth, such findings are surprising since other faster- growing heterotrophs tend to colonize similar ecological niches. Accordingly, Planctomycetes were suspected to produce antibiotics for habitat protection in response to the attachment on phototrophs. Recently, we demonstrated their genomic potential to produce non-ribosomal peptides, polyketides, bacteriocins, and terpenoids that might have antibiotic activities. In this study, we describe the development of a pipeline that consists of tools and procedures to cultivate Planctomycetes for the production of antimicrobial compounds in a chemically defined medium and a procedure to chemically mimic their interaction with other organisms such as for example cyanobacteria. We evaluated and adjusted screening assays to enable the hunt for planctomycetal antibiotics. As proof of principle, we demonstrate antimicrobial activities of planctomycetal extracts from Planctopirus limnophila DSM 3776, Rhodopirellula baltica DSM 10527, and the recently isolated strain Pan216. By combining UV/Vis and high resolution mass spectrometry data from high-performance liquid chromatography fractionations with growth inhibition of indicator strains, we were able to assign the antibiotic activity to candidate peaks related to planctomycetal antimicrobial compounds. The MS analysis points toward the production of novel bioactive molecules with novel structures. Consequently, we developed a large scale cultivation procedure to allow future structural elucidation of such compounds. Our findings might have implications for the discovery of novel antibiotics as Planctomycetes represent a yet untapped resource that could be developed by employing the tools and methods described in this study.

Permanent draft genome of ‘Rhodopirellula islandica’ strain K833

The planctomycete strain K833 was isolated from cold waters at the coast of Island and is tentatively named ‘Rhodopirellula islandica’. It has a lower temperature range for growth than other genome-sequenced Rhodopirellula strains affiliating to Rhodopirellula baltica and ‘Rhodopirellula europaea’. The permanent draft genome of strain K833 was obtained as part of a larger study on the biogeography of Rhodopirellula species in European marine waters. The genome consists of 55 contigs with a genome size of 7,433,200bp. With an average nucleotide identity of 81% to related genomes of R. baltica and ‘R. europaea’ and more than 4000 common genes, it will be a valuable source for the study of temperature adaptation of planctomycete genomes.

Phylogenetic and Taxonomic Analyses of Rhodopirellula caenicola Sp. Nov., a New Marine Planctomycetes Species Isolated from Iron Sand

A Gram-negative, strictly aerobic, pink-pigmented, non-motile, spherical bacterium designated YM26-125T was isolated from iron sand. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new strain was affiliated with the phylum Planctomycetes and shared 96.1% sequence similarity with Rhodopirellula baltica SH 1T. The strain could be also differentiated phenotypically from the Rhodopirellula baltica SH 1T. The major fatty acids of strain YM26-125T were identified as C16:0 and C18:1 ω9c as defined by the MIDI system. Amino acid analysis of cell wall hydrolysates indicated that the novel isolate did not contain diaminopimelic acid in their cell walls. The DNA G+C content was determined to be 57.5 mol%, the major respiratory quinone was identified as menaquinone 6 (MK-6) and a polar lipid profile was present consisting of a phosphatidylglycerol, a diphosphatidylglycerol and three unidentified phospholipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species for which the name Rhodopirellula caenicola sp. nov. is proposed. The type strain of R. caenicola is YM26-125T (=KCTC 32995T=NBRC 110016T).

The C‐terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli

The marine Gram-negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C-terminal regions similar to the YidC homologues in mitochondria and Gram-positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C-terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane. Here, we show in E. coli that an extended C-terminal region of YidC can compensate for a loss of SRP-receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C-terminally elongated YidC chimera. This complex was visualized at 8.6 Å by cryo-electron microscopy and shows a close contact of the ribosome and a YidC monomer.

Permanent draft genome of Rhodopirellula rubra SWK7

The genome of Rhodopirellula rubra strain SWK7 was sequenced as a permanent draft to complement the full genome sequence of the type strain Rhodopirellula baltica SH1T. This isolate is part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the fourth among a series of five publications reporting in a total of eight new permanent draft genomes of Rhodopirellula species.

Permanent draft genomes of the two Rhodopirellula europaea strains 6C and SH398

The genomes of two Rhodopirellula europaea strains were sequenced as permanent drafts to study the genomic diversity within this genus, especially in comparison with the closed genome of the type strain Rhodopirellula baltica SH1T. The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the second of a series of five publications describing a total of eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the Rhodopirellula maiorica strain SM1

The genome of Rhodopirellula maiorica strain SM1 was sequenced as a permanent draft to complement the full genome sequence of the type strain Rhodopirellula baltica SH1T. This isolate is part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the fifth of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genome of Rhodopirellula sallentina SM41

The genome of Rhodopirellula sallentina SM41 was sequenced as a permanent draft to supplement the full genome sequence of the type strain Rhodopirellula baltica SH1T. This isolate is part of a larger study to gain insights into the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the third of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the three Rhodopirellula baltica strains SH28, SWK14 and WH47

