The Marine-Origin Exopolysaccharide-Producing Bacteria Micrococcus Antarcticus HZ Inhibits Pb Uptake in Pakchoi (Brassica chinensis L.) and Affects Rhizosphere Microbial Communities.

Exopolysaccharides (EPSs) produced by microorganisms play an important role in biotolerance and reducing heavy metal (HM) contamination by limiting the migration of HMs into plants. However, research on the application of EPS-producing marine bacteria for soil heavy metal remediation remains limited, particularly regarding their mechanisms of HM immobilization in soil and impact on plant growth. In this study, the EPS-producing marine bacterium Micrococcus antarcticus HZ was investigated for its ability to immobilize Pb and produce EPSs in soil filtrate. The effects on the growth quality and biomass of pakchoi (Brassica chinensis L.), as well as bacterial communities in inter-root soil contaminated with Pb, were also investigated. The results indicated that HZ could reduce the Pb concentration in the soil filtrate, achieving a removal rate of 43.25-63.5%. The EPS content and pH levels increased in the presence of Pb. Pot experiments showed that adding HZ significantly increased the biomass of pakchoi (9.45-14.69%), vitamin C (Vc) (9.69-12.92%), and soluble protein content (22.58-49.7%). HZ reduced the Pb content in the roots (17.52-47.48%) and leaves (edible tissues) (43.82-52.83%) of pakchoi. HZ increased soil enzyme activities (alkaline phosphatase, dehydrogenase, and urease), and the contents of ammonium nitrogen and nitrate nitrogen. Additionally, HZ also increased the relative abundance of beneficial bacteria (e.g., Proteobacteria, Cyanobacteria, and Chlorobacteria) in the inter-root soil, which have prophylactic and heavy-metal fixation functions. In summary, HZ reduces effective Pb content in edible tissues, roots, and inter-root soil by regulating inter-root soil microbial community structure, increasing soil pH, nitrogen content, and soil enzyme activity, and altering dominant phylum abundance.

Widespread production of plant growth-promoting hormones among marine bacteria and their impacts on the growth of a marine diatom.

BACKGROUND: Reciprocal exchanges of metabolites between phytoplankton and bacteria influence the fitness of these microorganisms which ultimately shapes the productivity of marine ecosystems. Recent evidence suggests that plant growth-promoting hormones may be key metabolites within mutualistic phytoplankton-bacteria partnerships, but very little is known about the diversity of plant growth-promoting hormones produced by marine bacteria and their specific effects on phytoplankton growth. Here, we aimed to investigate the capacity of marine bacteria to produce 7 plant growth-promoting hormones and the effects of these hormones on Actinocyclus sp. growth. RESULTS: We examined the plant growth-promoting hormone synthesis capabilities of 14 bacterial strains that enhance the growth of the common diatom Actinocyclus. Plant growth-promoting hormone biosynthesis was ubiquitous among the bacteria tested. Indeed all 14 strains displayed the genomic potential to synthesise multiple hormones, and mass-spectrometry confirmed that each strain produced at least 6 out of the 7 tested plant growth-promoting hormones. Some of the plant growth-promoting hormones identified here, such as brassinolide and trans-zeatin, have never been reported in marine microorganisms. Importantly, all strains produced the hormone indole-3 acetic acid (IAA) in high concentrations and released it into their surroundings. Furthermore, indole-3 acetic acid extracellular concentrations were positively correlated with the ability of each strain to promote Actinocyclus growth. When inoculated with axenic Actinocyclus cultures, only indole-3 acetic acid and gibberellic acid enhanced the growth of the diatom, with cultures exposed to indole-3 acetic acid exhibiting a two-fold increase in cell numbers. CONCLUSION: Our results reveal that marine bacteria produce a much broader range of plant growth-promoting hormones than previously suspected and that some of these compounds enhance the growth of a marine diatom. These findings suggest plant growth-promoting hormones play a large role in microbial communication and broaden our knowledge of their fuctions in the marine environment. Video Abstract.

Application of the luminous bacterium Photobacterium phosphoreum for toxicity monitoring of selenite and its reduction to selenium(0) nanoparticles.

