Neonatal Elizabethkingia anophelis meningitis originating from the water reservoir of an automated infant milk dispenser, the Netherlands, February 2024.

Elizabethkingia anophelis is a multidrug-resistant pathogen causing high mortality and morbidity in adults with comorbidities and neonates. We report a Dutch case of E. anophelis meningitis in a neonate, clonally related to samples taken from an automated infant milk dispenser located at the family's residence. We inform about the emergence of E. anophelis and suggest molecular surveillance in hospitals and other health settings. This is the first case connecting an automated formula dispenser to an invasive infection in a neonate.

Endosymbiont and gut bacterial communities of the brown-banded cockroach, Supella longipalpa.

The brown-banded cockroach (Supella longipalpa) is a widespread nuisance and public health pest. Like the German cockroach (Blattella germanica), this species is adapted to the indoor biome and completes the entirety of its life cycle in human-built structures. Recently, understanding the contributions of commensal and symbiotic microbes to the biology of cockroach pests, as well as the applications of targeting these microbes for pest control, have garnered significant scientific interest. However, relative to B. germanica, the biology of S. longipalpa, including its microbial associations, is understudied. Therefore, the goal of the present study was to quantitatively examine and characterize both the endosymbiont and gut bacterial communities of S. longipalpa for the first time. To do so, bacterial 16S rRNA gene amplicon sequencing was conducted on DNA extracts from whole adult females and males, early instar nymphs, and late instar nymphs. The results demonstrate that the gut microbiome is dominated by two genera of bacteria known to have beneficial probiotic effects in other organisms, namely Lactobacillus and Akkermansia. Furthermore, our data show a significant effect of nymphal development on diversity and variation in the gut microbiome. Lastly, we reveal significant negative correlations between the two intracellular endosymbionts, Blattabacterium and Wolbachia, as well as between Blattabacterium and the gut microbiome, suggesting that Blattabacterium endosymbionts could directly or indirectly influence the composition of other bacterial populations. These findings have implications for understanding the adaptation of S. longipalpa to the indoor biome, its divergence from other indoor cockroach pest species such as B. germanica, the development of novel control approaches that target the microbiome, and fundamental insect-microbe interactions more broadly.

Whole-genome sequence and resistance determinants of four Elizabethkingia anophelis clinical isolates collected in Hanoi, Vietnam.

Four isolates of the opportunistic pathogen Elizabethkingia anophelis were identified for the first time in a Vietnamese hospital and underwent antimicrobial susceptibility testing and genomic characterization by whole-genome sequencing. Complete, fully circularized genome sequences were obtained for all four isolates. Average Nucleotide Identity analysis and single nucleotide polymorphism phylogenetic analysis on the core genome showed that three of the four isolates were genetically distinct, ruling out the hypothesis of a single strain emergence. Antibiotic susceptibility testing highlighted multi-resistant phenotypes against most antimicrobial families, including beta-lactams, carbapenems, aminoglycosides, quinolones, macrolides, amphenicols, rifamycins and glycopeptides. Additionally, in silico genomic analysis was used to correlate the phenotypic susceptibility to putative resistance determinants, including resistance genes, point mutations and multidrug efflux pumps. Nine different resistance genes were located inside a single resistance pocket predicted to be a putative Integrative and Conjugative Element (ICE). This novel ICE was shared by three isolates from two different lineages and displayed similarity with ICEs previously reported in various Elizabethkingia and Chryseobacterium species. The role of such ICEs in pathogenicity, genome plasticity and antimicrobial resistance gene spread within the Flavobacteriaceae family needs to be further elucidated.

Opportunistic Elizabethkingia miricola Infections in Intensive Care Unit, Spain.

In 2021, we identified a cluster of Elizabethkingia miricola cases in an intensive care unit in Spain. Because E. miricola is not considered a special surveillance agent in Spain, whole-genome sequencing was not performed. The bacterial source was not identified. All Elizabethkingia species should be listed as special surveillance bacteria.

Revealing the algicidal characteristics of Maribacter dokdonensis: An investigation into bacterial strain P4 isolated from Karenia mikimotoi bloom water.

