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Genome mining has revealed that myxobacteria contain a myriad of cryptic biosynthetic gene clusters (BGCs). Here, we report the characterization of a panel of myxobacterial promoters with variable strength that are applicable in the engineering of BGCs in myxobacteria. The screened strongest constitutive promoter was used to efficiently enhance the expression of two complex BGCs governing the biosynthesis of myxochromide and DKxanthene in the model myxobacterium Myxococcus xanthus DK1622. We also showcased the combination of promoter engineering and MS2-based spectral networking as an effective strategy to shed light on the previously overlooked chemistry in the family of myxochromide-type lipopeptides. The enriched promoter library substantially expanded the synthetic biology toolkit available for myxobacteria.

Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N (1) and O (2), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an “SOS-box” motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

Graphical abstract, Highlights • Obtained high accuracy AI-modeling structures of 22 GH families for the first time. • Glycoside hydrolases are structurally derived from 27 superfamilies and 16 folds. • The distribution of GHs is highly biased in only a few superfamilies and folds. • Most GH superfamilies are multifunctional, and a few are extremely versatile. • Most GH superfamilies exhibit narrow environmental microbiome distributions., Glycoside hydrolases (GHs) are greatly diverse in sequences and functions, but systematic studies of GH relationships based on structural information are lacking. Here, we report that GHs have multiple evolutionary origins and are structurally derived from 27 homologous superfamilies and 16 folds, but GHs are highly biased to distribute in a few superfamilies and folds. Six of these superfamilies are widely encoded by archaea, bacteria, and eukaryotes, indicating that they may be the most ancient in origin. Most superfamilies vary in enzyme function, and some, such as the superfamilies of (β/α)8-barrel and (α/α)6-barrel structures, exhibit extreme functional diversity; this is highly positively correlated with sequence diversity. More than one-third of glycosidase activities show a phenomenon of convergent evolution, especially the degradation functions of GHs on polysaccharides. The GHs of most superfamilies have relatively narrow environmental distributions, normally with the highest abundance in host-associated environments and a distribution preference for moderate low-temperature and acidic environments. Overall, our expanded analysis facilitates an understanding of complex GH sequence–structure–function relationships and may guide our screening and engineering of GHs.

A Gram-stain-negative, strictly aerobic, non-motile, orange-coloured bacterium, designated YR1-1T, was isolated from a soil sample collected from the Yellow River Delta wetlands (PR China). Growth was observed at a salinity of 1.0–15.0 % NaCl, 4–45 °C and pH 6.0–9.0. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that YR1-1T represented a member of the genus Psychroflexus , with the highest sequence similarity to Psychroflexus sediminis YIM-C238T (97.9 %), followed by Psychroflexus aestuariivivens (97.1 %) and Psychroflexus torquis (96.4 %). The average nucleotide identity and digital DNA–DNA hybridization values between YR1-1T and other closely related type strains of species of the genus Psychroflexus were 68.7–86.3% and 17.8–30.9 %. The genome of the strain was 2 899 374 bp in length with 39.8 % DNA G+C content. The predominant fatty acids (>10 %) were iso-C15 : 0 and anteiso-C15 : 0. The major respiratory quinone was menaquinone-6 (MK-6) and the major polar lipids were phosphatidylethanolamine, phospholipid, diphosphatidylglycerol, two unidentified aminolipids and four unidentified lipids. The combined genotypic and phenotypic data indicate that YR1-1T represents a novel species within the genus Psychroflexus , for which the name Psychroflexus aurantiacus sp. nov., is proposed. The type strain is YR1-1T (=KCTC 72794T=CGMCC 1.17458T).

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterium, designated CWB-1T, was isolated from a haloalkaline lake sediment sample collected from the bottom of Chaiwopu Lake, Urumchi, Xinjiang Province, PR China. Strain CWB-1T grew at 4–40 °C (optimum, 30–35 °C), pH 6.5–9.0 (optimum, pH 6.5–7.0) and with 0.5–5.5 % (w/v) NaCl (optimum, 2.5–3.0 %). Phylogenetic analyses based on the 16S rRNA gene sequence and the whole genome sequence both revealed that strain CWB-1T belonged to the family Flavobacteriaceae . The strain had the highest similarity of the 16S rRNA gene sequence to Psychroserpens jangbogonensis PAMC 27130T (92.8 %). The genome of strain CWB-1T was 3 548 011 bp long with 36.3 % DNA G+C content. The predominant fatty acids (>10 %) in the CWB-1T cells were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 1 (iso-C15 : 1 H/C13 : 0 3-OH). The major respiratory quinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Based on the phylogenetic analyses, as well as the phenotypic characteristics, a novel genus and species of the family Flavobacteriaceae , Paucihalobacter ruber gen. nov., sp. nov., is proposed. The type strain is CWB-1T (=KCTC 72450T=CGMCC 1.17149T).