Luminous marine bacteria are traditionally used as a bioassay due to the convenience and high rate of registering the intensity of their physiological function - luminescence. This study aimed to develop the application of Photobacterium phosphoreum in traditional and novel fields - toxicity monitoring and biotechnology. We demonstrated (1) effects of selenite ions on bioluminescence, and (2) biotransformation of selenite to selenium(0) in the form of nanoparticles. The effects of selenite (SeO32-) on the intensity of bacterial bioluminescence were studied, and its dependencies on exposure time and concentration of Na2SeO3 were analyzed. Bioluminescence activation and inhibition were revealed; dose-effect dependencies corresponded to the hormesis model. The toxicity of SeO32- was characterized by an effective concentration of 10-3 M. Effects of SeO32- on reactive oxygen species (ROS) in bacterial suspensions were studied. High positive correlations were found between the bioluminescence intensity and ROS content, which indicates the decisive role of ROS and associated redox processes in the bioeffects of selenite ions. Scanning and transmission electron microscopy revealed the presence of nano-structures in the bacteria exposed to selenite. The energy dispersion spectrum detected a high content of selenium in the nanoparticles. The particle size distribution depended on Na2SeO3 concentration; maxima of the distribution varied within 45-55 nm.

Genomics, Proteomics, and Antifungal Activity of Chitinase from the Antarctic Marine Bacterium Curtobacterium sp. CBMAI 2942.

This study aimed to evaluate the genomic profile of the Antarctic marine Curtobacterium sp. CBMAI 2942, as well as to optimize the conditions for chitinase production and antifungal potential for biological control. Assembly and annotation of the genome confirmed the genomic potential for chitinase synthesis, revealing two ChBDs of chitin binding (Chi C). The optimization enzyme production using an experimental design resulted in a 3.7-fold increase in chitinase production. The chitinase enzyme was identified by SDS-PAGE and confirmed through mass spectrometry analysis. The enzymatic extract obtained using acetone showed antifungal activity against the phytopathogenic fungus Aspergillus sp. series Nigri CBMAI 1846. The genetic capability of Curtobacterium sp. CBMAI 2942 for chitin degradation was confirmed through genomic analysis. The basal culture medium was adjusted, and the chitinase produced by this isolate from Antarctica showed significant inhibition against Aspergillus sp. Nigri series CBMAI 1846, which is a tomato phytopathogenic fungus. This suggests that this marine bacterium could potentially be used as a biological control of agricultural pests.

Impact of chitin-derived ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine on chitinase upregulation in Shewanella baltica.

The first steps in chitin degradation in marine bacteria involve chitinase, which produces N,N'-diacetylchitobiose (GlcNAc)2 from chitin. Moreover, in Vibrio bacteria, chitinase activity is enhanced by heterodisaccharide ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) produced from (GlcNAc)2 by chitin oligosaccharide deacetylase (COD). However, the role of COD in other marine bacteria, such as Shewanella, remains unexplored. This study investigates GlcNAc-GlcN's impact on chitinase gene expression and enzyme production in S. baltica ATCC BAA-1091, drawing parallels with Vibrio parahaemolyticus RIMD2210633. Using real-time quantitative PCR, the study assesses the upregulation of chitinase gene expression in S. baltica in response to GlcNAc-GlcN, informed by COD's known ability to produce GlcNAc-GlcN from (GlcNAc)2. In Vibrio, GlcNAc-GlcN considerably upregulates chitinase gene expression. This study posits a similar regulatory mechanism in S. baltica, with preliminary investigations indicating COD's capacity to produce GlcNAc-GlcN. This study highlights the importance of exploring GlcNAc-GlcN's regulatory role in chitin metabolism across diverse marine bacteria. The potential induction of chitinase production in S. baltica suggests broader ecological implications. Further research is crucial for a comprehensive understanding of chitin utilization and regulatory pathways in marine bacterial genera.

Marine Delivery Vehicles: Molecular Components and Applications of Bacterial Extracellular Vesicles.

In marine ecosystems, communication among microorganisms is crucial since the distance is significant if considered on a microbial scale. One of the ways to reduce this gap is through the production of extracellular vesicles, which can transport molecules to guarantee nutrients to the cells. Marine bacteria release extracellular vesicles (EVs), small membrane-bound structures of 40 nm to 1 µm diameter, into their surrounding environment. The vesicles contain various cellular compounds, including lipids, proteins, nucleic acids, and glycans. EVs may contribute to dissolved organic carbon, thus facilitating heterotroph growth. This review will focus on marine bacterial EVs, analyzing their structure, composition, functions, and applications.

A Novel Bacitracin-like Peptide from Mangrove-Isolated Bacillus paralicheniformis NNS4-3 against MRSA and Its Genomic Insights.

The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

Complete genome sequence of the 4-hydroxybenzoate-degrading bacterium Gymnodinialimonas sp. 57CJ19, a potential novel species from intertidal sediments.

A bacterium Gymnodinialimonas sp. 57CJ19, was isolated from the intertidal sediments of Aoshan Bay, and further assays showed that it has the ability to degrade the antibacterial preservative 4-hydroxybenzoate. The complete genome sequence was sequenced, and phylogenomic analyses indicated that strain 57CJ19 represents a potential novel species in the genus Gymnodinialimonas (family Rhodobacteraceae). Its genome contains a 3,861,607-bp circular chromosome with 61.25% G + C content. Gene prediction revealed 3716 protein-encoding genes, 41 tRNA genes, 3 rrn operons, and 3 non-coding RNA genes. Functional annotation revealed a complete metabolic pathway for 4-hydroxybenzoate. The genome sequence of strain 57CJ19 provides new insights into the potential and underlying genomic basis of aromatic compound pollutant degradation by marine bacteria.

Whole-Cell Biosensor for Iron Monitoring as a Potential Tool for Safeguarding Biodiversity in Polar Marine Environments.

Iron is a key micronutrient essential for various essential biological processes. As a consequence, alteration in iron concentration in seawater can deeply influence marine biodiversity. In polar marine environments, where environmental conditions are characterized by low temperatures, the role of iron becomes particularly significant. While iron limitation can negatively influence primary production and nutrient cycling, excessive iron concentrations can lead to harmful algal blooms and oxygen depletion. Furthermore, the growth of certain phytoplankton species can be increased in high-iron-content environments, resulting in altered balance in the marine food web and reduced biodiversity. Although many chemical/physical methods are established for inorganic iron quantification, the determination of the bio-available iron in seawater samples is more suitably carried out using marine microorganisms as biosensors. Despite existing challenges, whole-cell biosensors offer other advantages, such as real-time detection, cost-effectiveness, and ease of manipulation, making them promising tools for monitoring environmental iron levels in polar marine ecosystems. In this review, we discuss fundamental biosensor designs and assemblies, arranging host features, transcription factors, reporter proteins, and detection methods. The progress in the genetic manipulation of iron-responsive regulatory and reporter modules is also addressed to the optimization of the biosensor performance, focusing on the improvement of sensitivity and specificity.

The Phylogeny and Metabolic Potentials of an Aromatics-Degrading Marivivens Bacterium Isolated from Intertidal Seawater in East China Sea.

Lignocellulosic materials, made up of cellulose, hemicellulose, and lignin, constitute some of the most prevalent types of biopolymers in marine ecosystems. The degree to which marine microorganisms participate in the breakdown of lignin and their impact on the cycling of carbon in the oceans is not well understood. Strain LCG002, a novel Marivivens species isolated from Lu Chao Harbor's intertidal seawater, is distinguished by its ability to metabolize lignin and various aromatic compounds, including benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. It also demonstrates a broad range of carbon source utilization, including carbohydrates, amino acids and carboxylates. Furthermore, it can oxidize inorganic gases, such as hydrogen and carbon monoxide, providing alternative energy sources in diverse marine environments. Its diversity of nitrogen metabolism is supported by nitrate/nitrite, urea, ammonium, putrescine transporters, as well as assimilatory nitrate reductase. For sulfur assimilation, it employs various pathways to utilize organic and inorganic substrates, including the SOX system and DSMP utilization. Overall, LCG002's metabolic versatility and genetic profile contribute to its ecological significance in marine environments, particularly in the degradation of lignocellulosic material and aromatic monomers.

Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan.

A Gram-negative, aerobic, rod-shaped, non-motile, yellow-pigmented bacterium, KMM 9835T, was isolated from the sediment sample obtained from the Amur Bay of the Sea of Japan seashore, Russia. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences positioned the novel strain KMM 9835T in the genus Mariniflexile as a separate line sharing the highest 16S rRNA gene sequence similarities of 96.6% and 96.2% with Mariniflexile soesokkakense RSSK-9T and Mariniflexile fucanivorans SW5T, respectively, and similarity values of <96% to other recognized Mariniflexile species. The average nucleotide identity and digital DNA-DNA hybridization values between strain KMM 9835T and M. soesokkakense KCTC 32427T, Mariniflexile gromovii KCTC 12570T, M. fucanivorans DSM 18792T, and M. maritimum M5A1MT were 83.0%, 82.5%, 83.4%, and 78.3% and 30.7%, 29.6%, 29.5%, and 24.4%, respectively. The genomic DNA GC content of strain KMM 9835T was 32.5 mol%. The dominant menaquinone was MK-6, and the major fatty acids were iso-C15:0, iso-C15:1ω10c, and C15:0. The polar lipids of strain KMM 9835T consisted of phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, and six unidentified lipids. A pan-genome analysis showed that the KMM 9835T genome encoded 753 singletons. The annotated singletons were more often related to transport protein systems (SusC), transcriptional regulators (AraC, LytTR, LacI), and enzymes (glycosylases). The KMM 9835T genome was highly enriched in CAZyme-encoding genes, the proportion of which reached 7.3%. Moreover, the KMM 9835T genome was characterized by a high abundance of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), indicating its potential to catabolize pectin. This may represent part of an adaptation strategy facilitating microbial consumption of plant polymeric substrates in aquatic environments near shorelines and freshwater sources. Based on the combination of phylogenetic and phenotypic characterization, the marine sediment strain KMM 9835T (=KCTC 92792T) represents a novel species of the genus Mariniflexile, for which the name Mariniflexile litorale sp. nov. is proposed.

Production optimization and antioxidant potential of exopolysaccharide produced by a moderately halophilic bacterium Virgibacillus dokdonensis VITP14.

This study aimed to enhance the extracellular polymeric substances (EPS) production of Virgibacillus dokdonensis VITP14 and explore its antioxidant potential. EPS and biomass production by VITP14 strain were studied under different culture parameters and media compositions using one factor at a time method. Among different nutrient sources, glucose and peptone were identified as suitable carbon and nitrogen sources. Furthermore, the maximum EPS production was observed at 5% of inoculum size, 5 g/L of NaCl, and 96 h of fermentation. Response surface methodology was employed to augment EPS production and investigate the optimal levels of nutrient sources with their interaction. The strain was observed to produce actual maximum EPS of about 26.4 g/L for finalized optimum medium containing glucose 20 g/L, peptone 10 g/L, and NaCl 50 g/L while the predicted maximum EPS was 26.5 g/L. There was a nine fold increase in EPS production after optimization study. Additionally, EPS has exhibited significant scavenging, reducing, and chelating potential (>85%) at their higher concentration. This study imparts valuable insights into optimizing moderately halophilic bacterial EPS production and evaluating its natural antioxidant properties. According to findings, V. dokdonensis VITP14 was a promising isolate that will provide significant benefits to biopolymer producing industries.

Exploring Seaweed-Associated Marine Microbes: Growth Impacts and Enzymatic Potential for Sustainable Resource Utilization.

Seaweed, a valuable marine resource widely cultivated worldwide, can be vulnerable to stress and microbiome alterations, resulting in the decay of seaweeds and substantial economic losses. To investigate the seaweed-microbiome interaction, our study aimed to isolate marine bacteria and fungi that can cause Ice-Ice disease and evaluate their enzymatic characteristics for potential application in bioethanol production from seaweed biomass. Three red seaweed species (Gracilaria edulis, Kappaphycus alvarezii, and Eucheuma cottonii) were obtained for our study and placed in separate culture tanks. Among the 18 isolated marine microbial species, 12 tested positive for agar and carrageenan activity: six exhibited both activities, three displayed only agar activity, and three only carrageenan activity. DNA sequencing of the positive microbes identified ten bacteria and two yeast species. The 3,5-Dinitrosalicylic acid (DNSA) assay results revealed that the identified bacterial Caldibacillus kokeshiiformis strain FJAT-47861 exhibited the highest carrageenase activity (0.76 units/ml), while the yeast Pichia fermentans strain PM79 demonstrated the highest agarase activity (0.52 units/ml). Notably, Pichia fermentans strain PM79 exhibited the highest overall agarase and carrageenase activity, averaging 0.63 units/ml. The average carrageenase activity of all six positive microbes was 1.5 times higher than their agarase activity. These findings suggest that the 12 isolated microbes hold potential for bioethanol production from macroalgae, as their agarase and carrageenase activity indicates their ability to break down seaweed cell wall carbohydrates, causing ice-ice disease. Moreover, these results provide exciting prospects for harnessing the bioconversion capabilities of these microbes, paving the way for sustainable and efficient bioethanol production from seaweed resources. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01205-w.

Marine bacteria Alteromonas spp. require UDP-glucose-4-epimerase for aggregation and production of sticky exopolymer.

The physiology and ecology of particle-associated marine bacteria are of growing interest, but our knowledge of their aggregation behavior and mechanisms controlling their association with particles remains limited. We have found that a particle-associated isolate, Alteromonas sp. ALT199 strain 4B03, and the related type-strain A. macleodii 27126 both form large (>500 µm) aggregates while growing in rich medium. A non-clumping variant (NCV) of 4B03 spontaneously arose in the lab, and whole-genome sequencing revealed a partial deletion in the gene encoding UDP-glucose-4-epimerase (galEΔ308-324). In 27126, a knock-out of galE (ΔgalE::kmr) resulted in a loss of aggregation, mimicking the NCV. Microscopic analysis shows that both 4B03 and 27126 rapidly form large aggregates, whereas their respective galE mutants remain primarily as single planktonic cells or clusters of a few cells. Strains 4B03 and 27126 also form aggregates with chitin particles, but their galE mutants do not. Alcian Blue staining shows that 4B03 and 27126 produce large transparent exopolymer particles (TEP), but their galE mutants are deficient in this regard. This study demonstrates the capabilities of cell-cell aggregation, aggregation of chitin particles, and production of TEP in strains of Alteromonas, a widespread particle-associated genus of heterotrophic marine bacteria. A genetic requirement for galE is evident for each of the above capabilities, expanding the known breadth of requirement for this gene in biofilm-related processes. IMPORTANCE: Heterotrophic marine bacteria have a central role in the global carbon cycle. Well-known for releasing CO2 by decomposition and respiration, they may also contribute to particulate organic matter (POM) aggregation, which can promote CO2 sequestration via the formation of marine snow. We find that two members of the prevalent particle-associated genus Alteromonas can form aggregates comprising cells alone or cells and chitin particles, indicating their ability to drive POM aggregation. In line with their multivalent aggregation capability, both strains produce TEP, an excreted polysaccharide central to POM aggregation in the ocean. We demonstrate a genetic requirement for galE in aggregation and large TEP formation, building our mechanistic understanding of these aggregative capabilities. These findings point toward a role for heterotrophic bacteria in POM aggregation in the ocean and support broader efforts to understand bacterial controls on the global carbon cycle based on microbial activities, community structure, and meta-omic profiling.

Screening of inhibitors on successful covalent tyrosinase coupling with help from SpyBank.

Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag-TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag-TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1H NMR and 13C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems.

Genomic analysis of Alteromonas sp. M12 isolated from the Mariana Trench reveals its role in dimethylsulfoniopropionate cycling.

