Natural product biosynthetic potential reflects macroevolutionary diversification within a widely distributed bacterial taxon.

IMPORTANCE: This is the most comprehensive study performed thus far on the biosynthetic potential within the Flavobacteriaceae family. Our findings reveal intertwined taxonomic and natural product biosynthesis diversification within the family. We posit that the carbohydrate, peptide, and secondary metabolism triad synergistically shaped the evolution of this keystone bacterial taxon, acting as major forces underpinning the broad host range and opportunistic-to-pathogenic behavior encompassed by species in the family. This study further breaks new ground for future research on select Flavobacteriaceae spp. as reservoirs of novel drug leads.

Multifunctional Enzyme with Endoglucanase and Alginase/Glucuronan Lyase Activities from Bacterium Cellulophaga lytica.

Cellulophaga lytica is a Gram-negative aerobic bacterium in the genome of which there are many genes encoding polysaccharide degrading enzymes. One of the enzymes named ClGP contains a glycoside hydrolase domain from the GH5 family and a polysaccharide lyase domain from the PL31 family. The enzyme also contains the TAT signaling peptide and the TIGR04183 domain that indicates extracellular nature of the enzyme. Phylogenetic analysis has shown that the enzymes most closely related to ClGP and containing all four domains (TAT, GH5, PL31, TIGR04183) are widespread among bacterial species belonging to the Flavobacteriaceae family. ClGP produced by the recombinant strain of E. coli was purified and characterized. ClGP exhibited activity of endoglucanase (EC 3.2.1.4) and catalyzed hydrolysis of ß-D-glucan, carboxymethyl cellulose sodium salt (CMC-Na), and amorphous cellulose, but failed to hydrolyze microcrystalline cellulose and xylan. Products of CMC hydrolysis were cellobiose and cellotriose, whereas ß-D-glucan was hydrolyzed to glucose, cellobiose, cellotetraose, and cellopentaose. ClGP was more active against the poly-ß-D-mannuronate blocks than against the poly-α-L-glucuronate blocks of alginic acid. This indicates that the enzyme is a polyM lyase (EC 4.2.2.3). ClGP was active against polyglucuronic acid, so it displayed a glucuronan lyase (EC 4.2.2.14) activity. The enzyme had a neutral pH-optimum, was stable in the pH range 6.0-8.0, and displayed moderate thermal stability. ClGP effectively saccharified two species of brown algae, Saccharina latissima and Laminaria digitata, that suggests its potential for use in the production of biofuel from macroalgae.

Constantimarinum furrinae gen. nov., sp. nov., a marine bacterium isolated from saline volcanic rock aquifer (lava seawater) at Jeju Island, Republic of Korea.

A Gram-stain-negative, aerobic, rod-shaped (0.3-0.5 × 1.0-1.9 µm), non-motile marine bacterium designated as ALE3EIT was isolated from a saline volcanic rock aquifer (lava sea-water) on Jeju Island, Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain ALE3EIT showed high similarity to 'Altibacter lentus' JLT2010T (97.2%), followed by Marixanthomonas ophiurae KMM 3046T (94.5%). Growth was observed at 10-41°C (optimum, 30°C), at pH 6.0-8.5 (optimum, pH 7.5) and at 0.5-8% (optimum, 4.0%) NaCl. The predominant cellular fatty acids were iso-C15:0 (23.5%), iso-C16:0 (10.2%), iso-C16:0 3OH (10.5%), and iso-C17:0 3OH (16.8%). The DNA G + C contents was 40.4 mol%. The major respiratory quinone was MK-6. The major polar lipids were determined to be phosphatidylethanolamine, two unidentified glycolipids, and two unidentified aminolipids. Several phenotypic characteristics such as production of acetoin, activities of arginine dihydrolase and acid phosphatase, and utilization pattern of carbon sources differentiate strain ALE3EIT from 'A. lentus' JLT2010T. Activities of the lipase, trypsin, α-chymotrypsin and gelatinase and utilization pattern of carbon sources differentiate strain ALE3EIT from M. ophiurae KMM 3046T. The genome of strain ALE3EIT is 3.0 Mbp long and its ANI and AAI values against 'A. lentus' JLT2010T were 76.58 and 72.76, respectively, however, AAI values against members in other genera were lower than 72%. The phylogenomic tree inferred by PhyloPhlAn clearly differentiated the strain ALE3EIT together with strain JLT2010T from other genera in the Falvobacteriaceae. This polyphasic taxonomic data indicates that strain ALE3EIT should be identified as a novel species in the genus 'Altibacter', however, the name has not been validated. Therefore, the strain is classified as a novel genus and is proposed as Constantimarinum furrinae gen. nov., sp. nov. The type strain is ALE3EIT (= KCCM 43303T = JCM 33022T).

Ion transport activity and optogenetics capability of light-driven Na+-pump KR2.

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.

Two Novel Lyso-Ornithine Lipids Isolated from an Arctic Marine Lacinutrix sp. Bacterium.

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.

Transient Near-UV Absorption of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2: A Spectroscopic Marker for Retinal Configuration.

We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S0 → S3 and/or an S0 → S5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.

Identification of intermediate conformations in the photocycle of the light-driven sodium-pumping rhodopsin KR2.

The light-driven rhodopsin KR2 transports Na+via the M- and O-states. However, the mechanisms by which the retinal regulates Na+ pumping is unknown, in part because KR2 adopts both pentamer and monomer forms in crystal structures and in part because these structures show differences in the protein conformation near the Schiff base, even when they are of the same intermediate state within the photocycle. A particular open question is the nature of the H-bond networks and protonation state in the active site, including Asp116. Here, we analyze the protonation state and the absorption wavelength for each crystal structure, using a quantum mechanical/molecular mechanical approach. In the pentamer ground state, the calculated absorption wavelength reproduces the experimentally measured absorption wavelength (530 nm). The analysis also shows that ionized Asp116 is stabilized by the H-bond donations of both Ser70 and a cluster of water molecules. The absorption wavelength of 400 nm in the M-state can be best reproduced when the two O atoms of Asp116 interact strongly with the Schiff base, as reported in one of the previous monomer ground state structures. The absorption wavelengths calculated for the two Na+-incorporated O-state structures are consistent with the measured absorption wavelength (∼600 nm), which suggests that two conformations represent the O-state. These results may provide a key to designing enhanced tools in optogenetics.

Molecular mechanism of light-driven sodium pumping.

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.

Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2.

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.

Two New Alginate Lyases of PL7 and PL6 Families from Polysaccharide-Degrading Bacterium Formosa algae KMM 3553T: Structure, Properties, and Products Analysis.

A bifunctional alginate lyase (ALFA3) and mannuronate-specific alginate lyase (ALFA4) genes were found in the genome of polysaccharide-degrading marine bacterium Formosa algae KMM 3553T. They were classified to PL7 and PL6 polysaccharide lyases families and expressed in E. coli. The recombinant ALFA3 appeared to be active both on mannuronate- and guluronate-enriched alginates, as well as pure sodium mannuronate. For all substrates, optimum conditions were pH 6.0 and 35 °C; Km was 0.12 ± 0.01 mg/ml, and half-inactivation time was 30 min at 42 °C. Recombinant ALFA4 was active predominately on pure sodium mannuronate, with optimum pH 8.0 and temperature 30 °C, Km was 3.01 ± 0.05 mg/ml. It was stable up to 30 °C; half-inactivation time was 1h 40 min at 37 °C. 1H NMR analysis showed that ALFA3 degraded mannuronate and mannuronate-guluronate blocks, while ALFA4 degraded only mannuronate blocks, producing mainly disaccharides. Products of digestion of pure sodium mannuronate by ALFA3 at 200 µg/ml inhibited anchorage-independent colony formation of human melanoma cells SK-MEL-5, SK-MEL-28, and RPMI-7951 up to 17% stronger compared to native polymannuronate. This fact supports previous data and suggests that mannuronate oligosaccharides may be useful for synergic tumor therapy.

Allosteric Communication with the Retinal Chromophore upon Ion Binding in a Light-Driven Sodium Ion-Pumping Rhodopsin.

Krokinobacter rhodopsin 2 (KR2) serves as a light-driven sodium ion pump in the presence of sodium ion and works as a proton pump in the presence of larger monovalent cations such as potassium ion, rubidium ion, and cesium ion. Recent crystallographic studies revealed that KR2 forms a pentamer and possesses an ion binding site at the subunit interface. It is assumed that sodium ion bound at this binding site is not transported but contributes to the thermal stability. Because KR2 can convert its function in response to coexisting cation species, this ion binding site is likely to be involved in ion transport selectively. However, how sodium ion binding affects the structure of the retinal chromophore, which plays a crucial role in ion transport, remains poorly understood. Here, we observed the structure of the retinal chromophore under a wide range of cation concentrations using visible absorption and resonance Raman spectroscopy. We discovered that the hydrogen bond formed between the Schiff base of the retinal chromophore and its counterion, Asp116, is weakened upon binding of sodium ion. This allosteric communication between the Schiff base and the ion binding site at the subunit interface likely increases the apparent efficiency of sodium ion transport. In addition, this study demonstrates the significance of sodium ion binding: even though sodium ion is not transported, binding regulates the structure around the Schiff base and stabilizes the oligomeric structure.

Cadmium-affected synthesis of exopolysaccharides by rhizosphere bacteria.

AIM: Study is focused on the influence of cadmium addition to growth media on production yield, their size and molecular mass of exopolysaccharides (EPS) synthesized by three rhizosphere bacteria strains. Inhibition of bacterial growth by increasing concentrations of Cd2+ was also analysed. METHODS AND RESULTS: The highest impact of Cd2+ was noticed on the growth of Arthrobacter sp. and Rhizobium metallidurans. Chryseobacterium sp. and Arthrobacter sp. produced significantly lower when compared to R. metallidurans amounts of EPS under the influence of Cd2+ . In all bacterial strains both size and molecular mass decreased after addition of Cd2+ to growth media. It causes a change in EPS conformation to more planar, which minimizes the volume of liquid in the interglobular space next to the bacterial wall. Results confirmed strong effect of Cd2+ on the structure and synthesis of bacterial EPS what can be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal polluted soils. CONCLUSION: This work proves that due to the presence of cadmium ions, the size and conformation of EPS produced by selected bacterial strains is changed to minimize their impact on cell. We suggest that shifting in EPS conformation from bigger globular particles to the smaller planar ones could be one of the probable mechanisms of Cd resistance in metallotolerant bacteria, and finally explain increased efficiency of heavy metal phytoextraction by EPS-producing plant growth-promoting micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the most promising remediation technique for Cd-contaminated areas is the phytoremediation in which rhizosphere bacteria play an important role by protecting plants' roots from toxic condition thus enhancing efficiency of intake. EPS secretion by bacteria is one of the most common mechanisms to protect the cell from impact of unpleasant environmental conditions, for example, toxicity of heavy metals like Cd.

Engineered Passive Potassium Conductance in the KR2 Sodium Pump.

Light-driven sodium pumps (NaRs) are microbial rhodopsins that utilize light energy to actively transport sodium ions out of the cell. Here, we used targeted mutagenesis and electrophysiological methods in living cells to demonstrate that NaRs can be converted into light-activated cation channels by molecular engineering. Specifically, introduction of the R109Q mutation into the sodium ion pump of Dokdonia eikasta (KR2) results in passive ion conductance, with a high preference for potassium over sodium ions. However, in this mutant, residual active outward pumping of sodium ions competes with passive inward transport of potassium. Channel-like behavior could also be achieved by introduction of other mutations into the KR2 counterion complex, and further, these modifications were transferrable to other NaRs. Combining the R109Q replacement with modifications at position S70 removed the residual sodium pumping and greatly enhanced the channel-like activity. However, passive photocurrents were only observed in leak mutants if the KR2 counterions, D116 and D251, were deprotonated, which was only observed under alkaline conditions. Overall, our results reveal that interactions between R109 and the nearby residues, L75, S70, D116, and D251, prevent passive backflow during ion transport in NaRs.

Investigation and Characterization of Myroides odoratimimus Strain 3J2MO Aflatoxin B1 Degradation.

Aflatoxin B1 (AFB1), is a type I carcinogen that is one of the strongest naturally occurring aflatoxins and can be injurious to humans and livestock upon ingestion, inhalation, or skin contact, with carcinogenic and mutagenic effects. It causes significant hazardous effects to the food- and animal-production industries. We found a bacterial strain, 3J2MO, that degraded AFB1 well, and here we tested and characterized its AFB1-degradation ability. The strain degraded about 93.82% of the AFB1 after incubation for 48 h in Luria-Bertani (LB) medium at 37 °C with a final concentration of 100 ppb and an inoculation quantity of 1 × 107 cfu/mL. High-performance liquid chromatography-fluorescence detection (HPLC-FLD) was used to determine AFB1 amounts. The maximum degradation rates were 89.23% at pH 8.5; 55.78% at an inoculation quantity of 1 × 108 cfu/mL; and 71.50 and 71.21% at 34 and 37 °C, respectively. Treatment with sucrose and soluble starch as carbon sources and beef extract and ammonium acetate as nitrogen sources stimulated the degradation rate. Mg2+ and Ca2+ ions were activators for AFB1 degradation; however, Mn2+, Fe3+, Zn2+, and Cu2+ were strong inhibitors. This bacterial strain has potential in bioremediation and the detoxification of aflatoxin contamination for biocontrol strategies in both agricultural products and food-industry matrices.

Ulvibacterium marinum gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae, isolated from a culture of the green alga Ulva prolifera.

A Gram-stain negative, aerobic, rod-shaped, and non-motile bacterium, designated strain CCMM003T, was isolated from a culture of the green alga Ulva prolifera. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CCMM003T belongs to the family Flavobacteriaceae and exhibits a close relationship to Pseudozobellia thermophila DSM 19858T (92.5%). Optimal growth occurred in the presence of 4% (w/v) NaCl, at pH 7.0 and 30 °C. The polar lipids of strain CCMM003T consisted of phosphatidylethanolamine and six unidentified lipids. The predominant isoprenoid quinone was MK-6. The major fatty acids were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). The DNA G + C content of strain CCMM003T calculated on the basis of the genome sequence was 41.2 mol% and the genome size was 5.9 Mbp. On the basis of data from this polyphasic study, strain CCMM003T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Ulvibacterium marinum gen. nov., sp. nov. is proposed. The type strain is CCMM003T (= MCCC 1K03244T =KCTC 52639T).