Biological modification of natural products is an important approach to improve the pharmacological properties. 4′-d-β-Demethylepipodophyllotoxin (4′-d-β-DMEP), a typical natural lignin, is used as the aglycon of topoisomerase inhibitor etoposide (VP-16). In this study, 4′-d-β-DMEP was isomerized into the novel product 4′-d-α-DMEP with 43.09% conversion by P. purpurogenum ACCC 32170. 4′-d-α-DMEP was largely produced extracellularly in the late fermentation stage, which was accompanied with the decrease of dry cell weight and the increase of culture pH. A constitutively expressed cytosolic enzyme was involved in the transformation and functioned when culture pH ranged from 7.0–10.0. The IC50 value of 4′-d-α-DMEP was around 0.80 μM against tumor cells MCF-7, which was significantly reduced by 11.27 and 17.34 times than 4′-d-β-DMEP (i.e., 9.82 μM) and VP-16 (i.e., 14.67 μM). This study provided the novel podophyllotoxin derivative with higher anti-tumor activity and microbial transformation process, which would promote transferring lignans into the clinic trial.

Strain SDU3-2T was isolated from a soil sample collected in Shandong Province, PR China. Cells of SDU3-2T were spherical, Gram-stain-positive, aerobic and non-motile. Cellular growth of the strain occurred at 25–45 °C, pH 5.5–8.5 and with 0–1.5 % (w/v) of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SDU3-2T was closest to the type strain Deinococcus murrayi ALT-1bT with a similarity of 95.2 %. The draft genome was 3.49 Mbp long with 69.2 mol% G+C content. Strain SDU3-2T exhibited high resistance to gamma radiation (D10 >12 kGy) and UV (D10 >900 J m−2). The strain encoded many genes for resistance to radiation and oxidative stress, which were highly conserved with other Deinococcus species, but possessed interspecific properties. The major fatty acids of SDU3-2T cells were C15 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c, and C17 : 1 ω8c, the major menaquinone was menaquinone-8, and the major polar lipids were an unidentified phosphoglycolipid, four unidentified glycolipids and an unidentified phospholipid. The average nucleotide identity and DNA–DNA hybridization results further indicated that strain SDU3-2T represents a new species in the genus Deinococcus , for which the name Deinococcus terrestris sp. nov. is proposed. The type strain is SDU3-2T (=CGMCC 1.17147T=KCTC 43098T).

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Polyangium belongs to Polyangiaceae family of Myxococcales, a taxonomic group well-known for their extraordinary social lifestyle and diverse novel gene clusters of secondary metabolites. A yellow-golden strain, designated SDU3-1T, and two rose pink strains, designated SDU13 and SDU14T, were isolated from a soil sample. These three strains were aerobic, mesophilic, not salt-tolerant and were able to prey on living microorganisms. SDU13 and SDU14T formed solitary sporangioles under starvation conditions, while SDU3-1T had no fruiting body structures. They showed 95.9–97.0% (SDU3-1T) or 98.7–98.9% (SDU13 and SDU14T) 16S rRNA gene similarity with the type strains of Polyangium, but were phylogenetically separate from them based on the 16S rRNA gene and genome sequences. Their genomes were 12.3 Mbp (SDU3-1T), 13.9 Mbp (SDU13) and 13.8 Mbp (SDU14T) with the G + C content range of 68.3–69.4 mol%. The average nucleotide identity and DNA-DNA hybridization analyses of genomes further indicated that these three strains belonged to two new species in Polyangium. Their major fatty acids were C18:1ω9c, C16:0 and C18:0. The polyphasic taxonomic characterization suggest that the three strains represent two novel species in the genus Polyangium, for which the names Polyangium aurulentum sp. nov. and Polyangium jinanense sp. nov. are proposed, and the type strains are SDU3-1T (=CGMCC 1.16875T = KCTC 72136T) and SDU14T (=CCTCC AB 2021123T = KCTC 82625T), respectively.