Dimethylsulfoniopropionate (DMSP) is a ubiquitous organosulfur molecule in marine environments with important roles in stress tolerance, global carbon and sulfur cycling, and chemotaxis. It is the main precursor of the climate active gas dimethyl sulfide (DMS), which is the greatest natural source of bio­sulfur transferred from ocean to atmosphere. Alteromonas sp. M12, a Gram-negative and aerobic bacterium, was isolated from the seawater samples collected from the Mariana Trench at the depth of 2500 m. Here, we report the complete genome sequence of strain M12 and its genomic characteristics to import and utilize DMSP. The genome of strain M12 contains one circular chromosome (5,012,782 bp) with the GC content of 40.88%. Alteromonas sp. M12 can grow with DMSP as a sole carbon source, and produced DMS with DMSP as a precursor. Genomic analysis showed that strain M12 contained a set of genes involved in the downstream steps of DMSP cleavage, but no known genes encoding DMSP transporters or DMSP lyases. The results indicated that this strain contained novel DMSP transport and cleavage genes in its genome which warrants further investigation. The import of DMSP into cells may be a strategy of strain M12 to adapt the hydrostatic pressure environment in the Mariana Trench, as DMSP can be used as a hydrostatic pressure protectant. This study sheds light on the catabolism of DMSP by deep-sea bacteria.

ι-Carrageenan catabolism is initiated by key sulfatases in the marine bacterium Pseudoalteromonas haloplanktis LL1.

Marine bacteria contribute substantially to cycle macroalgae polysaccharides in marine environments. Carrageenans are the primary cell wall polysaccharides of red macroalgae. The carrageenan catabolism mechanism and pathways are still largely unclear. Pseudoalteromonas is a representative bacterial genus that can utilize carrageenan. We previously isolated the strain Pseudoalteromonas haloplanktis LL1 that could grow on ι-carrageenan but produce no ι-carrageenase. Here, through a combination of bioinformatic, biochemical, and genetic analyses, we determined that P. haloplanktis LL1 processed a desulfurization-depolymerization sequential pathway for ι-carrageenan utilization, which was initiated by key sulfatases PhSulf1 and PhSulf2. PhSulf2 acted as an endo/exo-G4S (4-O-sulfation-ß-D-galactopyranose) sulfatase, while PhSulf1 was identified as a novel endo-DA2S sulfatase that could function extracellularly. Because of the unique activity of PhSulf1 toward ι-carrageenan rather than oligosaccharides, P. haloplanktis LL1 was considered to have a distinct ι-carrageenan catabolic pathway compared to other known ι-carrageenan-degrading bacteria, which mainly employ multifunctional G4S sulfatases and exo-DA2S (2-O-sulfation-3,6-anhydro-α-D-galactopyranose) sulfatase for sulfate removal. Furthermore, we detected widespread occurrence of PhSulf1-encoding gene homologs in the global ocean, indicating the prevalence of such endo-acting DA2S sulfatases as well as the related ι-carrageenan catabolism pathway. This research provides valuable insights into the enzymatic processes involved in carrageenan catabolism within marine ecological systems.IMPORTANCECarrageenan is a type of linear sulfated polysaccharide that plays a significant role in forming cell walls of marine algae and is found extensively distributed throughout the world's oceans. To the best of our current knowledge, the ι-carrageenan catabolism in marine bacteria either follows the depolymerization-desulfurization sequential process initiated by ι-carrageenase or starts from the desulfurization step catalyzed by exo-acting sulfatases. In this study, we found that the marine bacterium Pseudoalteromonas haloplanktis LL1 processes a distinct pathway for ι-carrageenan catabolism employing a specific endo-acting DA2S-sulfatase PhSulf1 and a multifunctional G4S sulfatase PhSulf2. The unique PhSulf1 homologs appear to be widely present on a global scale, indicating the indispensable contribution of the marine bacteria containing the distinct ι-carrageenan catabolism pathway. Therefore, this study would significantly enrich our understanding of the molecular mechanisms underlying carrageenan utilization, providing valuable insights into the intricate roles of marine bacteria in polysaccharide cycling in marine environments.

Blue valorization of lignin-derived monomers via reprogramming marine bacterium Roseovarius nubinhibens.