Harmful algal blooms (HABs) are a global environmental concern, causing significant economic losses in fisheries and posing risks to human health. Algicidal bacteria have been suggested as a potential solution to control HABs, but their algicidal efficacy is influenced by various factors. This study aimed to characterize a novel algicidal bacterium, Maribacter dokdonensis (P4), isolated from a Karenia mikimotoi (Hong Kong strain, KMHK) HAB and assess the impact of P4 and KMHK's doses, growth phase, and algicidal mode and the axenicity of KMHK on P4's algicidal effect. Our results demonstrated that the algicidal effect of P4 was dose-dependent, with the highest efficacy at a dose of 25% v/v. The study also determined that P4's algicidal effect was indirect, with the P4 culture and the supernatant, but not the bacterial cells, showing significant effects. The algicidal efficacy was higher when both P4 and KMHK were in the stationary phase. Furthermore, the P4 culture at the log phase could effectively kill KMHK cells at the stationary phase, with higher algicidal efficacy in the bacterial culture than that of the supernatant alone. Interestingly, P4's algicidal efficacy was significantly higher when co-culturing with xenic KMHK (~90% efficacy at day 1) than that with the axenic KMHK (~50% efficacy at day 1), suggesting the presence of other bacteria could regulate P4's algicidal effect. The bacterial strain P4 also exhibited remarkable algicidal efficacy on four other dinoflagellate species, particularly the armored species. These results provide valuable insights into the algicidal effect of M. dokdonensis on K. mikimotoi and on their interactions.

Maribacter halichondriae sp. nov., isolated from the marine sponge Halichondria panicea, displays features of a sponge-associated life style.

A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-ß-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).

Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Characterization of an outbreak caused by Elizabethkingia miricola using Fourier-transform infrared (FTIR) spectroscopy.

Fourier-transform infrared (FTIR) spectroscopy has the potential to be used for bacterial typing and outbreak characterization. We evaluated FTIR for the characterization of an outbreak caused by Elizabethkingia miricola. During the 2020-2021 period, 26 isolates (23 clinical and 3 environmental) were collected and analyzed by FTIR (IR Biotyper) and core-genome MLST (cgMLST), in addition to antimicrobial susceptibility testing. FTIR spectroscopy and cgMLST showed that 22 of the isolates were related to the outbreak, including the environmental samples, with only one discordance between both methods. Then, FTIR is useful for E. miricola typing and can be easily implemented in the laboratory.

Antibiotic Combination to Effectively Postpone or Inhibit the In Vitro Induction and Selection of Levofloxacin-Resistant Mutants in Elizabethkingia anophelis.

Fluoroquinolones are potentially active against Elizabethkingia anophelis. Rapidly increased minimum inhibitory concentrations (MICs) and emerging point mutations in the quinolone resistance-determining regions (QRDRs) following exposure to fluoroquinolones have been reported in E. anophelis. We aimed to investigate point mutations in QRDRs through exposure to levofloxacin (1 × MIC) combinations with different concentrations (0.5× and 1 × MIC) of minocycline, rifampin, cefoperazone/sulbactam, or sulfamethoxazole/trimethoprim in comparison with exposure to levofloxacin alone. Of the four E. anophelis isolates that were clinically collected, lower MICs of levofloxacin were disclosed in cycle 2 and 3 of induction and selection in all levofloxacin combination groups other than levofloxacin alone (all p = 0.04). Overall, no mutations were discovered in parC and parE throughout the multicycles inducted by levofloxacin and all its combinations. Regarding the vastly increased MICs, the second point mutations in gyrA and/or gyrB in one isolate (strain no. 1) occurred in cycle 2 following exposure to levofloxacin plus 0.5 × MIC minocycline, but they were delayed appearing in cycle 5 following exposure to levofloxacin plus 1 × MIC minocycline. Similarly, the second point mutation in gyrA and/or gyrB occurred in another isolate (strain no. 3) in cycle 4 following exposure to levofloxacin plus 0.5 × MIC sulfamethoxazole/trimethoprim, but no mutation following exposure to levofloxacin plus 1 × MIC sulfamethoxazole/trimethoprim was disclosed. In conclusion, the rapid selection of E. anophelis mutants with high MICs after levofloxacin exposure could be effectively delayed or postponed by antimicrobial combination with other in vitro active antibiotics.

Flavivirga spongiicola sp. nov. and Flavivirga abyssicola sp. nov., Isolated from Marine Environments.

