A hotspot of diversity: novel Shewanella species isolated from Baltic Sea sediments delineate a sympatric species complex.

Two bacterial strains, SP1S1-4T and SP2S1-2T, were isolated from sediment samples collected in the Stockholm archipelago in November 2021. Following whole-genome sequencing, these strains were identified as tentatively belonging to two novel Shewanella genospecies, based on digital DNA-DNA hybridization, as implemented in the Type Strain Genome Server. Shewanella septentrionalis, Shewanella baltica and Shewanella hafniensis were, in this order and within a narrow genomic relatedness range, their closest genotypic relatives. Additional sampling and sequencing efforts led to the retrieval of distinct isolates that were monophyletic with SP1S1-4T and SP2S1-2T, respectively, based on phylogenomic analysis of whole-genome sequences. Comparative analyses of genome sequence data, which included blast-based average nucleotide identity, core genome-based and core proteome-based phylogenomics, in addition to MALDI-TOF MS-based protein profiling, confirmed the distinctness of the putative novel genospecies with respect to their closest genotypic relatives. A comprehensive phenotypic characterisation of SP1S1-4T and SP2S1-2T revealed only minor differences with respect to the type strains of S. septentrionalis, S. baltica and S. hafniensis. Based on the collective phylogenomic, proteomic, and phenotypic evidence presented here, we describe two novel genospecies within the genus Shewanella, for which the names Shewanella scandinavica sp. nov. and Shewanella vaxholmensis sp. nov. are proposed. The type strains are, respectively, SP2S1-2T (=CCUG 76457T=CECT 30688T), with a draft genome sequence of 5 041 805 bp and a G+C content of 46.3 mol%, and SP1S1-4T (=CCUG 76453T=CECT 30684T), with a draft genome sequence of 4 920147 bp and a G+C content of 46.0 mol%. Our findings suggest the existence of a species complex formed by the species S. baltica, S. septentrionalis, S. scandinavica sp. nov., and S. vaxholmensis sp. nov., with S. hafniensis falling in the periphery, where distinct genomic species clusters could be identified. However, this does not exclude the possibility of a continuum of genomic diversity within this sedimental ecosystem, as discussed herein with additional sequenced isolates.

Nitrogen Fixation and Microbial Communities Associated with Decomposing Seagrass Leaves in Temperate Coastal Waters.

Seagrass meadows play pivotal roles in coastal biochemical cycles, with nitrogen fixation being a well-established process associated with living seagrass. Here, we tested the hypothesis that nitrogen fixation is also associated with seagrass debris in Danish coastal waters. We conducted a 52-day in situ experiment to investigate nitrogen fixation (proxied by acetylene reduction) and dynamics of the microbial community (16S rRNA gene amplicon sequencing) and the nitrogen fixing community (nifH DNA/RNA amplicon sequencing) associated with decomposing Zostera marina leaves. The leaves harboured distinct microbial communities, including distinct nitrogen fixers, relative to the surrounding seawater and sediment throughout the experiment. Nitrogen fixation rates were measurable on most days, but highest on days 3 (dark, 334.8 nmol N g-1 dw h-1) and 15 (light, 194.6 nmol N g-1 dw h-1). Nitrogen fixation rates were not correlated with the concentration of inorganic nutrients in the surrounding seawater or with carbon:nitrogen ratios in the leaves. The composition of nitrogen fixers shifted from cyanobacterial Sphaerospermopsis to heterotrophic genera like Desulfopila over the decomposition period. On the days with highest fixation, nifH RNA gene transcripts were mainly accounted for by cyanobacteria, in particular by Sphaerospermopsis and an unknown taxon (order Nostocales), alongside Proteobacteria. Our study shows that seagrass debris in temperate coastal waters harbours substantial nitrogen fixation carried out by cyanobacteria and heterotrophic bacteria that are distinct relative to the surrounding seawater and sediments. This suggests that seagrass debris constitutes a selective environment where degradation is affected by the import of nitrogen via nitrogen fixation.

Biofilm formation and cell plasticity drive diazotrophy in an anoxygenic phototrophic bacterium.

IMPORTANCE: The contribution of non-cyanobacterial diazotrophs (NCDs) to total N2 fixation in the marine water column is unknown, but their importance is likely constrained by the limited availability of dissolved organic matter and low O2 conditions. Light could support N2 fixation and growth by NCDs, yet no examples from bacterioplankton exist. In this study, we show that the phototrophic NCD, Rhodopseudomonas sp. BAL398, which is a member of the diazotrophic community in the surface waters of the Baltic Sea, can utilize light. Our study highlights the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions and the role of cell plasticity in regulating these processes. Our findings have implications for the general understanding of the ecology and importance of NCDs in marine waters.

