Marinomonas rhodophyticola sp. nov. and Marinomonas phaeophyticola sp. nov., isolated from marine algae.

Two Gram-stain-negative, facultatively aerobic, and motile rod bacteria, designated as strains KJ51-3T and 15G1-11T, were isolated from marine algae collected in the Republic of Korea. Both strains exhibited catalase- and oxidase-positive activities. Optimum growth conditions for strain KJ51-3T were observed at 30 °C and pH 6.0-8.0, with 1.0-7.0 % (w/v) NaCl, whereas strain 15G1-11T exhibited optimal growth at 30 °C, pH 7.0, and 1.0-5.0 % NaCl. Major fatty acids detected in both strains included C16 : 0, C10 : 0 3-OH and summed features 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). As for polar lipids, strain KJ51-3T contained phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol, and two unidentified phospholipids, whereas strain 15G1-11T had PE, PG, and an unidentified aminolipid. Ubiquinone-8 was the predominant respiratory quinone in both strains, with minor detection of ubiquinone-9 in strain KJ51-3T. The genomic DNA G+C contents were 44.0 mol% for strain KJ51-3T and 40.5 mol% for strain 15G1-11T. Phylogenetic analyses based on both 16S rRNA gene and genome sequences placed strains KJ51-3T and 15G1-11T into distinct lineages within the genus Marinomonas, most closely related to Marinomonas arctica 328T (98.6 %) and Marinomonas algicola SM1966T (98.3 %), respectively. Strains KJ51-3T and 15G1-11T exhibited a 94.6 % 16S rRNA gene sequence similarity and a 70.7 % average nucleotide identity (ANI), with ANI values of 91.9 and 79.3 % between them and M. arctica 328T and M. algicola SM1966T, respectively, indicating that they represent novel species. In summary, based on their phenotypic, chemotaxonomic, and phylogenetic properties, strains KJ51-3T and 15G1-11T are proposed to represent novel species within the genus Marinomonas, for which the names Marinomonas rhodophyticola sp. nov. (KJ51-3T=KACC 22756T=JCM 35591T) and Marinomonas phaeophyticola sp. nov. (15G1-11T=KACC 22593T=JCM 35412T) are respectively proposed.

Marinomonas mediterranea synthesizes an R-type bacteriocin.

Prophages integrated into bacterial genomes can become cryptic or defective prophages, which may evolve to provide various traits to bacterial cells. Previous research on Marinomonas mediterranea MMB-1 demonstrated the production of defective particles. In this study, an analysis of the genomes of three different strains (MMB-1, MMB-2, and MMB-3) revealed the presence of a region named MEDPRO1, spanning approximately 52 kb, coding for a defective prophage in strains MMB-1 and MMB-2. This prophage seems to have been lost in strain MMB-3, possibly due to the presence of spacers recognizing this region in an I-F CRISPR array in this strain. However, all three strains produce remarkably similar defective particles. Using strain MMB-1 as a model, mass spectrometry analyses indicated that the structural proteins of the defective particles are encoded by a second defective prophage situated within the MEDPRO2 region, spanning approximately 13 kb. This finding was further validated through the deletion of this second defective prophage. Genomic region analyses and the detection of antimicrobial activity of the defective prophage against other Marinomonas species suggest that it is an R-type bacteriocin. Marinomonas mediterranea synthesizes antimicrobial proteins with lysine oxidase activity, and the synthesis of an R-type bacteriocin constitutes an additional mechanism in microbial competition for the colonization of habitats such as the surface of marine plants.IMPORTANCEThe interactions between bacterial strains inhabiting the same environment determine the final composition of the microbiome. In this study, it is shown that some extracellular defective phage particles previously observed in Marinomonas mediterranea are in fact R-type bacteriocins showing antimicrobial activity against other Marinomonas strains. The operon coding for the R-type bacteriocin has been identified.

Genomic analysis of Marinomonas algicola SM1966T reveals its role in marine sulfur cycling.

Dimethylsulfoniopropionate (DMSP) is a ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling, which is mainly produced by marine phytoplankton and macroalgae. Marinomonas algicola SM1966T, a Gram-negative, aerobic and rod-shaped bacterium, was isolated from the surface of Ulva pertusa (Chlorophyta) algal sample collected off the coastal areas of Rongcheng, China. Here, we report the complete genome sequence of strain SM1966T and its genomic characteristics to utilize DMSP, which may be produced by Ulva pertusa. The genome of strain SM1966T contains one circular chromosome (4.3 Mbp) and one circular plasmid (149,271 bp). Genomic analysis showed that strain SM1966T possesses a set of genes involved in DMSP transport, DMSP cleavage and the catabolism of acrylate, one product of DMSP cleavage. The results indicated that strain SM1966T has the capacity to utilize DMSP and produce dimethyl sulfide (DMS), a volatile infochemical with important roles in global sulfur cycling. This study provides genetic insights into DMSP catabolism by algae-associated bacteria.