Two novel Gram-stain-negative, strictly-aerobic, rod-shaped (1.2 ± 3.4 µm × 0.3 ± 0.7 µm), and non-motile marine bacterial species, designated MEBiC05379T and MEBiC07777T, were isolated from a marine sponge Pseudaxinella sp. in Gangneung City and deep-sea sediments of the Ulleung basin in the East Sea of Korea, respectively. The 16S rRNA gene sequence analysis revealed high levels of similarities between these strains and members of the genus Flavivirga (97.0-98.4% sequence identities). Both novel strains revealed as mesophilic, neutrophilic in pH and slightly halophilic. Similar to those of other Flavivirga members, the primary cellular fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C15:03-OH, and iso-C17:0 3-OH, with MEBiC05379T and MEBiC07777T containing relatively higher proportions of C12:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). In both taxa, the major isoprenoid quinone was MK-6. The DNA G + C contents of MEBiC05379T and MEBiC07777T genomes were 32.62 and 32.46 mol%, respectively. Compared to other members of Flavivirga, both strains exhibited similar DNA G + C ratio and fatty acids pattern, yet enzyme expression and carbon sources utilization pattern were different. Genomes of the genus Flavivirga showed enzyme preferences to fucoidan and sulfated galactans. Considering the monophyly rule, AAI values delineate the genus Flavivirga from adjacent genera calculated to be 76.0-78.7%. Based on the phenotypic, genomic and biochemical data, strains for MEBiC05379T and MEBiC07777T thus represent two novel species in the genus Flavivirga, for which the names Flavivirga spongiicola sp. nov. (MEBiC05379T [= KCTC 92527 T = JCM 16662 T]), and Flavivirga abyssicola sp. nov. (MEBiC07777T [= KCTC 92563 T = JCM 36477 T]) are proposed.

In situ community transcriptomics illuminates CO2-fixation potentials and supporting roles of phagotrophy and proton pump in plankton in a subtropical marginal sea.

Lineage-wise physiological activities of plankton communities in the ocean are important but challenging to characterize. Here, we conducted whole-assemblage metatranscriptomic profiling at continental shelf and slope sites in the South China Sea to investigate carbon fixation potential in different lineages. RuBisCO expression, the proxy of Calvin carbon fixation (CCF) potential, was mainly contributed by Bacillariophyta, Chlorophyta, Cyanobacteria, and Haptophyta, which was differentially affected by environmental factors among lineages. CCF potential exhibited positive or negative correlations with phagotrophy gene expression, suggesting phagotrophy possibly enhances or complements CCF. Our data also reveal significant non-Calvin carbon fixation (NCF) potential, as indicated by the active expression of genes in all five currently recognized NCF pathways, mainly contributed by Flavobacteriales, Alteromonadales, and Oceanospirillales. Furthermore, in Flavobacteriales, Alteromonadales, Pelagibacterales, and Rhodobacterales, NCF potential was positively correlated with proton-pump rhodopsin (PPR) expression, suggesting that NCF might be energetically supported by PPR. The novel insights into the lineage-differential potential of carbon fixation, widespread mixotrophy, and PPR as an energy source for NCF lay a methodological and informational foundation for further research to understand carbon fixation and the trophic landscape in the ocean.IMPORTANCEMarine plankton plays an important role in global carbon cycling and climate regulation. Phytoplankton and cyanobacteria fix CO2 to produce organic compounds using solar energy and mainly by the Calvin cycle, whereas autotrophic bacteria and archaea may fix CO2 by non-Calvin cycle carbon fixation pathways. How active individual lineages are in carbon fixation and mixotrophy, and what energy source bacteria may employ in non-Calvin carbon fixation, in a natural plankton assemblage are poorly understood and underexplored. Using metatranscriptomics, we studied carbon fixation in marine plankton with lineage resolution in tropical marginal shelf and slope areas. Based on the sequencing results, we characterized the carbon fixation potential of different lineages and assessed Calvin- and non-Calvin- carbon fixation activities and energy sources. Data revealed a high number of unigenes (4.4 million), lineage-dependent differential potentials of Calvin carbon fixation and responses to environmental conditions, major contributors of non-Calvin carbon fixation, and their potential energy source.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides.

BACKGROUND: Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. RESULTS: Prominent active 0.2-3 µm free-living clades comprised Aurantivirga, "Formosa", Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3-10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.

Preliminary identification and semi-quantitative characterization of a multi-faceted high-stability alginate lyase from marine microbe Seonamhaeicola algicola with anti-biofilm effect on Pseudomonas aeruginosa.