The genomes of three Rhodopirellula baltica strains were sequenced as permanent drafts to complement the full genome sequence of the type strain R. baltica SH1T. The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the first of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

High ultraviolet C resistance of marine Planctomycetes

Planctomycetes are bacteria with particular characteristics such as internal membrane systems encompassing intracellular compartments, proteinaceous cell walls, cell division by yeast-like budding and large genomes. These bacteria inhabit a wide range of habitats, including marine ecosystems, in which ultra-violet radiation has a potential harmful impact in living organisms. To evaluate the effect of ultra-violet C on the genome of several marine strains of Planctomycetes, we developed an easy and fast DNA diffusion assay in which the cell wall was degraded with papain, the wall-free cells were embedded in an agarose microgel and lysed. The presence of double strand breaks and unwinding by single strand breaks allow DNA diffusion, which is visible as a halo upon DNA staining. The number of cells presenting DNA diffusion correlated with the dose of ultra-violet C or hydrogen peroxide. From DNA damage and viability experiments, we found evidence indicating that some strains of Planctomycetes are significantly resistant to ultra-violet C radiation, showing lower sensitivity than the known resistant Arthrobacter sp. The more resistant strains were those phylogenetically closer to Rhodopirellula baltica, suggesting that these species are adapted to habitats under the influence of ultra-violet radiation. Our results provide evidence indicating that the mechanism of resistance involves DNA damage repair and/or other DNA ultra-violet C-protective mechanism.

Diversity of Planctomycetes in iron-hydroxide deposits from the Arctic Mid Ocean Ridge (AMOR) and description of Bythopirellula goksoyri gen. nov., sp. nov., a novel Planctomycete from deep sea iron-hydroxide deposits

Planctomycetes form a deep branching and distinct phylum of the domain Bacteria, and represent a fascinating group due to their unusual features such as intracellular compartmentalization and lack of peptidoglycan in their cell walls. The phylum Planctomycetes was described already in 1924, but still the diversity of this phylum represents an enigma and unexploited resource. In this study the diversity of the phylum Planctomycetes in low temperature iron-hydroxide deposits at the Mohns Ridge, a part of the Arctic Mid Ocean Ridge (AMOR), was characterised by descriptive analysis of 16S rRNA gene sequences in combination with isolation of planctomycetes strains. The 16S rRNA gene sequences were affiliated with three order within the phylum Planctomycetes namely the (i)Planctomycetales, (ii) "Candidatus Brocadiales" and (iii) Phycisphaerae in addition to sequences affiliating to hitherto unknown Planctomycetes. The majority of the sequences were affiliated with the CCM11a group (Phycisphaerae), and with the Pir4 group (Planctomycetaceae). Two strains from the order Planctomycetales were isolated. One strain (Plm2) showed high similarity to the previously isolated Planctomyces maris (99 % 16S rRNA sequence identity). The other strain (Pr1d) belonged to the Pir4 group, and showed highest identity with Rhodopirellula baltica (86 %), Blastopirellula marina (86 %) and Pirellula staleyi (85 %). Based on its physiological and biochemical properties, strain Pr1d(T) is considered to represent a new genus of the order Planctomycetales. We propose to classify the novel planctomycete in a new genus and species, Bythoypirellula goksoyri gen. nov., sp. nov., the type strain being Pr1d(T).

From genome mining to phenotypic microarrays: Planctomycetes as source for novel bioactive molecules

Most members of the phylum Planctomycetes share many unusual traits that are unique for bacteria, since they divide independent of FtsZ through asymmetric budding, possess a complex life cycle and comprise a compartmentalized cell plan. Besides their complex cell biological features Planctomycetes are environmentally important and play major roles in global matter fluxes. Such features have been successfully employed in biotechnological applications such as the anaerobic oxidation of ammonium in wastewater treatment plants or the utilization of enzymes for biotechnological processes. However, little is known about planctomycetal secondary metabolites. This is surprising as Planctomycetes have several key features in common with known producers of small bioactive molecules such as Streptomycetes or Myxobacteria: a complex life style and large genome sizes. Planctomycetal genomes with an average size of 6.9 MB appear as tempting targets for drug discovery approaches. To enable the hunt for bioactive molecules from Planctomycetes, we performed a comprehensive genome mining approach employing the antiSMASH secondary metabolite identification pipeline and found 102 candidate genes or clusters within the analyzed 13 genomes. However, as most genes and operons related to secondary metabolite production are exclusively expressed under certain environmental conditions, we optimized Phenotype MicroArray protocols for Rhodopirellula baltica and Planctomyces limnophilus to allow high throughput screening of putative stimulating carbon sources. Our results point towards a previously postulated relationship of Planctomycetes with algae or plants, which secrete compounds that might serve as trigger to stimulate the secondary metabolite production in Planctomycetes. Thus, this study provides the necessary starting point to explore planctomycetal small molecules for drug development.