Comparative genomic analysis of the genus Marinomonas and taxonomic study of Marinomonas algarum sp. nov., isolated from red algae Gelidium amansii.

Members of the genus Marinomonas are known for their environmental adaptation and metabolically versatility, with abundant proteins associated with antifreeze, osmotic pressure resistance, carbohydrase and multiple secondary metabolites. Comparative genomic analysis focusing on secondary metabolites and orthologue proteins was conducted with 30 reference genome sequences in the genus Marinomonas. In this study, a Gram-stain-negative, rod-shaped, non-flagellated and strictly aerobic bacterium, designated as strain E8T, was isolated from the red algae (Gelidium amansii) in the coastal of Weihai, China. Optimal growth of the strain E8T was observed at temperatures 25-30 °C, pH 6.5-8.0 and 1-3% (w/v) NaCl. The DNA G + C content was 42.8 mol%. The predominant isoprenoid quinone was Q-8 and the major fatty acids were C16:0, summed feature 3 and summed feature 8. The major polar lipids were phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Based on data obtained from this polyphasic taxonomic study, strain E8T should be considered as a novel species of the genus Marinomonas, for which the name Marinomonas algarum is proposed. The type strain is E8T (= KCTC 92201T = MCCC 1K07070T).

Genomic analysis of Marinomonas profundi M1K-6T reveals its adaptation to deep-sea environment of the Mariana Trench.

The Mariana Trench is the deepest site on earth with diverse extreme conditions such as high hydrostatic pressure, low temperature and lack of light. Organisms surviving in this extreme environment and their life strategies have been largely uninvestigated. Here, we report the complete genome of Marinomonas profundi M1K-6T, isolated from the Mariana Trench deep seawater. The assembled genome comprised 3,648,059 bp without any plasmid. Gene annotation showed that strain M1K-6T possesses a series of genes encoding cold-shock proteins, DEAD box RNA helicase and enzymes for biosynthesis of unsaturated fatty acids, implying its high cold tolerance. Abundant genes responsible for transports of ion, branched-chain amino acids and organic compatible solutes were detected, which could maintain cellular osmotic balance disturbed by high hydrostatic pressure. In addition, detected genes (related to storage carbon, transport systems and two-component regulatory systems) could help strain M1K-6T to improve its ecological fitness in the deep-sea microaerobic and nutrient-limiting environments. Genomic information on M. profundi M1K-6T, provides insights into the adaptation strategies of Marinomonas spp. in the extreme deep-sea environment of the Mariana Trench.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation.

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I-F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.

Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin.

The ω-ester-containing peptides (OEPs) are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs). The biosynthetic gene clusters of ω-ester-containing peptides commonly include ATP-grasp ligase coding genes and are distributed over the genomes of a wide variety of bacteria. A new biosynthetic gene cluster of ω-ester-containing peptides was found in the genome sequence of the marine proteobacterium Marinomonas fungiae. Heterologous production of a new tricyclic peptide named marinomonasin was accomplished using the biosynthetic gene cluster in Escherichia coli expression host strain BL21(DE3). By ESI-MS and NMR experiments, the structure of marinomonasin was determined to be a tricyclic peptide 18 amino acids in length with one ester and two isopeptide bonds in the molecule. The bridging patterns of the three intramolecular bonds were determined by the interpretation of HMBC and NOESY data. The bridging pattern of marinomonasin was unprecedented in the ω-ester-containing peptide group. The results indicated that the ATP-grasp ligase for the production of marinomonasin was a novel enzyme possessing bifunctional activity to form one ester and two isopeptide bonds. KEY POINTS: • New tricyclic peptide marinomonasin was heterologously produced in Escherichia coli. • Marinomonasin contained one ester and two isopeptide bonds in the molecule. • The bridging pattern of intramolecular bonds was novel.

Marinomonas vulgaris sp. nov., a marine bacterium isolated from seawater in a coastal intertidal zone of Zhoushan island.

A Marinomonas-like, Gram-stain-negative, strictly aerobic and rod to ovoid-shaped bacterium, designated as strain A79T, was isolated from the seawater mixtures of oyster shells and brown algae in a coastal intertidal zone of Zhoushan, China. The strain was positive for oxidase and catalase. Colonies grown on marine agar for 48 h were round, milky white, smooth and moist with the diameter of 2-3 mm. Growth was observed at 15-30 °C (optimum, 25℃), pH 5.5-9.5 (optimum, pH 8.5) and with 0.5-8% (w/v) NaCl (optimum, 2-2.5%). The G + C content based on the genome sequence was 46.0%. The only respiratory quinone was Q-8. The main polar lipids contained phosphatidylglycerol, phosphatidylethanolamine, unidentified glycolipids, unidentified phospholipid and three unidentified lipids. The major fatty acids (> 10%) were C16:0, Summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c). The 16S rRNA gene sequence similarity between strain A79T and Marinomonas pollencensis IVIA-Po-185T was 97.4%, the similarities with other type strains of the genus Marinomonas were 93.8-96.7%. Based on the results, Marinomonas vulgaris sp. nov. was proposed as a novel species. The type strain is A79T (= MCCC 1K05799T = KCTC 82519T = JCM 34473T).