Biological valorization of lignin, the second most abundant biopolymer on Earth, is an indispensable sector to build a circular economy and net-zero future. However, lignin is recalcitrant to bioupcycling, demanding innovative solutions. We report here the biological valorization of lignin-derived aromatic carbon to value-added chemicals without requesting extra organic carbon and freshwater via reprogramming the marine Roseobacter clade bacterium Roseovarius nubinhibens. We discovered the unusual advantages of this strain for the oxidation of lignin monomers and implemented a CRISPR interference (CRISPRi) system with the lacI-Ptrc inducible module, nuclease-deactivated Cas9, and programmable gRNAs. This is the first CRISPR-based regulatory system in R. nubinhibens, enabling precise and efficient repression of genes of interest. By deploying the customized CRISPRi, we reprogrammed the carbon flux from a lignin monomer, 4-hydroxybenzoate, to achieve the maximum production of protocatechuate, a pharmaceutical compound with antibacterial, antioxidant, and anticancer properties, with minimal carbon to maintain cell growth and drive biocatalysis. As a result, we achieved a 4.89-fold increase in protocatechuate yield with a dual-targeting CRISPRi system, and the system was demonstrated with real seawater. Our work underscores the power of CRISPRi in exploiting novel microbial chassis and will accelerate the development of marine synthetic biology. Meanwhile, the introduction of a new-to-the-field lineage of marine bacteria unveils the potential of blue biotechnology leveraging resources from the ocean.IMPORTANCEOne often overlooked sector in carbon-conservative biotechnology is the water resource that sustains these enabling technologies. Similar to the "food-versus-fuel" debate, the competition of freshwater between human demands and bioproduction is another controversial issue, especially under global water scarcity. Here, we bring a new-to-the-field lineage of marine bacteria with unusual advantages to the stage of engineering biology for simultaneous carbon and water conservation. We report the valorization of lignin monomers to pharmaceutical compounds without requesting extra organic substrate (e.g., glucose) or freshwater by reprogramming the marine bacterium Roseovarius nubinhibens with a multiplex CRISPR interference system. Beyond the blue lignin valorization, we present a proof-of-principle of leveraging marine bacteria and engineering biology for a sustainable future.

Genomic and phenotypic characterization of 26 novel marine bacterial strains with relevant biogeochemical roles and widespread presence across the global ocean.

Prokaryotes dominate global oceans and shape biogeochemical cycles, yet most taxa remain uncultured and uncharacterized as of today. Here we present the characterization of 26 novel marine bacterial strains from a large isolate collection obtained from Blanes Bay (NW Mediterranean) microcosm experiments made in the four seasons. Morphological, cultural, biochemical, physiological, nutritional, genomic, and phylogenomic analyses were used to characterize and phylogenetically place the novel isolates. The strains represent 23 novel bacterial species and six novel genera: three novel species pertaining to class Alphaproteobacteria (families Rhodobacteraceae and Sphingomonadaceae), six novel species and three new genera from class Gammaproteobacteria (families Algiphilaceae, Salinispheraceae, and Alteromonadaceae), 13 novel species and three novel genera from class Bacteroidia (family Flavobacteriaceae), and one new species from class Rhodothermia (family Rubricoccaceae). The bacteria described here have potentially relevant roles in the cycles of carbon (e.g., carbon fixation or energy production via proteorhodopsin), nitrogen (e.g., denitrification or use of urea), sulfur (oxidation of sulfur compounds), phosphorus (acquisition and use of different forms of phosphorus and remodeling of membrane phospholipids), and hydrogen (oxidation of hydrogen to obtain energy). We mapped the genomes of the presented strains to the Tara Oceans metagenomes to reveal that these strains were globally distributed, with those of the family Flavobacteriaceae being the most widespread and abundant, while Rhodothermia being the rarest and most localized. While molecular-only approaches are also important, our study stresses the importance of culturing as a powerful tool to further understand the functioning of marine bacterial communities.

The structure of a pectin-active family 1 polysaccharide lyase from the marine bacterium Pseudoalteromonas fuliginea.

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Šresolution X-ray crystal structure of PfPL1 reveals the compact parallel ß-helix fold of the PL1 family. The back side of the core parallel ß-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2.