Bacteria are globally distributed in various environments on earth, but a global view of the geographic diversity and distribution of a single taxon is lacking. The Earth Microbiome Project (EMP) has established a global collection of microbial communities, providing the possibility for such a survey. Myxococcales is a bacterial order with a potent ability to produce diverse natural products and have wide application potential in agriculture, biomedicine, and environmental protection. In this study, through a comparative analysis of the EMP data and public information, we determined that myxobacteria account for 2.34% of the total bacterial operational taxonomic units (OTUs), and are one of the most diverse bacterial groups on Earth. Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. Myxobacteria are among the least-investigated bacterial groups. The presently cultured and genome-sequenced myxobacteria are most likely environmentally widespread and abundant taxa, and account for approximately 10% and 7% of the myxobacterial community (>97% similarity), respectively. This global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently. The diversity and distribution of myxobacteria beyond the EMP data are further discussed. IMPORTANCE The diversity and distribution of bacteria are crucial for our understanding of their ecological importance and application potential. Myxobacteria are fascinating prokaryotes with multicellular behaviors and a potent capacity for producing secondary metabolites, and have a wide range of potential applications. The ecological importance of myxobacteria in major ecosystems is becoming established, but the global geographic diversity and distribution remain unclear. From a global survey we revealed that Myxococcales OTUs are globally distributed and prefer nonsaline soil and sediments, followed by saline environments, but rarely appear in host-associated environments. The global panoramic view of the geographic distribution and diversity of myxobacteria, as well as their cultured and genome-sequenced information, will enable us to explore these important bioresources more reasonably and efficiently.

The 4-O-β-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

Summary Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti‐tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC‐subbranch with 160 GT sequences. We demonstrated that the YjiC‐subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC‐subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high‐active GTs (Q66 and P77) but changed in two low‐efficient GTs. Site‐directed residues swapping at the two positions in the two low‐active GTs (BssGT and BamGT) and the high‐active GT BsGT‐1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Hsp70 proteins are among the most ubiquitous chaperones and play important roles in maintaining proteostasis and resisting environmental stress. Multiple copies of Hsp70s are widely present in eukaryotic cells with redundant and divergent functions, but they have been less well investigated in prokaryotes. Myxococcus xanthus DK1622 is annotated as having many hsp70 genes. In this study, we performed a bioinformatic analysis of Hsp70 proteins and investigated the functions of six hsp70 genes in DK1622, including two genes that encode proteins with the conserved PRK00290 domain (MXAN_3192 and MXAN_6671) and four genes that encode proteins with the cl35085 or cd10170 domain. We found that only MXAN_3192 is essential for cell survival and heat shock induction. MXAN_3192, compared with the other hsp70 genes, has a high transcriptional level, far exceeding that of any other hsp70 gene, which, however, is not the reason for its essentiality. Deletion of MXAN_6671 (sglK) led to multiple deficiencies in development, social motility, and oxidative resistance, while deletion of each of the other four hsp70 genes decreased sporulation and oxidative resistance. MXAN_3192 or sglK, but not the other genes, restored the growth deficiency of the E. colidnaK mutant. Our results demonstrated that the PRK00290 proteins play a central role in the complex cellular functions of M. xanthus, while the other diverse Hsp70 superfamily homologues probably evolved as helpers with some unknown specific functions. IMPORTANCE Hsp70 proteins are highly conserved chaperones that occur in all kingdoms of life. Multiple copies of Hsp70s are often present in genome-sequenced prokaryotes, especially taxa with complex life cycles, such as myxobacteria. We investigated the functions of six hsp70 genes in Myxococcus xanthus DK1622 and demonstrated that the two Hsp70 proteins with the PRK00290 domain play a central role in complex cellular functions in M. xanthus, while other Hsp70 proteins probably evolved as helpers with some unknown specific functions.