TnpAREP and REP sequences dissemination in bacterial genomes: REP recognition determinants.

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.

Complete genome sequence of Marinomonas arctica BSI20414, a giant antifreeze protein-producing bacterium isolated from Arctic sea ice.

Sea ice in the polar oceans is a dynamic and challenging environment for life to survive, with extreme gradients of temperature, salinity and nutrients etc., as well as formation of ice crystals. Bacteria surviving in sea ice attract broad attention from academia and industry, due to fascinating mechanisms for adaptation. Here we described the complete genome sequence of Marinomonas arctica BSI20414, isolated from Arctic sea ice. The strain tolerated high salinity and low temperature. Genetic features commonly related to adaptation to oxidative stress, osmotic stress and cold stress were detected in the genome. In addition, a large adhesion protein containing a putative antifreeze protein (AFP) domain was detected in the genome, similar with the giant AFP MpIBP from M. primoryensis. The presence of the putative AFP could facilitate M. arctica BSI20414 to bind to sea ice for favorable conditions and protect it from freezing. The genome sequence and the AFP reported here can provide insights into adaptation to sea ice and can be explored further for biotechnological applications.

Horizontal gene transfer and silver nanoparticles production in a new Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii.

We isolated a novel bacterial strain from a prokaryotic consortium associated to the psychrophilic marine ciliate Euplotes focardii, endemic of the Antarctic coastal seawater. The 16S rDNA sequencing and the phylogenetic analysis revealed the close evolutionary relationship to the Antarctic marine bacterium Marinomonas sp. BSw10506 and the sub antarctic Marinomonas polaris. We named this new strain Marinomonas sp. ef1. The optimal growth temperature in LB medium was 22 °C. Whole genome sequencing and analysis showed a reduced gene loss limited to regions encoding for transposases. Additionally, five genomic islands, e.g. DNA fragments that facilitate horizontal gene transfer phenomena, were identified. Two open reading frames predicted from the genomic islands coded for enzymes belonging to the Nitro-FMN-reductase superfamily. One of these, the putative NAD(P)H nitroreductase YfkO, has been reported to be involved in the bioreduction of silver (Ag) ions and the production of silver nanoparticles (AgNPs). After the Marinomonas sp. ef1 biomass incubation with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. The AgNPs were relatively small in size (50 nm) and had a strong antimicrobial activity against twelve common nosocomial pathogenic microorganisms including Staphylococcus aureus and two Candida strains. To our knowledge, this is the first report of AgNPs biosynthesis by a Marinomonas strain. This biosynthesis may play a dual role in detoxification from silver nitrate and protection from pathogens for the bacterium and potentially for the associated ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common pathogens.

Engineered Living Materials Based on Adhesin-Mediated Trapping of Programmable Cells.

Engineered living materials have the potential for wide-ranging applications such as biosensing and treatment of diseases. Programmable cells provide the functional basis for living materials; however, their release into the environment raises numerous biosafety concerns. Current designs that limit the release of genetically engineered cells typically involve the fabrication of multilayer hybrid materials with submicrometer porous matrices. Nevertheless the stringent physical barriers limit the diffusion of macromolecules and therefore the repertoire of molecules available for actuation in response to communication signals between cells and their environment. Here, we engineer a novel living material entitled "Platform for Adhesin-mediated Trapping of Cells in Hydrogels" (PATCH). This technology is based on engineered E. coli that displays an adhesion protein derived from an Antarctic bacterium with a high affinity for glucose. The adhesin stably anchors E. coli in dextran-based hydrogels with large pore diameters (10-100 µm) and reduces the leakage of bacteria into the environment by up to 100-fold. As an application of PATCH, we engineered E. coli to secrete the bacteriocin lysostaphin which specifically kills Staphyloccocus aureus with low probability of raising antibiotic resistance. We demonstrated that living materials containing this lysostaphin-secreting E. coli inhibit the growth of S. aureus, including the strain resistant to methicillin (MRSA). Our tunable platform allows stable integration of programmable cells in dextran-based hydrogels without compromising free diffusion of macromolecules and could have potential applications in biotechnology and biomedicine.

Insights into Thiotemplated Pyrrole Biosynthesis Gained from the Crystal Structure of Flavin-Dependent Oxidase in Complex with Carrier Protein.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

Heterologous Expression of a Thermostable ß-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides.