Alginate lyases with unique characteristics for degrading alginate into size-defined oligosaccharide fractions, were considered as the potential agents for disrupting Pseudomonas aeruginosa biofilms. In our study, a novel endolytic PL-7 alginate lyase, named AlyG2, was cloned and expressed through Escherichia coli. This enzyme exhibited excellent properties: it maintained more than 85% activity at low temperatures of 4 °C and high temperatures of 70 °C. After 1 h of incubation at 4 °C, it still retained over 95% activity, demonstrating the ability to withstand low temperature. The acid-base and salt tolerance properties shown it preserves more than 50% activity in the pH range of 5.0 to 11.0 and in a high salt environment at 3000 mM NacCl, indicating its high stability in several aspects. More importantly, AlyG2 in our research was revealed to be effective at removing mature biofilms and inhibiting biofilm formation produced by Pseudomonas aeruginosa, and the inhibition and disruption rates were 47.25 ± 4.52% and 26.5 ± 6.72%, respectively. Additionally, the enzyme AlyG2 promoted biofilm disruption in combination with antibiotics, particularly manifesting the synergistic effect with erythromycin (FIC=0.5). In all, these results offered that AlyG2 with unique characteristics may be an effective technique for the clearance or disruption of biofilm produced by P. aeruginosa.

Cation-π Interactions Greatly Influence Ion Transportability of the Light-Driven Sodium Pump KR2: A Molecular Dynamics Study.

Krokinobacter eikastus rhodopsin 2 (KR2) is a typical light-driven sodium pump. Although wild-type KR2 exhibits high Na+ selectivity, mutagenesis performed on the residues constituting the entrance enables permeation of K+ and Cs+, while the underlying mechanism remains elusive. This study presents a comprehensive molecular dynamics investigation, including force field optimization, metadynamics, and alchemical free energy methods, to explore the N61L/G263F mutant of KR2, which exhibits transportability for K+ and Cs+. The introduced Phe263 residue can directly promote ion binding at the entrance through cation-π interactions, while the N61L mutation can enhance ion binding at Phe46 by relieving steric hindrance. These results suggest that cation-π interactions may significantly influence the ion transportability and selectivity of KR2, which can provide important insights for protein engineering and the design of artificial ion transporters.

Exploring a novel ß-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

Aquimarina aquimarini sp. nov. and Aquimarina spinulae sp. nov., novel bacterial species with versatile natural product biosynthesis potential isolated from marine sponges.

This study describes two Gram-negative, flexirubin-producing, biofilm-forming, motile-by-gliding and rod-shaped bacteria, isolated from the marine sponges Ircinia variabilis and Sarcotragus spinosulus collected off the coast of Algarve, Portugal. Both strains, designated Aq135T and Aq349T, were classified into the genus Aquimarina by means of 16S rRNA gene sequencing. We then performed phylogenetic, phylogenomic and biochemical analyses to determine whether these strains represent novel Aquimarina species. Whereas the closest 16S rRNA gene relatives to strain Aq135T were Aquimarina macrocephali JAMB N27T (97.8 %) and Aquimarina sediminis w01T (97.1 %), strain Aq349T was more closely related to Aquimarina megaterium XH134T (99.2 %) and Aquimarina atlantica 22II-S11-z7T (98.1 %). Both strains showed genome-wide average nucleotide identity scores below the species level cut-off (95 %) with all Aquimarina type strains with publicly available genomes, including their closest relatives. Digital DNA-DNA hybridization further suggested a novel species status for both strains since values lower than 70 % hybridization level with other Aquimarina type strains were obtained. Strains Aq135T and Aq349T grew from 4 to 30°C and with between 1-5 % (w/v) NaCl in marine broth. The most abundant fatty acids were iso-C17 : 03-OH and iso-C15 : 0 and the only respiratory quinone was MK-6. Strain Aq135T was catalase-positive and ß-galactosidase-negative, while Aq349T was catalase-negative and ß-galactosidase-positive. These strains hold unique sets of secondary metabolite biosynthetic gene clusters and are known to produce the peptide antibiotics aquimarins (Aq135T) and the trans-AT polyketide cuniculene (Aq349T), respectively. Based on the polyphasic approach employed in this study, we propose the novel species names Aquimarina aquimarini sp. nov. (type strain Aq135T=DSM 115833T=UCCCB 169T=ATCC TSD-360T) and Aquimarina spinulae sp. nov. (type strain Aq349T=DSM 115834T=UCCCB 170T=ATCC TSD-361T).

The catabolic specialization of the marine bacterium Polaribacter sp. Q13 to red algal ß1,3/1,4-mixed-linkage xylan.