Legionella maioricensis sp. nov., a new species isolated from the hot water distribution systems of a hospital and a shopping center during routine sampling.

Two Legionella-like strains isolated from hot water distribution systems in 2012 have been characterized phenotypically, biochemically and genomically in terms of DNA relatedness. Both strains, HCPI-6T and EUR-108, exhibited biochemical phenotypic profiles typical of Legionella species. Cells were Gram-negative motile rods which grew on BCYEα agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for l-cysteine and testing catalase positive. Both strains were negative for oxidase, urease, nitrate reduction and hippurate negative, and non-fermentative. The major ubiquinone was Q12 (59.4 % HCPI-6T) and the dominant fatty acids were C16 : 1 ω7c (28.4 % HCPI-6T, ≈16 % EUR-108), C16 : 0 iso (≈22.5 % and ≈13 %) and C15 : 0 anteiso (19.5 % and ≈23.5 %, respectively). The percent G+C content of genomic DNA was determined to be 39.3 mol %. The 16S rRNA gene, mip sequence and comparative genome sequence-based analyses (average nucleotide identity, ANI; digital DNA-DNA hybridization, dDDH; and phylogenomic treeing) demonstrated that the strains represent a new species of the genus Legionella. The analysis based on the 16S rRNA gene sequences showed that the sequence similarities for both strains ranged from 98.8-90.1 % to other members of the genus. The core genome-based phylogenomic tree (protein-concatemer tree based on concatenation of 418 proteins present in single copy) revealed that these two strains clearly form a separate cluster within the genus Legionella. ANI and dDDH values confirmed the distinctiveness of the strains. Based on the genomic, genotypic and phenotypic findings from a polyphasic study, the isolates are considered to represent a single novel species, for which the name Legionella maioricensis sp. nov. is proposed. The type strain is HCPI-6T (=CCUG 75071T=CECT 30569T).

Everything Is Everywhere: Physiological Responses of the Mediterranean Sea and Eastern Pacific Ocean Epiphyte Cobetia Sp. to Varying Nutrient Concentration.

Bacteria are essential in the maintenance and sustainment of marine environments (e.g., benthic systems), playing a key role in marine food webs and nutrient cycling. These microorganisms can live associated as epiphytic or endophytic populations with superior organisms with valuable ecological functions, e.g., seagrasses. Here, we isolated, identified, sequenced, and exposed two strains of the same species (i.e., identified as Cobetia sp.) from two different marine environments to different nutrient regimes using batch cultures: (1) Cobetia sp. UIB 001 from the endemic Mediterranean seagrass Posidonia oceanica and (2) Cobetia sp. 4B UA from the endemic Humboldt Current System (HCS) seagrass Heterozostera chilensis. From our physiological studies, both strains behaved as bacteria capable to cope with different nutrient and pH regimes, i.e., N, P, and Fe combined with different pH levels, both in long-term (12 days (d)) and short-term studies (4 d/96 h (h)). We showed that the isolated strains were sensitive to the N source (inorganic and organic) at low and high concentrations and low pH levels. Low availability of phosphorus (P) and Fe had a negative independent effect on growth, especially in the long-term studies. The strain UIB 001 showed a better adaptation to low nutrient concentrations, being a potential N2-fixer, reaching higher growth rates (µ) than the HCS strain. P-acquisition mechanisms were deeply investigated at the enzymatic (i.e., alkaline phosphatase activity, APA) and structural level (e.g., alkaline phosphatase D, PhoD). Finally, these results were complemented with the study of biochemical markers, i.e., reactive oxygen species (ROS). In short, we present how ecological niches (i.e., MS and HCS) might determine, select, and modify the genomic and phenotypic features of the same bacterial species (i.e., Cobetia spp.) found in different marine environments, pointing to a direct correlation between adaptability and oligotrophy of seawater.

Toxicity of inorganic nanoparticles and commercial sunscreens on marine bacteria.

The Balearic Islands, a top tourist destination for sunny beaches, face physical and chemical pressures from human activities, impacting keystone species like the endemic seagrass Posidonia oceanica and its associated microbiome. This study evaluated the effects of ZnO and TiO2 nanoparticles and three commercial sunscreens with varying protection factors (50 or 90) and chemical complexities (1- SPF50_E "eco-friendly"; 2- SPF50 not "eco-friendly"; 3- SPF90 not "eco-friendly") on five heterotrophic bacteria (Pseudomonas azotifigens, Marinobacterium litorale, Thiothrix nivea, Sedimenticola thiotaurini and Cobetia sp) and two autotrophic cyanobacteria (Halothece sp. and Fischerella muscicola) associated to P. oceanica, as well as a natural leaf epiphytic community. Results indicated that TiO2 affected all heterotrophic bacteria, while ZnO was toxic to only two species, while autotrophs were unaffected. Commercial sunscreens impacted three heterotrophs and the natural epiphytic community, while autotrophs were only affected by SPF50. SPF50_E reduced phosphorus uptake, and both SPF50 and SPF90 decreased alkaline phosphatase activity. Reactive oxygen species production was mainly induced by SPF90, followed by SPF50_E and SPF50. Generally, the smallest bacteria were most sensitive to UV-filters (UVFs). This study indicates that UVFs exposure may alter the epiphytic community structure of P. oceanica.

Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds.

Stutzerimonas balearica (Pseudomonas balearica) has been found principally in oil-polluted environments. The capability of S. balearica to thrive from the degradation of pollutant compounds makes it a species of interest for potential bioremediation applications. However, little has been reported about the diversity of S. balearica. In this study, genome sequences of S. balearica strains from different origins were analyzed, revealing that it is a diverse species with an open pan-genome that will continue revealing new genes and functionalities as the genomes of more strains are sequenced. The nucleotide signatures and intra- and inter-species variation of the 16S rRNA genes of S. balearica were reevaluated. A strategy of screening 16S rRNA gene sequences in public databases enabled the detection of 158 additional strains, of which only 23% were described as S. balearica. The species was detected from a wide range of environments, although mostly from aquatic and polluted environments, predominantly related to petroleum oil. Genomic and phenotypic analyses confirmed that S. balearica possesses varied inherent capabilities for aromatic compounds degradation. This study increases the knowledge of the biology and diversity of S. balearica and will serve as a basis for future work with the species.

Cell size matters: Nano- and micro-plastics preferentially drive declines of large marine phytoplankton due to co-aggregation.

Marine plastic pollution represents a key environmental concern. Whilst ecotoxicological data for plastic is increasingly available, its impact upon marine phytoplankton remains unclear. Owing to their predicted abundance in the marine environment and likely interactions with phytoplankton, here we focus on the smaller fraction of plastic particles (~50 nm and ~2 µm polystyrene spheres). Exposure of natural phytoplankton communities and laboratory cultures revealed that plastic exposure does not follow traditional trends in ecotoxicological research, since large phytoplankton appear particularly susceptible towards plastics exposure despite their lower surface-to-volume ratios. Cell declines appear driven by hetero-aggregation and co-sedimentation of cells with plastic particles, recorded visually and demonstrated using confocal microscopy. As a consequence, plastic exposure also caused disruption to photosynthetic functioning, as determined by both photosynthetic efficiency and high throughput proteomics. Negative effects upon phytoplankton are recorded at concentrations orders of magnitude above those estimated in the environment. Hence, it is likely that impacts of NPs and MPs are exacerbated at the high concentrations typically used in ecotoxicological research (i.e., mg L-1).

Differential Effects of Varying Concentrations of Phosphorus, Iron, and Nitrogen in N2-Fixing Cyanobacteria.

Diazotrophs or N2-fixers are one of the most ecologically significant groups in marine ecosystems (pelagic and benthic). Inorganic phosphorus (PO4 3-) and iron (Fe) can limit the growth and N2-fixing capacities of cyanobacteria. However, studies investigating co-limitation of these factors are lacking. Here, we added different concentrations of PO4 3- and Fe in two cyanobacterial species whose relatives can be found in seagrass habitats: the unicellular Halothece sp. (PCC 7418) and the filamentous Fischerella muscicola (PCC 73103), grown under different nitrate (NO3 -) concentrations and under N2 as sole N source, respectively. Their growth, pigment content, N2-fixation rates, oxidative stress responses, and morphological and cellular changes were investigated. Our results show a serial limitation of NO3 - and PO4 3- (with NO3 - as the primary limiting nutrient) for Halothece sp. Simultaneous co-limitation of PO4 3- and Fe was found for both species tested, and high levels of Fe (especially when added with high PO4 3- levels) inhibited the growth of Halothece sp. Nutrient limitation (PO4 3-, Fe, and/or NO3 -) enhanced oxidative stress responses, morphological changes, and apoptosis. Furthermore, an extensive bio-informatic analysis describing the predicted Pho, Fur, and NtcA regulons (involved in the survival of cells to P, Fe, and N limitation) was made using the complete genome of Halothece sp. as a model, showing the potential of this strain to adapt to different nutrient regimes (P, Fe, or N).