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.

Genomic reconstructions and potential metabolic strategies of generalist and specialist heterotrophic bacteria associated with an estuary Synechococcus culture.

Interactions between photoautotrophs and heterotrophs are central to marine microbial ecosystems. Synechococcus are dominant marine phototrophs, and they are frequently associated with heterotrophic bacteria. These co-cultures provide a useful research system to investigate photoautotroph-heterotroph interactions in marine systems. Bacteria within the Roseobacter clade and Flavobacteria are two of the main bacterial lineages that exhibit intimate associations with Synechococcus populations. We conducted metagenomic analyses of a Synechococcus culture, followed by genomic binning of metagenomic contigs, and recovered five nearly complete genomes, including members of the Roseobacter clade (i.e. Marivita sp. XM-24) and Flavobacteria (i.e. Fluviicola sp. XM-24). Marivita sp. XM-24 is an ecological generalist of the Roseobacter clade and displays diverse metabolic capacities for the acquisition of nutrients and energy sources. Specifically, the genome contained numerous gene complements involved in the uptake and metabolism of nitrogen- and phosphorus-containing inorganic and organic compounds, in addition to the potential for aerobic anoxygenic photosynthesis, oxidation of carbon monoxide, inorganic sulfur oxidation, DMSP demethylation and PHA metabolism. The genome of the Flavobacteria representative, Fluviicola sp. XM-24, contained numerous peptidases, glycoside hydrolases, adhesion-related proteins and genes involved in gliding motility. Fluviicola sp. XM-24 likely specialize in the degradation of high molecular weight compound exudates from Synechococcus cells, including polysaccharides and polypeptides via attachment to particles, surfaces or cells. The distinct metabolic strategies identified within several heterotrophic bacteria that are associated with Syneochococcus cells provide insights into their lifestyles and nutrient utilization patterns, in addition to their interactions with photoautotrophs. Biological interactions, including mutualism, competition and antagonism, shape the microbial community structure of marine environments and are critical for understanding biogeochemical cycling in the ocean. These results provide valuable insights into the nature of interactions between dominant marine photoautotrophs and associated bacterial heterotrophs.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.

Confluentibacter flavum sp. nov., Isolated from the Saline Lake.

A Gram-stain-negative, rod-shaped, non-motile, bacterial isolate designated 3BT, was isolated from a saline lake, and subjected to a polyphasic taxonomic investigation. The phylogenetic analysis based on 16S rRNA gene sequence clearly showed an allocation to the genus Confluentibacter with similarity ranging from 95.1 to 98%. OrthoANI values between strain 3BT and related strains of Confluentibacter (< 90%) were lower than the threshold value of 95% ANI relatedness recommended for species demarcation. Strain 3BT grew at 4-35 °C and pH 6.0-8.0 (optimum, 28 °C and pH 6.5) and with 0-3% (w/v) NaCl (optimum, 0.5%). The predominant respiratory quinone was menaquinone-6 (MK-6) and the major fatty acids were iso-C15:0, iso-C15:1 G, iso-C15:0 3-OH, and iso-C17:0 3-OH. The polar lipid profile of strain 3BT comprised phosphatidylethanolamine, one unidentified aminolipid, one aminophospholipid, and three unidentified lipids (L1-3). The DNA G+C content was 33.1 mol%. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain 3BT is described as a novel species in genus Confluentibacter, for which the name Confluentibacter flavum sp. nov. (type strain 3BT = CGMCC115960T = KCTC52969T) is proposed.

Codon usage clusters correlation: towards protein solubility prediction in heterologous expression systems in E. coli.

Production of soluble recombinant proteins is crucial to the development of industry and basic research. However, the aggregation due to the incorrect folding of the nascent polypeptides is still a mayor bottleneck. Understanding the factors governing protein solubility is important to grasp the underlying mechanisms and improve the design of recombinant proteins. Here we show a quantitative study of the expression and solubility of a set of proteins from Bizionia argentinensis. Through the analysis of different features known to modulate protein production, we defined two parameters based on the %MinMax algorithm to compare codon usage clusters between the host and the target genes. We demonstrate that the absolute difference between all %MinMax frequencies of the host and the target gene is significantly negatively correlated with protein expression levels. But most importantly, a strong positive correlation between solubility and the degree of conservation of codons usage clusters is observed for two independent datasets. Moreover, we evince that this correlation is higher in codon usage clusters involved in less compact protein secondary structure regions. Our results provide important tools for protein design and support the notion that codon usage may dictate translation rate and modulate co-translational folding.