A thermostable ß-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme ß-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg-1 at 37 °C using ONPG (o-nitrophenyl-ß-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3'-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with ß-1,4 linkage and ß-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with ß-1,3 linkage.

A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

Characterization of Marinomonas algicida sp. nov., a novel algicidal marine bacterium isolated from seawater.

A novel Marinomonas-like, aerobic, Gram-reaction-negative, moderately halophilic, acidophilic, motile by a single polar flagellum, non-spore-forming, rod-shaped bacterium that showed algalytic activity, designated strain Yeongu 1-4T, was isolated from surface seawater of Geoje Island in the South Sea, Republic of Korea. The strain was oxidase-negative and weakly positive for catalase. Growth of this bacterium was observed at temperatures from 4 to 42 °C, at salinities from 0 to 12 % and at pH from 4.5 to 9.0, and it was not able to degrade starch, gelatin, casein or Tween 80. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Yeongu 1-4T was related most closely to Marinomonas spartinae SMJ19T with similarity of 99.3 %. However, levels of DNA-DNA relatedness between strain Yeongu 1-4T and the most closely related species were lower than 70 %, confirming that they represent distinct genomic species. The genomic DNA G+C content of strain Yeongu 1-4T was 44.2 mol%. The organism used Q-8 as the predominant respiratory quinone, and C16 : 1ω7c, C18 : 1ω7c and C16 : 0 as major cellular fatty acids. Based on data from this polyphasic taxonomic study, strain Yeongu 1-4T belongs to a novel species of the genus Marinomonas, within the family Oceanospirillaceae, for which the name Marinomonas algicida is proposed. The type strain is Yeongu 1-4T (=KEMB 9005-327T=MCCC 1K00609T).

Marinomonas epiphytica sp. nov., isolated from a marine intertidal macroalga.

A novel Gram-stain-negative, aerobic marine bacterial strain, SAB-3T, was isolated from brown macroalgae (Dictyota sp.) growing in the Arabian sea, Goa, India. The strain grew optimally at 30 °C, with 2.0-4.0 % (w/v) NaCl and at pH 7.0 on marine agar medium. Strain SAB-3T was unable to hydrolyse aesculin and did not grow in the presence of rifamycin but showed resistance to antibiotics such as cefadroxil and co-trimoxazole. The major fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 0, and Q-8 was the major ubiquinone. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 41.0 mol%. 16S rRNA gene sequencing and phylogenetic analysis indicated that the strain was a member of the genus Marinomonas with Marinomonas aquiplantarum IVIA-Po-159T (97.6 % similarity), Marinomonas posidonica IVIA-Po-181T (97.5 %) and Marinomonas dokdonensis DSM 17202T (97.4 %) as the closest relatives. Whole genome relatedness determined through DNA-DNA hybridization revealed values of 40-50 % (below the 70 % threshold recommended for species delineation) with the above three species, thus confirming it as representing a distinct and novel species of the genus Marinomonas for which the name Marinomonas epiphytica sp. nov. is proposed. The type strain is SAB-3T (=JCM 31365T=KCTC 52293T=MTCC 12569T).

Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems.

CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea. One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an 'arms race' in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems.

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 µm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ß-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

Biochemical and Structural Insights into a Novel Thermostable ß-1,3-Galactosidase from Marinomonas sp. BSi20414.

A novel ß-1,3-galactosidase, designated as MaBGA (ß-galactosidase from Marinomonas sp. BSi20414), was successfully purified to homogeneity from Marinomonas sp. BSi20414 isolated from Arctic sea ice by ammonium sulfate precipitation and anion exchange chromatography, resulting in an 8.12-fold increase in specific activity and 9.9% recovery in total activity. MaBGA displayed its maximum activity at pH 6.0 and 60 °C, and maintained at least 90% of its initial activity over the pH range of 5.0-8.0 after incubating for 1 h. It also exhibited considerable thermal stability, which retained 76% of its initial activity after incubating at 50 °C for 6 h. In contrast to other ß-galactosidases, MaBGA displayed strict substrate specificity, not only for the glycosyl group, but also for the linkage type. To better understand the structure-function relationship, the encoding gene of MaBGA was obtained and subject to bioinformatics analysis. Multiple alignments and phylogenetic analysis revealed that MaBGA belonged to the glycoside hydrolase family 42 and had closer genetic relationships with thermophilic ß-galactosidases of extremophiles. With the aid of homology modeling and molecular docking, we proposed a reasonable explanation for the linkage selectivity of MaBGA from a structural perspective. On account of the robust stability and 1,3-linkage selectivity, MaBGA would be a promising candidate in the biosynthesis of galacto-oligosaccharide with ß1-3 linkage.