"The Good, the Bad and the Double-Sword" Effects of Microplastics and Their Organic Additives in Marine Bacteria.

Little is known about the direct effects of microplastics (MPs) and their organic additives on marine bacteria, considering their role in the nutrient cycles, e.g., N-cycles through the N2-fixation, or in the microbial food web. To fill this gap of knowledge, we exposed marine bacteria, specifically diazotrophs, to pure MPs which differ in physical properties (e.g., density, hydrophobicity, and/or size), namely, polyethylene, polypropylene, polyvinyl chloride and polystyrene, and to their most abundant associated organic additives (e.g., fluoranthene, 1,2,5,6,9,10-hexabromocyclododecane and dioctyl-phthalate). Growth, protein overproduction, direct physical interactions between MPs and bacteria, phosphorus acquisition mechanisms and/or N2-fixation rates were evaluated. Cyanobacteria were positively affected by environmental and high concentrations of MPs, as opposed to heterotrophic strains, that were only positively affected with high concentrations of ~120 µm-size MPs (detecting the overproduction of proteins related to plastic degradation and C-transport), and negatively affected by 1 µm-size PS beads. Generally, the organic additives had a deleterious effect in both autotrophic and heterotrophic bacteria and the magnitude of the effect is suggested to be dependent on bacterial size. Our results show species-specific responses of the autotrophic and heterotrophic bacteria tested and the responses (beneficial: the "good," deleterious: the "bad" and/or both: the "double-sword") were dependent on the type and concentration of MPs and additives. This suggests the need to determine the threshold levels of MPs and additives concentrations starting from which significant effects can be observed for key microbial populations in marine systems, and these data are necessary for effective environmental quality control management.

The Role of Iron in the P-Acquisition Mechanisms of the Unicellular N2-Fixing Cyanobacteria Halothece sp., Found in Association With the Mediterranean Seagrass Posidonia oceanica.

Posidonia oceanica, an endemic seagrass of the Mediterranean Sea harbors a high diversity of N2-fixing prokaryotes. One of these is Halothece sp., a unicellular N2-fixing cyanobacteria detected through nifH analysis from the epiphytes of P. oceanica. The most related strain in culture is Halothece sp. PCC 7418 and this was used as the test organism in this study. In the Mediterranean Sea, phosphorus (P) and iron (Fe) can be the major limiting nutrients for N2 fixation. However, information about the mechanisms of P-acquisition and the role of metals (i.e., Fe) in these processes for N2-fixing bacteria is scarce. From our genomic analyses of the test organism and other phylogenetically related N2-fixing strains, Halothece sp. PCC 7418 is one of the strains with the greatest number of gene copies (eight copies) of alkaline phosphatases (APases). Our structural analysis of PhoD (alkaline phosphatase type D) and PhoU (phosphate acquisition regulator) of Halothece sp. PCC 7418 showed the connection among metals (Ca2+ and Fe3+), and the P-acquisition mechanisms. Here, we measured the rates of alkaline phosphatase activity (APA) through MUF-P hydrolysis under different combinations of concentrations of inorganic P ( P O 4 3 - ) and Fe in experiments under N2-fixing (low NO 3 - availability) and non-N2 fixing (high NO 3 - availability) conditions. Our results showed that APA rates were enhanced by the increase in Fe availability under low levels of P O 4 3 - , especially under N2-fixing conditions. Moreover, the increased P O 4 3 - -uptake was reflected in the increased of the P-cellular content of the cells under N2 fixation conditions. We also found a positive significant relationship between cellular P and cellular Fe content of the cells (r 2 = 0.71, p < 0.05). Our results also indicated that Fe-uptake in Halothece sp. PCC 7418 was P and Fe-dependent. This study gives first insights of P-acquisition mechanisms in the N2-fixing cyanobacteria (Halothece sp.) found in P. oceanica and highlights the role of Fe in these processes.