Catabolism of algal polysaccharides by marine bacteria is a significant process of marine carbon cycling. ß1,3/1,4-Mixed-linkage xylan (MLX) is a class of xylan in the ocean, widely present in the cell walls of red algae. However, the catabolic mechanism of MLX by marine bacteria remains elusive. Recently, we found that a marine Bacteroidetes strain, Polaribacter sp. Q13, is a specialist in degrading MLX, which secretes a novel MLX-specific xylanase. Here, the catabolic specialization of strain Q13 to MLX was studied by multiomics and biochemical analyses. Strain Q13 catabolizes MLX with a canonical starch utilization system (Sus), which is encoded by a single xylan utilization locus, XUL-Q13. In this system, the cell surface glycan-binding protein SGBP-B captures MLX specifically, contributing to the catabolic specificity. The xylanolytic enzyme system of strain Q13 is unique, and the enzymatic cascade dedicates the stepwise hydrolysis of the ß1,3- and ß1,4-linkages in MLX in the extracellular, periplasmic, and cytoplasmic spaces. Bioinformatics analysis and growth observation suggest that other marine Bacteroidetes strains harboring homologous MLX utilization loci also preferentially utilize MLX. These results reveal the catabolic specialization of MLX degradation by marine Bacteroidetes, leading to a better understanding of the degradation and recycling of MLX driven by marine bacteria.IMPORTANCERed algae contribute substantially to the primary production in marine ecosystems. The catabolism of red algal polysaccharides by marine bacteria is important for marine carbon cycling. Mixed-linkage ß1,3/1,4-xylan (MLX, distinct from hetero-ß1,4-xylans from terrestrial plants) is an abundant red algal polysaccharide, whose mechanism of catabolism by marine bacteria, however, remains largely unknown. This study reveals the catabolism of MLX by marine Bacteroidetes, promoting our understanding of the degradation and utilization of algal polysaccharides by marine bacteria. This study also sets a foundation for the biomass conversion of MLX.

Metagenomic insights into the dynamic degradation of brown algal polysaccharides by kelp-associated microbiota.

Marine bacteria play important roles in the degradation and cycling of algal polysaccharides. However, the dynamics of epiphytic bacterial communities and their roles in algal polysaccharide degradation during kelp decay are still unclear. Here, we performed metagenomic analyses to investigate the identities and predicted metabolic abilities of epiphytic bacterial communities during the early and late decay stages of the kelp Saccharina japonica. During kelp decay, the dominant epiphytic bacterial communities shifted from Gammaproteobacteria to Verrucomicrobia and Bacteroidetes. In the early decay stage of S. japonica, epiphytic bacteria primarily targeted kelp-derived labile alginate for degradation, among which the gammaproteobacterial Vibrionaceae (particularly Vibrio) and Psychromonadaceae (particularly Psychromonas), abundant in alginate lyases belonging to the polysaccharide lyase (PL) families PL6, PL7, and PL17, were key alginate degraders. More complex fucoidan was preferred to be degraded in the late decay stage of S. japonica by epiphytic bacteria, predominantly from Verrucomicrobia (particularly Lentimonas), Pirellulaceae of Planctomycetes (particularly Rhodopirellula), Pontiellaceae of Kiritimatiellota, and Flavobacteriaceae of Bacteroidetes, which depended on using glycoside hydrolases (GHs) from the GH29, GH95, and GH141 families and sulfatases from the S1_15, S1_16, S1_17, and S1_25 families to depolymerize fucoidan. The pathways for algal polysaccharide degradation in dominant epiphytic bacterial groups were reconstructed based on analyses of metagenome-assembled genomes. This study sheds light on the roles of different epiphytic bacteria in the degradation of brown algal polysaccharides.IMPORTANCEKelps are important primary producers in coastal marine ecosystems. Polysaccharides, as major components of brown algal biomass, constitute a large fraction of organic carbon in the ocean. However, knowledge of the identities and pathways of epiphytic bacteria involved in the degradation process of brown algal polysaccharides during kelp decay is still elusive. Here, based on metagenomic analyses, the succession of epiphytic bacterial communities and their metabolic potential were investigated during the early and late decay stages of Saccharina japonica. Our study revealed a transition in algal polysaccharide-degrading bacteria during kelp decay, shifting from alginate-degrading Gammaproteobacteria to fucoidan-degrading Verrucomicrobia, Planctomycetes, Kiritimatiellota, and Bacteroidetes. A model for the dynamic degradation of algal cell wall polysaccharides, a complex organic carbon, by epiphytic microbiota during kelp decay was proposed. This study deepens our understanding of the role of epiphytic bacteria in marine algal carbon cycling as well as pathogen control in algal culture.

A repertoire of alginate lyases in the alginate polysaccharide utilization loci of marine bacterium Wenyingzhuangia fucanilytica: biochemical properties and action pattern.

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T , and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35-50 °C and pH 8.0-9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M-specific enzyme (Aly17A). Ultra-performance liquid chromatography-mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M-blocks of oligosaccharide products, as well as the M-blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry.