The new genus stands as a Cretaceous chimera among diverse dustywings by having a combination of plesiomorphic and apomorphic characters.The Cretaceous dustywings (Neuroptera: Coniopterygidae) are valuable for understanding the early evolution of this specialized lacewing family. Here, a new genus with two new species of Coniopterygidae is described from the mid-Cretaceous Kachin amber of northern Myanmar. The wing venation of the new genus is remarkably regarding the unique configuration of ScP, RA, and RP, the presence of forewing A3, and the distal gradate crossveins. Paradoxoconis szirakii gen. et sp. nov. and Paradoxoconis longipalpa gen. et sp. nov. The new genus possesses a peculiar combination of wing characters, e.g., the terminal fusion or connection between ScP and RA, the terminal connection of RA to RP, the presence of forewing A3, and the presence of a distal gradate series of crossveins. Despite uncertain subfamilial placement, this new genus morphologically resembles the extant genus Coniocompsa Enderlein, 1905 of the subfamily Aleuropteryginae and the extant genus Flintoconis Sziráki, 2007 of the subfamily Brucheiserinae. Our finding highlights the palaeodiversity of dustywings from the Cretaceous.The species and morphological diversity of dustywings (Neuroptera: Coniopterygidae) from the Cretaceous, of which the knowledge is rapidly increasing by recent studies on the species from the mid-Cretaceous Kachin amber, provide valuable evidence for understanding the phylogeny and early evolution of this highly specialized lacewing lineage. Here we describe a new genus and two new species of this genus in Coniopterygidae from the mid-Cretaceous (lowermost Cenomanian) of northern Myanmar, namely  The lacewing family Coniopterygidae (dustywings), currently with ca. 580 extant species worldwide , represeParadoxoconisgen. nov., shows a set of peculiar wing characters and resembles the extant genera Coniocompsa Enderlein, 1905 (Aleuropteryginae) and Flintoconis Sziráki, 2007 (Brucheiserinae). The subfamilial placement of this new genus is discussed although it remains unclear.Here we report a new genus with two new species of Coniopterygidae from the mid-Cretaceous of northern Myanmar. The new genus, namely The amber specimens for this study come from the Hukwang Valley in Tanai Township, Myitkyina District of Kachin State, : A, anaOrder Neuroptera Linnaeus, 1758Family Coniopterygidae Burmeister, 1839Subfamily incertae sedisParadoxoconis gen. nov.Paradoxoconis longipalpa sp. nov. (by present designation)Type species: Diagnosis. Large-sized dustywings (forewing length 4.9–5.5 mm). Antenna slenderly filiform  and “konis” . The gender of the name is feminine. The genus is registered under urn:lsid:zoobank.org:act:EA8889B1-9650-4EED-8FCF-6B6A7578768E.Paradoxoconis szirakii sp. nov.Diagnosis. Besides the generic diagnostic characters, the new species is also characterized by the following characters: frons well-sclerotized between antennal insertions; antenna with ca. 23 segments; terminal portion of subcostal space with acute angle toward RA; no radial crossvein connecting stem of RP ± MA; four crossveins near wing apex between branches of RP, MA, MP, and CuA forming a gradate series.Description of holotype. Female. Body , Lowermost Cenomanian, Tanai Village, Hukawng Valley, northern Myanmar.Etymology. The new species is dedicated to Dr. György Sziráki, who made tremendous contributions to the systematics of Coniopterygidae.Paradoxoconis longipalpa sp. nov.Diagnosis. Besides the generic diagnostic characters, the new species is also characterized by the following characters: frons depressed between antennal insertions; antenna with ca. 30 segments; terminal portion of subcostal space with acute angle toward ScP; one radial crossvein present at stem of RP ± MA; three crossveins near wing apex between branches of RP, MA, and MP forming a gradate series; distal-most mp-cua crossvein distinctly apart from and proximad former gradate crossveins.Description of holotype. Female. Body , Lowermost Cenomanian, Tanai Village, Hukawng Valley, northern Myanmar.Etymology. The specific epithet “longipalpa” refers to the strongly elongated maxillary palpi in the new species. The species is registered under urn:lsid:zoobank.org:act:C673F0EB-92BC-4F60-B2EC-755E8B3AF9E3.Paradoxoconisgen. nov. differs from all genera of Aleuropteryginae and Coniopteryginae by the presence of rp-ma and mp1-mp2 crossveins, and the presence of forewing A3. Moreover, the new genus undoubtedly does not belong to Coniopteryginae by lacking the subfamilial apomorphies, i.e., the distal origin of hind wing RP ± MA and the presence of single rp ± ma-mp crossvein. So far, the autapomorphies of Aleuroteryginae are unclear [Paradoxoconisgen. nov. However, it is notable that the new genus shares some particular wing characters with the aleuropterygine genus Coniocompsa, i.e., the terminal fusion between ScP and RA, and the distinctly widened forewing subcostal space , although these two genera share the relatively small head dorsally covered by the large pronotum, the presence of plicatures, and some genital characters [Flintoconis is the only extant dustywing genus known to date having the third anal vein (A3) in the forewing .Notably, the new genus resembles the extant genus n et al. , in haviaracters . Flintoc Sziráki ), while Paradoxoconisgen. nov. Nevertheless, the new genus stands a Cretaceous chimera among diverse dustywings by having a combination of plesiomorphic and apomorphic characters. The plesiomorphic characters comprise the basal origin of RP ± MA in both fore- and hind wings, the presence of forewing A3, and the dense crossvenation, while the apomorphies include the strongly elongated maxillary palpi, the highly modified ScP and RA, the strongly widened subcostal space, and the large concaved sclerite of the female genitalia. Future phylogenetic analysis combining fossil and extant dustywings may recover the placement of this enigmatic genus in the family.As discussed above, there has been no strong evidence to determine the subfamilial affiliation of Paradoxoconisgen. nov. from the mid-Cretaceous of Myanmar represents a hitherto unknown lineage of dustywings with unclear subfamilial placement by having peculiar wing characters. The finding highlights the species and morphological diversity of the Cretaceous dustywings.In conclusion,


In this review, we summarize the current information on the nuts and bolts of T9SS. Torque generation by T9SS, its role in gliding motility of bacteria, and the mechanism via which a T9SS-driven swarm shapes the microbiota are discussed. The knowledge gaps in our current understanding of the T9SS machinery are outlined.The F The hydrophobic lipid bilayer of the bacterial cell membrane stops proteins from diffusing outside the cell. However, physiological processes such as the degradation and uptake of complex extracellular nutrition sources, adhesion to biotic and abiotic surfaces, colonization, motility, virulence, and interbacterial antagonism require transport of proteins across membranes. Protein secretion systems serve this purpose and form a pore that provides a gateway to a select group of proteins. Thus far, eleven bacterial protein secretion systems have been discovered . AdditioType 1 to Type 11 bacterial secretion systems are known to facilitate extracellular transport of proteins. Some bacterial secretion systems work in conjunction with either the Sec or the Tat transporters, whereas others work independently. The bacterial Sec protein export pathway allows transport of unfolded proteins through the cytoplasmic membrane  while thvia Sec or Tat pathway. The multiprotein piston-like pseudopilus component of the T2SS pushes its folded protein substrates out of the outer membrane secretin in a process powered by ATP hydrolysis  is comprised of an ABC transporter-like protein complex spanning the cytoplasmic membrane. It is coupled with a viral fusion protein and a β-barrel channel that spans the outer membrane. T1SS enables secretion of unfolded protein substrates from the cytoplasm to the extracellular milieu . The T2Sdrolysis . The T3Sdrolysis . The cordrolysis . The Typdrolysis . Additiodrolysis . The Typdrolysis . The Typdrolysis . The Typane pore . The Typane pore . The Typane pore . Recentlane pore .Flavobacterium johnsoniae and Porphyromonas gingivalis. Transposon mutagenesis led to the discovery that F. johnsoniae GldK, GldL, GldM, and GldN are required for motility and chitin utilization   were discovered around similar timelines in two model organisms lization . Similarlis PorT  and SprAlis PorT . Later,  (PorSS) . It was  (PorSS)  and they (PorSS) . In rece (PorSS)  and we k (PorSS) . Additio (PorSS) . T9SS se (PorSS) , 2019.Via the Sec system, T9SS substrates reach the periplasm and the N-terminal Sec signal peptide is cleaved. T9SS CTD containing periplasmic proteins are exported either to the extracellular milieu or they attach to the outer membrane (OM)  and one Type B CTD-containing protein. In contrast, another prominent human oral microbe Capnocytophaga ochracea ATCC 27872 encodes 2 Type A CTD containing proteins and 8 Type B CTD containing proteins.Type 9 secretion system driven protein export occurs in conjunction with the Sec transport machinery. T9SS substrates have a N-terminal Sec signal peptide and a C-terminal T9SS signal sequence (CTD). ane (OM) , 2019. Tane (OM)  and it iane (OM) . Super fane (OM) , 2019 whane (OM) . We founane (OM)  179,000 ins SprB  and RemAins SprB . Additiohnsoniae , 2015. Shnsoniae . SprB mohnsoniae  and is dhnsoniae . Adhesiohnsoniae . The humF. johnsoniae and P. gingivalis were given different names . These nuts and bolts of T9SS enable both gliding motility and protein secretion  is attached to the outer membrane via a lipid anchor and is localized in the periplasmic space  forms a ring and is associated with GldK/PorK in a 1:1 stoichiometry  is a cytoplasmic membrane protein (Flavobacterium johnsoniae cells containing amino acid substitution of protonatable residues of GldL/PorL (E49Q and E49D) are completely deficient for secretion ( protein  and is f protein . Flavobaecretion . Additioecretion . Aspartaecretion . GldL/Poecretion .GldM/PorM (Fjoh_1855/PGN_1674) interacts with GldL/PorL to form an asymmetric complex with two subunits of GldL/PorL surrounded by five subunits of GldM/PorM. It has one transmembrane domain that contains a protonable residue. Y17A substitution in the transmembrane domain of GldM/PorM resulted in reduced secretion. GldM/PorM has four periplasmic domains D1–D4. X-ray crystallography of GldM/PorM showed a hinge between D2 and D3 , supports stable rotation of the rod, and reduces friction . The Gldvia the GldN/PorN bushing, and it can induce rotation of the outer membrane associated GldK/PorK ring. However, one caveat to this model is that GldN/PorN ring is shown to directly interact with the GldK/PorK ring  is a periplasmic protein that is anchored to inner leaflet of outer membrane  of P. gingivalis has been reported to interact individually with PorE, Gld/PorK and GldM/PorM (ldM/PorM  and coulF. johnsoniae SprF encoding gene (Fjoh_0978) is localized downstream of the mobile cell-surface adhesin SprB encoding gene (Fjoh_0979). SprF/PorP is required for secretion of SprB, and it might be possible that SprF interacts with SprB during transport. If correct, this might imply that the interaction of SprF/PorP with GldK/PorK and GldM/PorM happens solely during transport and that it might help the T9SS pore recognize TIGR04131 containing substrates.SprF/PorP is required for secretion of TIGR04131 domain containing proteins. In the genomic context, a cell often has multiple copies of SprF/PorP and genes encoding SprF/PorP are often localized next to genes encoding TIGR04131 domain containing proteins . As an eSprA/Sov (Fjoh_1653/PGN_0832) is required for secretion SprB, RemA, chitinase, and gingipains (ngipains . It formngipains .PorV (Fjoh_1555/PGN_0023) is bound to the outer membrane pore of SprA and acts as a gate. There are highly conserved packing interactions between PorV and the lateral opening of SprA. When in complex with SprA, the PorV barrel is tilted by 25°. While in a free state, it is proposed that PorV adopts a more vertical position in the membrane bilayer  binds to the lateral face of SprA opposite to PorV. PPIases are chaperones which catalyze the cis/trans isomerization of proline and act as a regulatory switch during protein folding (Peptidyl-prolyl  folding . Deletio folding . HoweverPlug (Fjoh_1759/putative PGN_0144) can be bound to SprA ß-barrel when SprA is not in a complex with PorV. The plug protein mutant neither has gliding defect nor is defective in T9SS secretion. When PorV is not bound to SprA, the plug prevents leakage of small molecules through the SprA pore (prA pore .SprT/PorT (Fjoh_1466/PGN_0778) is an outer membrane protein  is required for the secretion of an array of T9SS proteins  is a lipoprotein that appears to form helical tracks on the cell-surface  but it lacks the active site of SUMF1 (-surface  and it m-surface . However of GldJ . Cells l of GldJ . GldJ haof SUMF1 .GldA (Fjoh_1516) belongs to the ATP binding cassette family of transport proteins (ABC transporter proteins) essential for gliding motility. GldA has Walker “A” and “B” motifs, which are characteristics of an ATP hydrolyzing domain of an ABC transporter (R (site of point mutation underlined) shows that a functional GldA is required for the stability of both GldJ and GldK/PorK (nsporter . FurtherldK/PorK .GldB (Fjoh_1793) is a lipoprotein essential for gliding motility and cells lacking GldB have a defective T9SS (ive T9SS . GldB isive T9SS .GldD (Fjoh_1540) is a lipoprotein essential for gliding motility (F. johnsoniae cells lacking GldD do not propel latex beads. GldD is required for the stability of both GldJ and GldK/PorK (motility . FurtherldK/PorK .GldF (Fjoh_2722) is essential for gliding motility (motility . Both Glmotility . GldF ismotility .GldG (Fjoh_2721). Cells lacking GldG are non-motile (n-motile . GldG isn-motile .GldH (Fjoh_0890) is a lipoprotein essential for gliding motility. Cell lacking GldH do not propel latex beads and are unable to utilize chitin (e chitin . GldH ise chitin .GldI (Fjoh_2369) mutants form non-spreading colonies on agar. GldI has a PPI domain  PorU of P. gingivalis is a part of the attachment complex that also involves PorZ, PorV, and PorQ. This complex modifies the T9SS substrates with an anionic lipopolysaccharide (A-LPS) and attaches them to the cell surface  and PorQ (Fjoh_2755/PGN_0645) PorZ of P. gingivalis interacts with A-LPS and PorU. Hence, it may play a role in providing A-LPS substrate to PorU  for either gliding motility or chitin utilization  displays a bright brilliant green structural coloration, which is caused by the 2D close-packed cell arrangement aided by gliding motility (F. johnsoniae and its dependence on swarm behavior is currently in its nascent stage. Future investigations in this area might reveal details about bacterial cooperation and behavior.It has long been known that r plates . A recenr plates . A F. joar plate . Notablyar plate . This twar plate . It is har plate . A threear plate . It has  biofilm . The colmotility . This mamotility . The rolPrevotella, Bacteroides, Parabacteroides, Capnocytophaga, Tanerella, and Porphyromonas. Capnocytophaga sp. exhibit robust gliding motility (C. ochracea that lack T9SS are non-motile and are deficient for biofilm formation (Type 9 secretion systems are found in bacterial genera that are abundant in the human oral and gut microbiota. Examples include the genera motility  and are motility . In contormation .Capnocytophaga gingivalis swarm in a multilayered counter clockwise vortex pattern (C. gingivalis cells transport non-motile bacteria as cargo and shape the spatial organization of a polymicrobial community (C. gingivalis swarm can also transport bacteriophages as cargo (Via a combination of vortexing, layering, transportation of other bacteria, and phage transportation, T9SS-driven microbes play a crucial role in shaping a polymicrobial community. This amazing molecular machinery holds many secrets, which once unraveled, can significantly enrich our understanding of microbiology. pattern . C. gingommunity . A C. gias cargo  and can AT and AS contributed all sections besides the ones described below and created The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Microalgae Chlorella spp. are a large group of eukaryotic, photosynthetic, unicellular microorganisms with high adaptability to environmental variations. Over the past decades, Chlorella has been used for the large-scale production of biomass. In addition, Chlorella has been actively used in various food industries for improving human health because of its antioxidant, antidiabetic, and immunomodulatory functions. However, the major restrictions in microalgal biofuel technology are the cost-consuming cultivation, processing, and lipid extraction processes. Therefore, various trials have been performed to enhance the biomass productivity and the lipid contents of Chlorella cells. This study provides a comprehensive review of lipid enhancement strategies mainly published in the last five years and aimed at regulating carbon sources, nutrients, stresses, and expression of exogenous genes to improve biomass production and lipid synthesis.Heavy reliance on fossil fuels has been associated with increased climate disasters. As an alternative, microalgae have been proposed as an effective agent for biomass production. Several advantages of microalgae include faster growth, usage of non-arable land, recovery of nutrients from wastewater, efficient CO Microalgae have recently drawn considerable attention for their high potential to produce valuable compounds as well as their applications in biodiesel production, phycoremediation, and dietary supplements. As a source of bioenergy raw materials that can be used to produce biofuels, microalgae are a unique bioresource that has been proposed as a solution to combat energy shortages and alleviate problems associated with global warming , 2. CompChlorella belongs to the Chlorophyta division and consists of small, non-motile, spherical unicellular microalgae with a single chloroplast [Chlorella mainly lives in freshwater, but a few species are also found in the marine environment. They are autospores and mainly reproduce asexually by mitosis [Chlorella species have been used as a bioresource because of their valuable molecules. Although they were initially considered a food resource owing to their high protein content [Chlorella species have high adaptability to environmental variations [Chlorella the most effective microalgae for the generation of bioresources. In addition, Chlorella species can survive and accumulate products using wastewater, making them an exciting target of study in the phytoremediation field [oroplast . Chlorel mitosis . Chlorel content , they ha content . Chlorelriations , and theon field , 16.Chlorella biomass and lipid production, published mainly in the last five years, and discusses the subsequent progress and perspectives.For example, lipids accumulated by microalgae can be used as feedstock for biodiesel production, and microalgal oils can be used in the food industry , 18. ManChlorella strains can grow under photoautotrophic, heterotrophic, and mixotrophic conditions, allowing them to shift in response to changes in the environment. Many studies have been performed to reveal the cultivation method with high efficiency under either photoautotrophic, mixotrophic, or heterotrophic conditions [Photoautotrophic growth of microalgae requires inorganic carbon as a carbon source for growth, which relies on light as a sole energy source. The application of organic carbon sources can be divided into two types depending on light's presence (mixotrophic) or absence (heterotrophic). nditions -27.2 , respectively, in a medium with reduced nutrient availability [et al. developed a two-stage photoautotrophic lipid production strategy in a sintered disk photobioreactor [C. pyrenoidosa was incubated with sufficient nutrients and further treated with nitrogen starvation for lipid induction, resulting in 410 mg/l/day lipid productivity [2 levels is also essential in photoautotrophic cultivation. C. sorokiniana AM-02 was cultured under high photosynthetic photon flux density (PPFD) conditions and CO2 gas levels. The preferred high PPFD and optimal CO2 levels were 1,000-1,400 μmol photons/m2/s and 0.5-2.0% (v/v), respectively [Chlorella cultivation within these five years due to low biomass and lipid productivity compared to heterotrophic or mixotrophic conditions.In photoautotrophic cultivations, the only source of carbon for photosynthesis comes from the available atmospheric CO2 . The pholability . Singh eoreactor . Initialuctivity . This liuctivity ). Investectively . Accordiet al. optimized the growth conditions of naturally isolated C. vulgaris strain in BG-11 medium in a flask and bioreactor. The total biomass and lipid content in heterotrophic culture with glucose were improved 3.5-fold and 9.3-fold, respectively, compared to the basal photoautotrophic culture condition in the shake flask experiment [et al. developed heterotrophic cultivation conditions using statistical assessment to explore the full potential of Chlorella sp. HS2, which was isolated for the ability to achieve extraordinary culture density (5.91 g/l) and biomass productivity (656.7 mg/l/day) under photoautotrophic conditions [Although microalgae can utilize inorganic carbon sources for photosynthesis, the biomass productivity of microalgae is low and limited . Biomassperiment . In the periment . Kim et nditions . The culnditions . Their tnditions ). Thus, nditions .Chlorella biomass and lipid productivity by using various organic carbon sources for cultivation, such as glucose, acetate, or glycerol  in C. sorokiniana [In the mixotrophic mode, photoautotrophic metabolism is integrated with heterotrophic metabolism. Recent studies successfully increased the glycerol . Of thesnditions . As a reokiniana . Ward anokiniana . Thus, get al. used 100 mM (6 g/l) acetic acid from the oxidized wine waste lees for mixotrophic cultivation of C. sorokiniana, and lipid productivity was 193.37 mg/l/day [et al. used 10 g/l sodium acetate as organic carbon for mixotrophic cultivation of C. pyrenoidosa to obtain the maximum production of total lipid [Acetic acid is preferentially adsorbed by the microalgal cells and directly converted into acetyl-CoA, achieving higher efficiency of lipid production. León-Vaz mg/l/day . Liu et al lipid .Chlorella cultivation in a mixotrophic mode with glycerol enhanced the overall biomass concentration and lipid accumulation [C. pyrenoidosa compared to control  [mulation -44. Recet basis) .et al. compared the effect of four monosaccharides  on Chlorella growth. Chlorella medium with fructose promoted C. sorokiniana growth to a much lesser extent than glucose, whereas supplementation with galactose had no effect, and supplementation with xylose inhibited growth [Chai d growth .et al. evaluated eleven known carbon sources for the cultivation of C. vulgaris (Beij.) and found that the best growth rate was provided by acetate cultivation and the second by glucose cultivation [et al. proposed Glucose-Acetate-Phosphorus (GAP) medium, which can maintain pH during cultivation [The question is which carbon source will provide the best lipid productivity. Glucose is first catabolized into glucose-6-phosphate and converted to pyruvate through an anaerobic glycolysis process. Furthermore, it is converted into acetyl-CoA, which is subsequently utilized in the TCA cycle for energy production or as a precursor for fatty acid synthesis ; therefotivation , 48. In tivation , 50, whitivation , 52. To tivation , and migChlorella biomass or lipid productivity. Li et al. reported that heterotrophic cultivation produced the maximum biomass productivity of 134.9 mg/l/day and maximum lipid productivity (42.4 mg/l/day), which were much higher than those under mixotrophic condition [et al. found that the heterotrophic mode maximized lipid productivity (32.7% DCW), and the mixotrophic condition induced a nutritionally favorable fatty acid profile and higher concentrations of carotenoids and phenolics with less lipid quantity (24.2% DCW) [et al. reported that Chlorella biomass productivity and lipid yield under mixotrophic conditions  were higher than the heterotrophic conditions  [Some researchers compared the effects of conditions in heterotrophic and mixotrophic cultivation modes for ondition . Canelli.2% DCW) . Applyin.2% DCW) . On the ctively) .Chlorella on a global industrial scale. The Chlorella market is mainly segmented into autotrophic and heterotrophic, and the heterotrophic segment is expected to grow at the highest compound annual growth rate (CAGR) during the forecast period [It remains a question as to which trophic mode is best for growing t period . It is m3-, nitrite NO2-, ammonium NH4+, and urea CO(NH2)2. Although ammonium (NH4+) can be directly assimilated into amino acids via the GS/GOGAT cycle [3-) needs to be reduced to nitrite (NO2-) in the cytosol, after which it is immediately reduced to ammonium in chloroplasts or plastids [Chlorella.Nitrogen is one of the essential nutrients for microalgal cultivation. Nitrogen can be delivered in various forms to the culture, such as nitrate NOAT cycle , 59, nitplastids  Fig. 2)3-, nitriplastids , 62. Heret al. cultivated C. vulgaris using mixed nitrate and nitrite as a nitrogen source to optimize the ratio of nitrate to nitrite. Nitrite addition triggered a growth rate inhibition, and nitrite uptake remained constant at a low level [et al. previously showed that cultivations with nitrate produced higher protein content (51%) compared to those with nitrite (41%) [Chlorella strains also reported better growth with nitrate supplementation [Pozzobon ow level . On the ow level . Mutlu ete (41%) . Recentlentation .et al. previously reported that nitrite-nitrogen (NO2-N) was the best among the nitrogen sources for the HQ growth and lipid accumulation potential of Chlorella sp. Nitrate-nitrogen (NO3-N) and urea-nitrogen (Urea-N) also contributed to algal growth and lipid accumulation potential at a lesser level, but ammonium nitrogen (NH4-N) and N-deficiency instead caused inhibitory effects in this Chlorella strain [et al. also reported that 200 μmog/l nitrite provided 3.0 mg/l/day lipid productivity in Chlorella sp. L38. By contrast, the average lipid productivity in the medium containing 200 μmog/l nitrite + nitrate resulted in 1.15 mg/l/day [Chlorella cells [A few studies showed better lipid productivity using nitrite as a nitrogen source. Zhan a strain . Li et amg/l/day . Such inmg/l/day , since nla cells .et al. reported that glycine and urea were organic nitrogen sources without a drastic increase in pH fluctuations in the medium [Chlorella species could grow faster in urea than in nitrate and ammonium as nitrogen sources. When urea was the nitrogen source, the highest dry cell weight (2.86 g/l), biomass productivity (345 mg/l), and specific growth rate (1.903/day) were obtained in Chlorella sp. GN1 culture [et al. reported that urea best promoted the biomass production, specific growth rate, and biomass productivity of Chlorella sp. HS2 among all nitrogen sources [The consumption of either nitrate or ammonium by microalgae causes a change in medium pH as they grow. Davis e medium . Several culture . Additio sources .4+) and unionized form . Unlike nitrate NO3-, when ammonium NH4+ is utilized, microalgae spend less energy on its assimilation, and ammonium is directly incorporated into amino acids [Ammonia nitrogen includes the ionized  was observed in modified Bold’s basal medium (BBM) with ammonium, while the growth rate in BBM with nitrate was only 1.07/day [Chlorella strains seem tolerant to ammonium, the degree of growth inhibition by ammonium varies between the strains [et al. compared the tolerance of green algae to ammonium using ten Chlorella strains. As a result, FACHB-1563 had the highest tolerance to ammonium among all strains tested, suggesting that FACHB-1563 might be able to remove excess ammonium from wastewater for bioremediation [Chlorella strains.ammonium . Zigansh1.07/day . Thus, a strains , 77, 78.ediation . Thus, dNRT1 (nitrate NO3- transporter), NRT2 (nitrate NO3- nitrite NO2- transporter), and NAR1 (nitrite NO2- transporter) [Chlorella sp. NC64A has a complete set of all the genes needed for nitrate assimilation, the strain can use ammonium or amino acids but not nitrate or nitrite [Chlorella strains. Chlorella NC64A contains two NRT1 and two NRT2 genes, but C. paradoxa conserves two NRT2 genes  . Interes nitrite . Additioet al. used 10 g/l glucose from food waste hydrolysis for mixotrophic cultivation of Chlorella sp. GY-H4, resulting in a 6.1 g/l biomass yield with a 2.5 g/l lipid yield [C. sorokiniana in palm oil mill effluent (POME) using a novel designed photobioreactor has brought enhancement in biomass production (409 mg/l/day), excellent lipid content , as well as effective POME remediation [et al. investigated the effects of sugarcane bagasse hydrolysate (SCBH) carbon sources on cell growth and fatty acid accumulation in Chlorella protothecoides. With the medium containing SCBH (20 g/l sugar concentration), the highest biomass and fatty acid yield were 10.7 g/l and 0.55 g/l, respectively, which was significantly higher than that in the culture using glucose [Chlorella biomass and lipid production.Phycoremediation refers to remediation with the help of algae. Using wastewater to grow microalgae as a nutrient source would decrease the cultivation costs and purify polluted water. Food waste also represents a valuable carbon source for algal cultivation and can improve the production of microalgal biomass and valuable oleochemicals. Arora and Philippidis utilized 25% sweet sorghum bagasse (SSB) hydrolysate and achieved the highest biomass and lipid productivity  under mixotrophic conditions compared to heterotrophic and photoautotrophic conditions . Wang etid yield . The culediation . Chen et glucose . Taken tet al. utilized cellulose-rich pretreated solids from spruce biomass to grow and produce lipids in oleaginous microalgae. They cultivated microalgae in a medium containing (20 g/l) glucose obtained from spruce hydrolysate, which resulted in the production of biomass (8.28 g/l at C/N 60) and lipid synthesis (3.61 g/l at C/N 60) after 72 h of cultivation [Using forest residues for biofuel production has attracted interest due to the generation of additional revenue and reduction of greenhouse gas emissions. Vyas tivation .et al. cultivated C. vulgaris in SPW under mixotrophic conditions [2 supply [et al. treated Chlorella sp. with aerated seafood processing wastewater, and higher biomass productivity (77.7 mg/l/day) and higher lipid productivity (20.4 mg/l/day) were obtained compared to those in SPW [The commercial seafood processing industry generates large quantities of solids and wastewater. Seafood processing wastewater (SPW) usually contains high concentrations of nutrients, indicating that SPW could be an alternative nutrient source for microalgae cultivation. Jain nditions . The bio2 supply . Gao et e in SPW , indicatC. vulgaris and Pseudochlorella pringsheimii, respectively. Between the two species, P. pringsheimii showed high lipid accumulation potential of 25.4% (dry weight basis) compared to C. vulgaris (9.3%) at 20% tannery effluent diluted with sewage , which contributed to the biomass (60.1 and 56.5 g/l) and lipid content (20.8 and 17.5 mg/l/day) in C. vulgaris and C. pyrenoidosa, respectively [Chlorella sp. UJ-3 was achieved in the butyrate-type fermentation, and the fatty acid compositions were considerably different for these three fermentation systems [Sewage wastewater treatment with microalgae cultivation is an eco-friendly process. Saranya and Shanthakumar evaluated the remediation of combined sewage and tannery effluent under different dilutions. The maximum biomass yield was achieved at 20% tannery effluent and 80% sewage effluent (20% tannery effluent diluted with sewage), resulting in 3.25 g/l and 2.84 g/l in ectively . Anaerob systems .C. vulgaris MBFJNU-1 in natural swine wastewater (RSW) with 3% CO2 resulted in the highest microalgal biomass (478.5 mg/l/day) and lipid (9.1 mg/l/day) productivities [Chlorella sp. cultivation, where the highest productivities of biomass (288.84 mg/l/day) and lipid (104.89 mg/l/day) were achieved [Using livestock wastewater for microalgal cultivation seems to be another alternative solution. tivities . The livachieved .et al. reported that aeration pretreatment of SPW reduced the amount of toxic unionized ammonia to solve this problem. At the same time, most of the nutrients were retained in the wastewater [et al. investigated the effect of pretreatment with a BAF on microalgae culture with dairy liquid digestate. They found that the BAFs can rapidly nitrify ammonia nitrogen, eliminating ammonia inhibition for C. pyrenoidosa [It is worth mentioning that free ammonia in wastewater has been demonstrated as the primary stress factor suppressing microalgal activities. Gao stewater . An aerastewater . BAFs hastewater . Qin et enoidosa .The contaminated biomass generated during phytoremediation poses a threat to our environment. Therefore, proper management is essential to dispose of the wastes to prevent them from further entering the food chain. The use and safe disposal of algal biomass after phytoremediation has been addressed by some researchers. For instance, the integration of algal bio-fertilizer production is recently gathering attention only when we use wastewater with a high level of safety to obtain pollutant-free biomass, such as wastewater from food or feed industries . On the et al. proposed a symbiotic microalgal-bacterial system using C. vulgaris and Pseudomonas putida. They revealed that the cell density of the microalgal-bacterial system was considerably increased compared to that of monoculture microalgae [et al. reported that Chlorella sp. HL and three indigenous bacteria  had synergistic effects on nutrient removal [Co-cultivation of algae and bacteria can enhance the efficiency of nutrient utilization in wastewater, and the growth rate of microalgal cells can be improved . Since vcroalgae . Moreove removal .et al., three bacterial strains  were inoculated into a medium to form a floc with the microalgal strain C. sorokiniana. Among the bacterial strains tested, M. jejuensis formed the largest floc with C. sorokiniana, with the highest sedimentation ability. Furthermore, the M. jejuensis co-culture improved biomass and lipid productivity compared with the pure algal culture [Generally, the biomass produced through microalgae cultivation is harvested using processes such as centrifugation and filtration . To redu culture .et al. developed a co-culture system that incubates Chlorella with yeast Rhodotorula glutinis placed on immobilized beads to enhance algal growth using sucrose [et al. found that a co-culture system can enhance Chlorella growth using sucrose at both heterotrophic and mixotrophic modes when mix-cultured with yeast Cryptococcus sp. [Disaccharides or polysaccharides, such as sucrose and lactose, are difficult to utilize for microalgae under heterotrophic conditions , 102. So sucrose . Tian etccus sp. .et al. revealed that the co-cultivation system among different species of algae improved their growth. When the growth of C. vulgaris and a unicellular green algae Scenedesmus dimorphus in the landfill leachate was compared, the co-culture biomass in 10% landfill leachate demonstrated improved nutrient utilization efficiency in microalgae [Hu croalgae .C. vulgaris, accounting for 3.93-fold, 3.33-fold, or 2.07-fold increases, respectively [et al. attempted to improve lipid production using silica-coated magnetic nanoparticles. As a result, silica-coated magnetic nanoparticles induced 4-fold lipid production (98 mg/l) compared to the control (28 mg/l) [4 was evaluated as a magnesium source for lipid production by C. vulgaris. The application of MgSO4 nanoparticles was found to improve lipid production [Chlorella might impact various physiological or molecular changes, thereby increasing the growth rate, biomass, and lipid production.Nanotechnology is currently a hot topic for its various applications and prospects for providing solutions to the various needs of many industries . Nanoparectively . Vashist28 mg/l) . Moreoveoduction . Thus, mChlorella produces large amounts of biomass with essential lipid contents. Further induction of lipid biosynthesis by environmental stresses is a crucial step for lipid production using Chlorella. There has been a wide range of studies to identify and develop efficient lipid induction techniques in microalgae, such as nutrient stress , osmotic stress, light, pH, temperature, heavy metals, and other chemicals  on lipids yield, CO2 fixation rate, and water quality for further reuse after first cultivation. They concluded that the initial 6 mM nitrate was found optimum for the growth and overall lipid productivity of C. vulgaris [et al. observed that the effects of the initial concentrations of nitrate in the medium varied between 2.5 to 15 mM on biomass generation and lipid production of Chlorella sp. ABC-001, a newly isolated strain with advantageous characteristics for CO2 fixation and biofuel production [Nitrogen is one of the essential nutrients for the growth of microalgae. Nitrogen deprivation in microalgae is widely studied during cultivation to induce lipid productions  compared to the conditions of phosphorus deficiency (36.53% DCW), high light (34.44% DCW), high salt (28.75% DCW), and control (25.14% DCW) [et al. selected the best salinity conditions for better growth, biomass accumulation, and lipid productivity of microalgae. Chlorella sp. showed the maximal lipid content of 32.19% DCW and lipid productivity of 10.27 mg/l/day at 160 mM NaCl in BG-11 media. The results indicate the feasibility of enhancing the lipid content and productivity through the salinity-induced stepwise cultivation strategy [et al. treated Chlorella sp. HS2 with heat shock (50ºC) for 12 h and found that the algal biomass and lipid productivity were enhanced up to 4% and 17%, respectively . Thus, heat shock was successfully adopted in the novel Chlorella sp. HS2 cultivation for lipid induction [C. vulgaris [et al. treated C. pyrenoidosa with ferrous ions to induce reactive oxygen species (ROS) via the catalytic decomposition of hydrogen peroxide (Fenton reaction), increasing the total lipid content [Chlorella sp. FACHB-8 strain, changing fatty acids afflux from polyunsaturated fatty acids to monounsaturated and saturated fatty acids, which were suitable for diesel application [et al. used pyrene pollutants (50-500 ppm) to evaluate the impact on metabolites and the induction of lipid biosynthesis to produce renewable biodiesel. They concluded that pyrene concentration at 230 ppm caused 1.24-fold higher lipid biosynthesis compared to the control medium [Dong 14% DCW) . Gour etstrategy . Kim et nduction . Treatmevulgaris . Zhang e content . Abscisilication . Pyrene l medium .C. kessleri [Chlorella fusca [C. vulgaris [et al. investigated the influence of different intensities and exposure times of magnetic fields on the stimulation of lipid synthesis by the microalga C. homosphaera. Lipid productivity reached 39.5 mg/l/day (1.52-fold) with the magnetic fields  with a slight reduction in biomass productivity [et al. optimized conditions of a magnetic flux density and yielded a maximum dry cell weight of 0.61 g/l, two-fold higher than the normal condition, with lipid content of 55.2% DCW, suggesting the enhancement of growth and lipid of C. vulgaris by magnetic fields [Research in magnetic fields has significantly affected the growth and production of proteins, carbohydrates, and lipids in microalgae. The magnetic field has been shown to have significant effects on the growth and production of proteins, carbohydrates, and lipids with kessleri , Chlorella fusca , and C. vulgaris . Costa euctivity . Baldev c fields .et al. [Chlorella transformation methods are currently considered the most severe limitation in Chlorella genetic engineering. Here, we introduce the latest successful examples of genetic engineering of Chlorella for better lipid production  compared to control [LEAFY COTYLEDON1 (LEC1) is a central regulator that controls many aspects of seed development, including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation in Arabidopsis thaliana [LEC1 in C. ellipsoidea enhanced the total fatty acid content (1.33-fold) and total lipid content (1.30-fold) with upregulation of key genes in the lipid synthesis pathway, such as ACCase, GPDH, PDAT1, and DGAT1 [et al., The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production, resulting in the production of 1.5-fold higher neutral lipid content compared to control cells in C. vulgaris [Chlorella. The carbonic anhydrases (CA) can catalyze the rapid conversion of carbon dioxide to bicarbonate and play a key role in CO2 transfer for cell respiration and photosynthesis [2, You et al. introduced carbonic anhydrase fused with dockerin to immobilize protein on the surface of C. vulgaris. As a result, C. vulgaris showed 1.6-fold rapid growth and 1.7-fold lipid production, suggesting that the CA complex can enhance CO2 fixation [Chlorella strains harbor significant advantages for biomass and lipid production. Further improvement of Chlorella transformation techniques remains to be developed to provide Chlorella biomass as feedstock for oils, antioxidants, or other bioactive benefits by genetic engineering.As discussed in Yang et al. , Chloreloduction . Transcr control . LEAFY Cthaliana . Overexpnd DGAT1 . In the vulgaris . These synthesis . To incrfixation . AlthougChlorella market was worth $269.6 million in 2021, and it is expected to grow at a CAGR of 6.3% from 2021 to 2028 to reach $412.3 million by 2028 [Chlorella market is expected to reach a market value of$198.5 million in 2022 and ultimately $427.7 million by registering a CAGR of 8% in the forecast period 2022-2032 [Chlorella market was estimated at $263.49 million in 2021. It is projected to reach $431.91 million by 2028, exhibiting a CAGR of 7.32% during the forecast period [Chlorella market growth over the forecasted period might be due to the rapidly increasing numbers of the vegan population and health-conscious consumers. According to the market report above, a high number of key participants now compete in the Chlorella industry. Currently, global players such as Sun Chlorella Corp. , Vedan Enterprise , FEMICO , and Taiwan Chlorella Manufacturing Co.  together account for a considerably large market, where Chlorella is mainly sold as a dried powder, capsules, or pressed pills [Chlorella market [According to a market report of the Research and Market, the  by 2028 . In addi022-2032 . Accordit period . This aced pills , 143. NuC. vulgaris, omega-3 fatty acids, such as α-linolenic acid (18:3), are dominant at around 28%, and omega-6 fatty acids account for 10% of total fatty acids [et al. revealed that C. sorokiniana accumulated EPA and DHA at 16.5 and 35%, respectively, with the addition of urea [Chlorella is an important source of essential fatty acids.Omega-3 and omega-6 fatty acids are essential to human health, and we must consume them through food; therefore, they are called essential fatty acids. Omega-3 polyunsaturated fatty acids (PUFAs) include a-linolenic acid , eicosapentaenoic acid , and docosahexaenoic acid , which are efficient at preventing cardiovascular diseases in humans, due to their characteristics that alter membrane fluidity and decrease triacylglycerols (TAGs) , 145. Inty acids . Toumi e of urea . Thus, Cet al. revealed that C. vulgaris and C. pyrenoidosa contain 138 mg/ml and 118 mg/ml of flavonoids, respectively, and these Chlorella species contain quercetin, catechin, and p-coumaric acid [Flavonoids, secondary metabolites of plants, are involved in defense against pathogens, photosynthesis, and growth . Also, fric acid .Chlorella species contain various types of pigment, including astaxanthin (red), β-carotene and violaxanthin (orange), lutein (yellow), chlorophyll-a and chlorophyll-b (green), and pheophytin-a (green-gray)[Chlorella can change by various factors, such as photoperiod, light intensity, carbon source, nitrogen source, and nanoparticles [153The demand for natural colorants has significantly increased due to health and environmental issues . Becauseeen-gray), 153. Tharticles , 153-156Chlorella species contain xanthophylls. C. protothecoides, C. sorokiniana, and C. vulgaris contain lutein [C. zofingiensis contains astaxanthin [C. luteoviridis contains zeaxanthin [C. vulgaris, C. sorokiniana, and C. ellipsoidea contain β-carotene, the precursor of vitamin A [Carotenoids have two important functions in human health as antioxidants and a precursor of vitamin A. However, humans cannot synthesize carotenoids in the de novo pathway, so it is important to consume foods containing carotenoids . Carotenn lutein , C. zofiaxanthin , and C. axanthin . Also, Citamin A -164.C. vulgaris contains eleven essential vitamins that humans cannot synthesize, including vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B9 (folate), vitamin B12 , vitamin C, vitamin E, and vitamin K [C. vulgaris [Chlorella supplementation can provide various vitamin and mineral sources.itamin K -168. Alsvulgaris , 167. ThChlorella, there are already many clinical trials on how Chlorella affects human health. Chlorella supplementation in humans has been shown to have antioxidant [Chlorella intake resulted in noticeable reductions in body fat percentage, total blood serum cholesterol, and fasting blood glucose levels [Chlorella-derived multicomponent supplementation decreases arterial stiffness in young people [Chlorella increased the level of several dicarboxylic acids in feces and propionate concentrations for individuals with low concentrations of fecal propionate [Chlorella-derived compounds might provide substitutes for synthetic compounds or drugs.Based on the valuable compounds of ioxidant , antidiaioxidant , immunomioxidant , and antioxidant . Chlorele levels . Chlorelg people  and middg people . The conopionate . These sChlorella species were used in this field. C. pyrenoidosa can accumulate 27%poly hydroxybutyrate (PHB), a type of poly hydroxy alkanoate that is categorized as biodegradable bioplastic [C. fusca accumulates 17.4% PHB with the addition of xylose [Bioplastics are being actively studied to eliminate the dependency on fossil fuels to produce plastics and avoid endocrine disruptors , and somoplastic . C. fuscf xylose .Chlorella is one of the most nutrient-dense superfoods on the earth. Chlorella products contain high-quality and high-quantity proteins  [Chlorella is rich in proteins, vitamins, minerals, and dietary fiber, it has been reported to have a rather bland flavor profile dominated by “grassy, vegetable, cucumber” aromas [Chlorella powder as an ingredient or take Chlorella tablets. Recently, Coleman et al. analyzed the taste of eight phototrophic microalgae, including C. vulgaris, to be used as flavor ingredients in plant-based seafood alternatives. According to their odor evaluation of eight microalgae, C. vulgaris has the highest earthy odor  among the algal species they analyzed [C. vulgaris contains higher odor activity values of alkyl aldehydes  and benzaldehydes  compared to the other microalgae and seafood. Although C. vulgaris seems to have a relatively low seafood aroma due to a lack of dimethyl sulfide, different taste evaluations disclosed that C. vulgaris has an intermediate level of umami (8 g MSG/100 g DW) among the algae examined with less bitterness [Chlorella strains or develop culture conditions to reduce their off-taste (earthy odor) seems necessary to use Chlorella as a flavor ingredient or plant-based seafood alternative.51%-58%) , 181. Al” aromas . Thus, manalyzed . Their atterness . Thus, bChlorella strains to improve its taste, such as white and honey Chlorella , Duplaco Gold , white Chlorella , and Chlorella Colors . Thus, Chlorella seems to be a promising candidate for meeting the food needs of the vegan diet and the world’s rapidly growing population.Indeed, several companies have recently developed Chlorella, a photosynthetic unicellular microorganism, can accelerate lipid accumulation under various environmental conditions, and has also received significant attention for biofuel production. This review has shown how Chlorella cultivation environments involve Chlorella biomass and lipid productivity, such as cultivation modes, carbon or nitrogen sources, and stress conditions. Heterotrophic cultivation with 10-20 g/l glucose seems to be used for industrial biomass production with many Chlorella strains. Additional glycerol will accelerate lipid production in Chlorella cells. Chlorella cultivation with a light source will induce more antioxidant content. Although Chlorella will assimilate nitrogen from either nitrate or ammonium, ammonium can be assimilated with less energy in Chlorella cells. Moreover, it is worth mentioning that we must carefully select nitrogen sources based on the genetic information of each Chlorella strain. Nitrogen starvation is an efficient environmental pressure for boosting lipid accumulation in Chlorella cells.Chlorella biomass production, harvesting, and processing. To solve these issues, we need to develop a cost-effective method of culturing Chlorella species, such as Chlorella with high biomass productivity, high photosynthetic efficiency with high density and high tolerance to various harsh environments . Also, we need to consider how to increase the production of value-added ingredients that can cover high manufacturing costs. Development of Chlorella genomic databases might also support genetic engineering approaches for those strain developments. Although Chlorella has come under the spotlight for its potential as a sustainable, nutrient-rich future food solution, it has not yet won over the consumer’s taste buds due to its earthy and grassy smell and taste. It is essential to provide Chlorella strains without an unpleasant taste for use in food.The current severe bottleneck includes the high manufacturing cost of Chlorella is a valuable alga that has two attractive consumption purposes as a potential source of renewable energy and nutrient-rich food. The knowledge and techniques accumulated in both fields will be utilized to develop innovative applications and culturing methods.Overall,


Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer.Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF.A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.The online version contains supplementary material available at 10.1186/s12866-022-02527-9. Glycine max (L.) Merrill] is one of the most important agricultural commodities worldwide due to its use in animal and human food and oil and biofuel production. Currently, the legume is the major crop in Brazil, with over 135 million tons of grains harvested in the 2020/2021 growing season [Bradyrhizobium elite strains selected by the research over the past decades provides highs rates of fixed atmospheric nitrogen (N2), supplying the entire N needs from the plants [Soybean [2 fixation than its parental, and since 1992 it composes the group of four Bradyrhizobium strains used in Brazilian inoculants [Strain SEMIA 5079 (= CPAC 15) was isolated in 1992 from a Cerrado soil that originally had no compatible soybean rhizobia and was inoculated with strain SEMIA 566; as both strains share identical rep-PCR profiles and differ from all other strains from the germplasm bank, SEMIA 5079 is considered a natural variant of SEMIA 566 , and belnod, noe) and fixation (fix and nif) genes, 247 hypothetical ORFS, and 90 mobile elements. Additionally, the symbiosis island encompasses a probable non-functional hydrogenase operon, cytochromes for energy supply, ABC-transporters, and operons for secretion systems.The genome of SEMIA 5079 was estimated at 9.58 Mbp, and is composed of one circular chromosome with two ribosomal operons and G + C content of 63.54%. The annotation predicted 4,203 putative genes with assigned function (48.6%) and 4,445 hypothetical CDSs (coding DNA sequences), totaling 8,648 predicted genes [Bradyrhizobium-soybean symbiosis is highly specific, with exchange of molecular signals between the plant and its symbiotic rhizobium. In the symbiosis, isoflavonoids (mainly genistein and daidzein) secreted by the soybean seeds and roots attract the specific Bradyrhizobium and activate the NodD family of transcription activators [nodD gene activates the expression of common and host-specific nod genes. Following, rhizobia synthesize and secrete Nod factors , which are signal molecules that interact with the soybean plant, inducing signal transduction cascades [nod gene expression, isoflavonoids are also related to chemotactic signals [The tivators , 8. The cascades . The reccascades . Besides signals , inducti signals , with th signals , promoti signals , and act signals . Altoget signals , 17.Bradyrhizobium strains are publicly available, including several B. japonicum strains [Bradyrhizobium species have been described and their genomes released in the last two years [Bradyrhizobium genomes, there is still a lack of transcriptional studies supporting gene expression levels, their molecular functions during symbiosis, and their interactions with isoflavonoids.Nowadays, more than 187 genomes of wo years . DespiteB. japonicum, and later reclassified as the type strain of B. diazoefficien) as a model in micro and macro-arrays studies to assess the whole-genome expression profiling of soybean bradyrhizobia, evaluating gene expression levels in both bacteroids and free-living conditions [The majority of the transcriptome studies has used strain USDA 110 , T3SS effectors, efflux pump system in B. diazoefficiens USDA 110 [B. diazoefficiens strain CPAC 7 (= SEMIA 5080), also used in commercial inoculants in Brazil [nodC, nodW, and nodP genes in B. japonicum SEMIA 5079 [nodD1, nodD2, and nodA genes in Bradyrhizobium liaoningense CCBAU 05525 [Bradyrhizobium elkanii strains [Quantitative reverse transcription PCR (qRT-PCR) has been broadly chosen to evaluate the expression of target genes in rhizobia. Previous studies reported effects of isoflavonoids in the expression of several genes related to BNF  genes in the symbiosis island of bjs_07605 is the nodulation noeI, with a distance of 8,615 bp, followed by noeE . The bjs_07621 is located close to several BNF genes, the closest being fixA. The bjs_07647 is located at 1,698 bp from nolZ gene, while bjs_08317 is located at 2,361 bp from the nopP gene (type III secreted effector). Following, bjs_08160 has as nearest gene the conjugal transfer gene trbL , and bjs_08216 is closer to fixW gene . The bjs_08251 and bjs_08254 are located near to each other , and are close to the 6-O-carbamoyl transferase (nodU gene), with a distance of 3,381 bp and 1,022 bp, respectively. We also selected the aspC gene (bjs_08258) in order to compare the expression of an annotated gene in our set of HP, and the gene is close to nodU (315 bp). Close to the bjs_08267, an enoyl-CoA hydratase  and a LacI family transcription regulator coding (lacI) were identified. Several ABC transporters spanning a ~ 5 kb region are also located close to bjs_08267 , followed by strains of B. ottawaense (14 proteins) B. elkanii (12 proteins). In contrast, B. betae, B. guangxiense, B. guangzhouense, B. mercantei, B. neotropicale, B; rifense, and B. symbiodeficiens showed only one protein. On average, species showed from five to seven hypothetical proteins of the set of 15 genes selected ; B. ottawaense USDA 4; and four strains of Bradyrhizobium spp., CCBAU 15,615, CCBAU 15,635, CCBAU 43,298, SG09. Some variance regarding the proteins retrieved from B. japonicum strains was observed. While USDA 38 and J5 strains showed 13 proteins, CCBAU 25,435 carried 12 HPs, other B. japonicum strains  showed nine proteins. In contrast, 44 strains showed only one out of the 15 proteins. For example, B. mercantei strain SEMIA 6399 carried only the BJS_8160 protein. Similarly, strains CCBAU 51,670, CCBAU 53,424, CCBAU 53,426 of B. guangzhouense carried only BJS_08267. As both species do not nodulate soybean, we may suggest that the absence of HP might be explained because the symbiosis islands have been adapted to specific host plants. Other examples of strains showing only one protein were: B. neotropicale strain BR 10,247 (BJS_07536), B. symbiodeficiens strain 65S1MB (BJS_08160), and B. rifense strain CTAW71 (BJS_08160).Following with the analysis, eight strains of aspC gene of the symbiosis island of SEMIA.Several bioinformatics tools and resources were used to assign functions to the 14 HPs and to confirm the annotation of the 5079. The tools included Pfam, UniProtKB, InterPro, SMART, and CDD databases and all the results are shown in Supplementary File SAccording to the functional annotation, 11 HPs were assigned as enzymes playing roles in different pathways, such as in the metabolism of hopanoid, methionine, queuosine biosynthesis, oxidation-reduction, and proteolysis. The majority of the enzymes were categorized as peptidases and hydrogenases. Two HPs were identified as membrane transporters, one as a DNA binding protein and another as a transcription regulator  were predicted as potential T3SS effectors.Using the EffectiveDB database, we analyzed the HPs that could be related to virulence and competitiveness, by investigating type III secretion signals, conserved binding sites of type III chaperones, type IV secretion peptides, eukaryotic-like domains, and subcellular targeting signals in the host Table . The pepBradyrhizobium icense LMTR 13 and Pseudonocardia acacia, isolated from roots of Acacia auriculiformis. Two trypsin-like peptidases (BJS_08251 and BJS_08254) were present in eight bacterial species, including B. elkanii USDA 76 (16 proteins showing this domain). Surprisingly, the GFA enzyme (BJS_07621) and the non-homologous end-joining protein Ku (BJS_08160) showed several proteins with their domains in species of the Order Rhizobiales. The remaining proteins identified with the EffectiveELD tool, pyridoxal phosphate-dependent enzyme (BJS_0858), succinate-semialdehyde dehydrogenase (BJS_08261), and MFS transporter (BJS_08267) were present in the nitrogen-fixing Paraburkholderia nodosa strain DSM 21,604, but also in pathogenic Burkholderia. Bradyrhizobium species were not enriched of the last three proteins. GFA enzyme showed several proteins with GFA domain in different strains of Bradyrhizobium  through the EffectiveELD tool Table . These dB. japonicum type strain USDA 6 or in B. diazoefficiens type strain USDA 110, considering BLAST of both nucleotides and proteins, but other soybean symbionts were detected.Following the criteria of threshold of ≥ 70% of identity and ≥ 50% of coverage for BLAST searches, we performed the phylogenetic analysis of all the 14 HPs and of the BJS_08258 (AspC)  and B. diazoefficiens Y21, isolated from a root nodule of common bean (Phaseolus vulgaris) in China, but there was no information about their ability to nodulate and fix nitrogen with soybean. All the remaining strains in this cluster are reported as capable of nodulating soybean. Interestingly, other species reported as nodulating soybean plants were closer to symbionts of other legumes instead of B. japonicum or B. diazoefficiens. For example, B. yuanmingense strains were grouped with strains associated with cowpea (Vigna unguiculate) and peanut (Arachis hypogaea). Also, almost all B. elkanii strains  were grouped in the same clade and were closer to symbiotic strains of common bean (Phaseolus vulgaris), and pigeon pea (Cajanus cajan). Strains associated with other beans, such as lima bean (Phaseolus lunatus), hyacinth bean (Lablab purpureus) and least snout bean (Rhynchosia minima) were grouped together, and were closer to other legumes, as Retama monosperma and Stylosanthes viscosa. In general, the results indicated that GFA proteins are present in several species associated with a large range of legume plants, with low host specificity.We chose to explore in more details the evolutionary relationships and functions of the GFA, since this protein showed higher number of copies compared to the Ku protein  were higher than the other 14 genes. Similar expression level patterns, ranging from 26.72 to 32.64-fold were observed in the set composed by HxlR-type HTH transcription regulator (bjs_08317), pyridoxal phosphate-dependent enzyme , succinate-semialdehyde dehydrogenase (bjs_08261), MFS transporter (bjs_08267), acetylcholinesterase (bjs_08774), and peptidase U49 (bjs_08240).In the next step, we validated the relative expression levels of all 15 selected genes by quantitative RT-PCR. Significant expression levels were detected in all 15 genes after the induction with 2.5 µM of genistein. The induction levels ranged from 10.15- to 42.58-fold  and 7-cyano-7-deazaguanine synthase (bjs_08523), with about 15.80-fold.In contrast, other genes showed lower gene expression levels than the group described above. This group included both trypsin-like peptidases, B. japonicum USDA 110. From the initial set of 1,210 RpoN-like promoters, 910 were identified, with 199 RpoN-like promoters identified in the symbiosis island; from the initial set of 4,007 RpoD-like promoters, 2,683 were identified in SEMIA 5079, with 481 located in the symbiosis island. It is worth to mentioned that several USDA 110 promoters aligned into the same regions in SEMIA 5079 and for those, only one promoter was counted.To get more insights about gene expression, we identified RpoN and RpoD-like promoters on the SEMIA 5079 genome through a list of promoters predicted in bjs_08317 and bjs_07605 genes were identified , in which, bjs_07536 apparently belongs. Several RpoN-like promoters were identified in the region of nod and fix genes (close to bjs_07647 and bjs_07621). For instance, a RpoN-like promoter upstream of nodD1, which is followed by the nod box genes. Also, other RpoN-like promoters were found upstream of fixR and fixA genes. For the bjs_08774 region, a RpoN-like promoter was predicted upstream the nodM gene; however, a potential operon of three HPs, which contains the bjs_08774 could be observed preceded by a RpoN-like promoter. RpoD-like promoters were predicted upstream of the bjs_07647, and bjs_07621 (which contain multiple RpoD and RpoN promoters in the region). For the following genes, bjs_08261, bjs_08267, bjs_08251, bjs_08216, bjs_08254, and aspC (bjs_08258), neither RpoD or RpoN-like promoters were identified in their regions. While for bjs_08240, bjs_08160, and bjs_08523, RpoD-like promoters were predicted between genes upstream of their location and no clear operon organization was observed between those genes. In general, as expected, more RpoD-like promoters were identified than RpoN, in special in the bjs_07647, bjs_07621 and bjs_07605 gene regions. All the RpoN and RpoD-like promoter sequences predicted in SEMIA 5079 and their location are provided in Supplementary File SRpoN-like promoters upstream of the Bradyrhizobium spp. [We submitted our sequences to several protein databases and bioinformatic tools that have been used to assign functions to hypothetical proteins in bacterial species, including ium spp. , 35, creium spp. . AlthougWe found 11 HP proteins as potential enzymes, and they were classified in six classes. Transferases and hydrolases, each one containing four and three HPs, respectively, another protein was categorized as lyase, one as oxidoreductase, one as hydrogenase (chaperone), and one as ligase. Enzymes play several critical roles in rhizobia, being necessary for bacterial survival and nutrition , nodulatPseudomonas putida [Saccharopolyspora spinosa [metE has been identified in transcriptomes studies in both bacteroids and free-living conditions in USDA 110 [Ralstonia solanacearum, metE is under the control of the pathogenicity regulator HrpG, and metE mutants showed reduced disease symptoms on tomato (Solanum lycopersicum) [metE has also been related to stress conditions tolerance, e.g. to acetate and temperature in Escherichia coli [Rhodotorula mucilaginosa [metH  in S. meliloti causes inability of fix nitrogen in alfalfa plants. The expression of metE in metH mutants complements the Fix− phenotype, as the average plant height, nitrogenase activity and the percentage of pink nodules are dramatically improved [Transferases catalyze the transfer of a specific group from one substance to another , and we s putida , and Sac spinosa . In rhizUSDA 110 , 36. In ersicum) . The methia coli , and coplaginosa . Thus, wimproved .S. meliloti 4R3, alteration in aspartate transaminase resulted in mutants unable to catabolize aspartate and to induce effective nodules on M. sativa [aspC gene was found expressed in nitrogen-fixing bacteroids in the S. meliloti-Medicago truncatula symbiosis [aspC mutant of S. meliloti showed a nitrogen-starvation phenotype during plant infection assays and significantly reduction in nitrogenase activity [B. japonicum SEMIA 5079 [The BJS_08258 protein was predicted as an aminotransferase class I/II-fold pyridoxal phosphate (PLP)-dependent enzyme, also named aspartate aminotransferase . Almost all PLP-dependent enzymes are involved in biochemical pathways with amino compounds, mainly amino acids . In S. m. sativa , 51. Theymbiosis . Also, aactivity . FinallyMIA 5079 , supportBradyrhizobium spp. [Bradyrhizobium sp. strain BTAi1, an shc mutant showed slower-growing under optimal growth conditions, lower resistance to stresses, and reduced ability to survive intracellularly in its host plant Aeschynomene [The BJS_07536 was predicted as a squalene-hopene cyclase  involved in the hopanoid biosynthesis pathway (secondary metabolite biosynthesis) . Hopanoiium spp. , 58, andhynomene .In general, transferases play roles in spore germination, synthesis of lipoproteins, and virulence in bacteria , 61. CorBradyrhizobium, as the protein was majorly identified in Bradyrhizobium symbionts of this legume  cleaves host-encoded signaling peptides and mediates symbiotic compatibility [Trypsin peptidases (STS) have been described as markers in phytopathogenic fungi genomes . Secretonditions  and in bnditions , but theetli CE3 , and in d stress . Also, eeratures , and mutinosarum . In the tibility . Little tibility . We may B. diazoefficiens [B. diazoefficiens USDA 110, gfa was induced in cells grown with vanillin (aromatic compound) as sole source of carbon [M. loti MAFF303099 in response to salt stress [R. tropici CIAT 899 to acid stress [2 [The BJS_07621 protein was predicted as a lyase, a glutathione-dependent formaldehyde-activating (GFA) enzyme , of the glutathione-dependent formaldehyde oxidation pathway, involved in C1 metabolism and methanol oxidation in f carbon , in M. lt stress , in R. td stress , and in tress [2 . TherefoR. leguminosarum bv. viciae strain 3841 was mutated in aminotransferase and succinate semialdehyde dehydrogenases, the mutants were still able to fix nitrogen on pea plants [R. etli, gabD is required for glutamine utilization, and mutations affected growth in culture medium with glutamine as only carbon source [R. leguminosarum SRDI565 cultivated with sulfosugar sulfoquinovose (SQ) as a carbon source [B. diazoefficiens USDA 110 cells grown in HM (Mueller-Hinton) medium [R. tropici CIAT 899 induced by apigenin and salt stress [R. favelukesii LPU83 in response to acid stress [S. meliloti to map regions associated with symbiosis phenotypes [gabD and queC) were annotated and validated in our study. Therefore, GabD in SEMIA 5079 may be associated with the utilization of different carbon sources and also with tolerance to environmental stresses.The BJS_08261 protein was identified as an oxidoreductase enzyme, a succinate-semialdehyde dehydrogenase  involved in the GABA (γ-aminobutyric acid) metabolism . When R.n source . Proteom) medium , R. tropt stress , and R. d stress . In an aenotypes , two of hypA gene is part of hydrogenase uptake (hup) operon located in the symbiosis island of several rhizobia, such as SEMIA 5079 [R. tropici CIAT 899, and PRF 81 [B. diazoefficiens USDA 110 has two sets of the hup gene cluster, a complete set of functional hup genes located outside the symbiosis island and an incomplete set of non-functional hup genes within the symbiosis island [B. japonicum USDA 6 and SEMIA 5079 the genes are duplicated and the operon incomplete, or are pseudogenes; therefore, also non-functional [− phenotype has been reported in B. japonicum SEMIA 5079 [R. leguminosarum a mutation in hypA confirmed that the gene is essential for hydrogenase activity, as it is required for the correct processing of the large hydrogenase subunit [hyp and hup genes, including hypA, were induced in USDA 110 cells during chemoautotrophic growth [hypA was expressed in SEMIA 5079 even though apparently the hydrogenase is not functional [The BJS_08216 protein was predicted as the hydrogenase maturation factor HypA, a nickel metalloenzyme, an accessory protein for nickel incorporation into hydrogenases . The hypMIA 5079 , R. tropd PRF 81 . Hydroged PRF 81 . HoweverMIA 5079 . In R. l subunit . Neverthc growth . Similarnctional , what miqueC of S. meliloti resulted in ineffective BNF [S. meliloti, queC was identified in one of the regions mapped [Shigella flexneri, full expression of the virulence VirF was shown to require queuosine Q34 [queC gene might be relative both with the infection process and the effectiveness of the BNF process.The BJS_08523 protein was predicted as a 7-cyano-7-deazaguanine synthase, QueC protein (EC 2.7.7) involved in the biosynthesis pathway of queuosine . Mutatiotive BNF , and in s mapped . In Shigsine Q34 . TherefoBradyrhizobium sp. (Lupinus) in plants treated with high doses of glyphosate doses [R. etli CFN4 [The BJS_07647 was predicted as a porin, a class of proteins that are β-barrel channels located in Gram-negative bacterial outer membranes and allow the diffusion of different molecules, being also associated with multidrug-resistant . Environte doses , requirete doses , and invtli CFN4 , 103. Altli CFN4 , 69, 92.Rhizobium sp. RC1, mediating the uptake of haloacids into the cells [S. meliloti [R. leguminosarum bv. viciae [The BJS_08267 was predicted as a major facilitator transporter (MFS), a superfamily composed of multidrug resistance efflux pumps (MDR). Besides the roles in multidrug resistance, the genes of this family have been primarily described in bacteria and might play a general role in detoxification of toxic compounds and in rhizobia actuating in competitiveness in the rhizosphere . MFS trahe cells . In relameliloti  and in R. viciae . Thus, tS. meliloti Ku proteins have been identified in both free-living conditions and bacteroids [S. meliloti, Ku protein was not essential to the symbiotic interaction with M. truncatula [The BJS_8160 was identified as a non-homologous end-joining protein Ku (Ku protein); these proteins are specialized in repairing DNA damages  In S. mecteroids , and relcteroids ; howeveruncatula . Our reshxlAB operon in Bacillus subtilis, which is part of the ribulose monophosphate pathway involved in formaldehyde fixation [P. fluorescens in the presence of heavy metals [Lactobacillus acetotolerans in response to ethanol stress [Bacillus atrophaeus UCMB-5137 stimulated by maize root exudates [A probable HxlR-type HTH domain, present in putative transcription regulators with a winged helix-turn-helix (wHTH) structure, was found in BJS_08317. This DNA-binding protein acts as a positive regulator of the formaldehyde-inducible fixation . HxlR try metals , Lactobal stress , and Bacexudates . As othenod, fix, and nif genes and are positioned in symbiosis islands in Bradyrhizobium and some Mesorhizobium strains [nod genes are usually organized in operons, as nodABC in S. meliloti [nodYABCSUIJnolMNO in USDA 110 [We applied some parameters to choose the hypothetical protein-coding genes for further functional annotation, followed by confirmation by qRT-PCR. Firstly, to identify potential new genes related to BNF traits, we selected genes from the symbiosis island of SEMIA 5079, which contains several predicted hypothetical protein-coding genes . As the  strains –119, we meliloti  and nodYUSDA 110 , indicatUSDA 110 , 123. ThBradyrhizobium [bjs_07647) was close to several nodulation genes previously identified as induced by genistein, as nodC in both B. japonicum SEMIA 5079 (1 µM) [B. diazoefficiens 5080 (5 µM) [nodD1, nodD2 and nodA genes, all induced in B. liaoningense CCBAU05525 (0.5 µM) [bjs_08317), close to the nopP gene, which was also previously induced by genistein in SEMIA 5079 [bjs_07536-hopanoid biosynthesis pathway; bjs_08240-peptidase, bjs_08774- acetylcholinesterase, bjs_08267- MFS transporter and bjs_08261- succinate-semialdehyde dehydrogenase), reinforcing the hypothesis of major roles in BNF. These results also support our strategy of choosing genes close to known BNF genes, followed by a functional workflow of annotation and curation, and the transcription validation in the presence of a nod-gene inducer.We quantified the gene expression levels of the 15 selected protein-coding genes in the presence of 2.5 µM of genistein, a known inducer of nodulation genes in hizobium , 8, and hizobium , 32. For9 (1 µM)  and SEMI0 (5 µM) , and als54) have been identified as associated with nitrogen-fixing ability [70) associated with free-living conditions [bjs_08261, bjs_08267 and aspC) are located close in the genome and showed high gene expression levels compared to the remained genes. The bjs_08267 gene (MFS transporter) was preceded by several transporters-coding genes  and aspC are enzymes associated with biosynthesis and oxidation process, with roles in bacterial growth and BNF ability, respectively. Therefore, further investigation in order to identify other types of promoters regulating those genes should be performed. Also, other hypothesis could be the regulation of those genes by the two sigma-54 factors copies present in B. japonicum located in the symbiosis island: bjs_08297  and y4pA gene . These genes have been reporting regulating several genes with diverse functions, including genes related to nitrogen fixation, free-living conditions, and transport on rhizobia species genes [We predicted RpoN and RpoD-like promoters in SEMIA 5079 genome to get insights about gene expression regulation. We chose to select those types of promoters since RpoN promoters , which seems to belong to the P450 operon preceded by a RpoN promoter, as previously reported in B. japonicum USDA 110 [bjs_08317 (HTH transcription regulator) and bjs_07605 (metE) showed an RpoN promoter in their upstream region, and in special metE gene has its expression associated with the NifA-RpoN regulon in M. loti [bjs_07647 (porin protein) and bjs_07621 (GFA enzyme) were preceded by RpoD-like promoters, which makes sense, since both genes are associated with bacterial growth and tolerance to different environment stresses. In the bjs_08160 gene region, which was annotated as a protein Ku associated with free-living conditions and bacteroids, only RpoD-like promoters were predicted. In a similar way, the bjs_07647 (porin) region was predicted with a enrichment of RpoD-like promoters, thus, raising the idea the others HPs located in those regions could play important roles to free-living conditions in B. japonicum as bjs_07647 and bjs_08169. In general, the prediction the RpoN and RpoD-like promoters in SEMIA 5079 was similar to the predictions in the symbiosis island of USDA 110 [Interestingly, the highest gene expression level was observed to USDA 110 , 131 and M. loti . The genUSDA 110 . Also, iB. japonicum strain SEMIA 5079 contains a large proportion of hypothetical protein-coding genes, especially in its main symbiosis island, a key region in the circular genome where the nodulation and nitrogen fixation genes are located. Our study brings a workflow of bioinformatics tools/databases to correctly assign the function of hypothetical protein-coding genes present in SEMIA 5079 symbiosis island and that may be applied to other rhizobia. Fourteen hypothetical protein-coding genes and another one later identified as aspC were studied in more detailed and were induced by the nod-gene inducer genistein, from 10.15- up to 42.58-fold. Some proteins were specific to Bradyrhizobium, while others were present in several rhizobia, and the analysis of orthologues helped to predict roles in BNF, including competitiveness and infectiveness. Although the results are descriptive and efforts should be done to identify the role of HP genes on symbiosis island, the study contributed to improve the knowledge about the genomics features of this outstanding strain used in more than 70 million doses of i soybeaninoculants yearlyin Brazil and in other countries of South America.The genome of B. japonicum SEMIA 5079 (CP007569.1 - GenBank) was retrieved from the NCBI Genome database [nod, fix, nif genes), and with size range of 600 to 2,500 bp (avoiding truncated genes or pseudo-genes). The hypothetical protein-coding gene sequences were taken separately to assign their accurate function based on several bioinformatic tools.The annotated genome of database . Hypothehttp://pfam.xfam.org/); Kyoto Encyclopedia of Genes and Genomes v98.1 (KEGG) (http://www.genome.jp/kegg/); Universal Protein Resource Knowledgebase (UniProtKB) (http://www.uniprot.org/); Rhizobase (genome.microbedb.jp/rhizobase); NCBI-CDD v3.18 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi); InterPro (https://www.ebi.ac.uk/interpro/); SMART (http://smart.embl-heidelberg.de/) and Gene Ontology – GO (http://geneontology.org/).To assign the best function to HP genes, we analyzed the chosen protein sequences against sequences deposited in several databases: PFAM 34.0 (https://web.expasy.org/protparam/). Sub-cellular localization of the HPs was predicted using online tools as TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/); CELLO v.2.5 (http://cello.life.nctu.edu.tw/); PSORTb version 3.0.3 (https://www.psort.org/psortb/), and PSLpred (http://crdd.osdd.net/raghava/pslpred/). Presence of classical signal peptide in hypothetical proteins was predicted using SignalP-5.0 Server , and for non-classical protein secretion the SecretomeP 2.0 Server (http://www.cbs.dtu.dk/services/SecretomeP/). The 3D modeling of hypothetical proteins was performed by the online serve SWISS-MODEL (https://swissmodel.expasy.org/).For each protein, physical and chemical parameters as molecular weight, theoretical pI, amino acid composition, isoelectric point, aliphatic index, grand average of hydropathicity, among others were performed with the ProtParam tool from Expasy server  and VirulentPred (http://203.92.44.117/virulent/index.html/) tools that are based on PSI-Blast and the support vector machine (SVM) method based on patterns, amino acid and dipeptide composition of bacterial protein sequences.In order to predict proteins related to soybean infection by Bradyrhizobium was verified by similarity searches using BLASTX , at the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Further, only sequences from Reference Sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/), which provides a non-redundant collection of sequences representing genomic data, transcripts and proteins were kept. Also, in cases where more than one protein per genome was identified, only the protein with more similarity with the hypothetical protein-coding gene from SEMIA 5079 was used. Protein alignments between HPs queries and the resulting protein sequences obtained from BLAST steps were performed using the CLUSTAL W program in MEGA version 11 [The occurrence of the studied hypothetical proteins in genomes of other strains of rsion 11 . Phylogersion 11 , 136, wi54 RpoN-dependent promoters, mainly activated in symbiosis and promoter motifs similar to σ70 RpoD-dependent promoters, associated with free-living conditions. Through BLASTN, we searched on SEMIA 5079 genome for 1,201 RpoN-like promoters and 4,007 RpoN-like promoter sequences upstream of TSSs  predicted in B. japonicum USDA 110. Only promoters located in intergenic regions were kept and analyzed.We searched in SEMIA 5079 genome for both promoter motifs similar to σBradyrhizobium japonicum strain SEMIA 5079 , was obtained from the “Diazotrophic and Plant Growth Promoting Bacteria Culture Collection” of Embrapa Soja . The strain was cultured at 28 °C in modified-YM solid medium supplemented with Congo red (0.025%) [600 of 0.4, 1 mL was inoculated into 100 mL of TY broth supplied with genistein at a final concentration of 2.5 µM, followed by incubation at 28 °C under shaking (100 rpm and 28 °C) for approximately 1 h, up to the exponential phase (OD600 0.5 ~ 0.6). The cells were then recovered and used for RNA isolation and expression analysis. The experiment was performed with a randomized block design, with three biological replicates. The control treatment received only methanol, which was the solvent used to prepare the genistein solution.(0.025%) . Bacteri(0.025%) . When re® reagent  following the manufacturer’s instructions, with some modifications [Total RNA was extracted using TRIzolications . Thirty → III First-Strand Synthesis System  and random primers, according to the manufacturer’s instructions.RNA concentration was estimated in a NanoDrop™ 8000 Spectrophotometer  and integrity was assessed in 1.0% agarose gel by electrophoresis. RNA samples were treated with DNAse I , according to the manufacturer’s instructions. Following, cDNA strands were synthesized from 5 µg of treated RNA using the SuperScript® software . The default parameters for qRT-PCR were applied, with an amplicon range of 50–150 bp size. The primers sets were checked against SEMIA 5079 genome by BLASTN tool to verify their specificity, and the list of the primers used is shown in Table Primers were designed using the Primer Express 3.0nodC gene was used as positive control [Reactions were performed on a qRT-PCR thermocycler 7500 (Applied Biosystems), according to the manufacturer’s recommendations, using a Platinum SYBR Green qPCR SuperMix-UDG with ROX kit . Initially, the PCR primers efficiency was determined using a quantitative standard curve for each pair of primers . The Ct (cycle threshold) values obtained in each dilution were plotted as a function of logarithm of the cDNA dilutions, with the slope of regression curve used to estimate primer efficiency values . All qPC control , 141.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Gene set enrichment analysis (GSEA) is a commonly used algorithm for characterizing gene expression changes. However, the currently available tools used to perform GSEA have a limited ability to analyze large datasets, which is particularly problematic for the analysis of single-cell data. To overcome this limitation, we developed a GSEA package in Python (GSEApy), which could efficiently analyze large single-cell datasets.We present a package (GSEApy) that performs GSEA in either the command line or Python environment. GSEApy uses a Rust implementation to enable it to calculate the same enrichment statistic as GSEA for a collection of pathways. The Rust implementation of GSEApy is 3-fold faster than the Numpy version of GSEApy (v0.10.8) and uses >4-fold less memory. GSEApy also provides an interface between Python and Enrichr web services, as well as for BioMart. The Enrichr application programming interface enables GSEApy to perform over-representation analysis for an input gene list. Furthermore, GSEApy consists of several tools, each designed to facilitate a particular type of enrichment analysis.https://pypi.org/project/gseapy/. The GSEApy source code is freely available at https://github.com/zqfang/GSEApy. Also, the documentation website is available at https://gseapy.rtfd.io/.The new GSEApy with Rust extension is deposited in PyPI: Bioinformatics online. Gene set enrichment analysis (GSEA) . It provides better memory security, comparable speed to that of C/C++ and the same API as the previous version (≤ v0.10.8). Furthermore, the Rust extension of GSEApy can take full advantage of modern computers' multi-threading parallel computing capabilities because Python’s global interpreter lock was released. When small gene set libraries are analyzed (e.g. 278 pathways in this article), it runs 3-fold faster but with four times less memory usage . For larThe GSEApy internal is written in Rust, and it consists of six tools:gsea: Perform enrichment analysis using the GSEA statistical methodology.prerank: Pre-ranked GSEA, particularly for datasets with a small number of replicates.ssgsea: Single sample GSEA (ssGSEA) methodology.replot: Re-generate enrichment plots from the GSEA-P output files.enrichr: Perform an over-representation analysis on a list of genes. It supports several organisms, including human, mouse, fly, yeast, zebrafish and C.elegans.biomart: Convert gene ids with the BioMart API.The GSEApy will automatically obtain gene set libraries from the Enrichr web service for analysis. To facilitate the interpretation of enrichment results, GSEApy provides several visualization methods . We also provide a complete documentation website, including a user guide describing all of these tools, examples and frequently asked questions.P-value calculation. GSEA performs random permutations of either the samples or the gene labels to obtain the null distribution. One thousand permutations are set as the default parameter, which produces 1000 ES. An observed ES is then compared with the 1000 shuffled ES to calculate a P-value. We previously implemented a Numpy version that performed the GSEA. However, the Numpy version consumed a huge amount of memory while using multi-CPUs, an issue reported by many GSEApy users. To address this, we re-implemented the GSEA algorithm in Rust. When we compared the time and memory cost between the Numpy (v0.10.8) and Rust version with a gene permutation experiment , we found that the Rust implementation was 3- to 80-fold faster in run time with one thread  for the e thread . The rune thread . We alsoe thread . While ie thread , and thee thread .+ monocytes (2215 control and 2147 stimulated cells). We performed GSEA  on the 4362 transcriptomes of the CD14+ monocytes. The interferon β production pathway was significantly enriched  < 0.01, NES = 3.262) in the stimulated CD14+ monocytes  of peripheral blood mononuclear cells (PBMCs) obtained from control and interferon β-treated subjects (onocytes . Next, wonocytes , while tonocytes . This anGSEApy provides a fast and straightforward way to perform enrichment analysis. It requires minimal arguments, provides clear output and operates from both the command line and Python environment, which maximizes the ease of use, accommodates novice programmers and supports large-scale dataset analysis. It also can quickly generate high-quality, publication-ready plots. GSEApy will be one of the fundamental packages for enrichment analysis in Python.btac757_Supplementary_DataClick here for additional data file.