Bacterial proline-alanine-alanine-arginine (PAAR) proteins are located at the top of the type VI secretion system (T6SS) nanomachine and carry and deliver effectors into neighboring cells. Many PAAR proteins are fused with a variable C-terminal extended domain (CTD). Here, we report that two paar-ctd genes (MXAN_RS08765 and MXAN_RS36995) located in two homologous operons are involved in different ecological functions of Myxococcus xanthus MXAN_RS08765 inhibited the growth of plant pathogenic fungi, while MXAN_RS36995 was associated with the colony-merger incompatibility of M. xanthus cells. These two PAAR-CTD proteins were both toxic to Escherichia coli cells, while MXAN_RS08765, rather than MXAN_RS36995, was also toxic to Saccharomyces cerevisiae cells. Their downstream adjacent genes, i.e., MXAN_RS08760 and MXAN_RS24590, protected against the toxicities. The MXAN_RS36995 protein was demonstrated to have nuclease activity, and the activity was inhibited by the presence of MXAN_RS24590. Our results highlight that the PAAR proteins diversify the CTDs to play divergent roles in M. xanthus IMPORTANCE The type VI secretion system (T6SS) is a bacterial cell contact-dependent weapon capable of delivering protein effectors into neighboring cells. The PAAR protein is located at the top of the nanomachine and carries an effector for delivery. Many PAAR proteins are extended with a diverse C-terminal sequence with an unknown structure and function. Here, we report two paar-ctd genes located in two homologous operons involved in different ecological functions of Myxococcus xanthus: one has antifungal activity, and the other is associated with the self-identification phenotype. The PAAR-CTD proteins and the proteins encoded by their downstream genes form two toxin-immunity protein pairs. We demonstrated that the C-terminal diversification of the PAAR-CTD proteins enriches the ecological functions of bacterial cells.

Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Background Multiple genes involving in a complex pathway are often clustered into a giant operon with no transcription terminator before the end, and this leads to frangibility of the transcriptional process and arduous engineering work to control the transcription of operon genes. Internal promoters might occur in operon to coordinate the transcription of individual genes, but their effects on the transcription of operon genes have been less investigated. Results Epothilones are a kind of polyketides synthesized by seven multifunctional enzymes, which are encoded by a 56-kb operon of the myxobacterium Sorangium cellulosum. In this study, we determined that the epothilone operon contained multiple internal promoters. These promoters were activatable by the CRISPRa technique, and the yields of epothilones were accordingly increased. However, the activation efficiencies of promoters in operon and separate forms were greatly different. Further, we found that the transcriptional levels of the epothilone genes were always increased at a greater extent than the epothilone yields, which suggested that the transcriptional activation of single genes probably had a weak effect on the final epothilone yield, and higher yield required an overall transcriptional increase of the multiple operonic genes. Finally, we combined the activation of the starting promoter PepoA together with internal promoters in different epothilone-producing strains, and obtained the highest 15-fold increase of epothilone yield in Myxococcus xanthus ZE5. Conclusions This is the first time to report the internal promoters in epothilone gene clusters in Myxococcus xanthus and the first time to assay the activation effects of these internal promoters by CRISPR-dCas9. Our results highlight that tuning internal promoter activities is critical to control the transcription of operon genes and the production efficiency of microbial secondary metabolites.

Estuaries connect rivers with the ocean and are considered transition regions due to the continuous inputs from rivers. Microbiota from different sources converge and undergo succession in these transition regions, but their assembly mechanisms along environmental gradients remain unclear. Here, we found that salinity had a stronger effect on planktonic than on benthic microbial communities, and the dominant planktonic bacteria changed more distinctly than the dominant benthic bacteria with changes in salinity. The planktonic bacteria in the brackish water came mainly from seawater, which was confirmed in the laboratory, whereas the benthic bacteria were weakly affected by salinity, which appeared to be a mixture of the bacteria from riverine and oceanic sediments. Benthic bacterial community assembly in the sediments was mainly controlled by homogeneous selection and almost unaffected by changes in salinity, the dominant assemblage processes for planktonic bacteria changed dramatically along the salinity gradient, from homogeneous selection in freshwater to drift in seawater. Our results highlight that salinity is the key driver of estuarine microbial succession and that salinity is more important in shaping planktonic than benthic bacterial communities in the Yellow River estuary.

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

The ParABS partitioning system, a main driver of DNA segregation in bacteria, employs two proteins, ParA and ParB, for plasmid partition. The pMF1 plasmid from Myxococcus fulvus 124B02 has a par operon encoding a small acidic protein, ParC, in addition to type I ParA and ParB homologs. Here, we show that expression of parC upstream of parA (as in the natural case), but not ectopic expression, is essential for the plasmid inheritance in Myxococcus cells. Co-expression of parC upstream of parA was determined to form a soluble ParC–ParA heterodimer at a 1:1 ratio, while individual expression of parA or co-expression of parA with ectopic parC formed insoluble ParA proteins. Purified ParA proteins alone had no ATPase activity and was easily dimerized, while mixing ParA with ParC formed the ParC–ParA heterodimer with the ATPase and polymerization activities. Fusing ParC and ParA also produced soluble proteins and some chimeras restored the ATPase activity and plasmid inheritance. The results highlight that proximal location of parC before parA is critical to realize the functions of ParA in the partition of Myxococcus plasmid pMF1 and shed light on a new mechanism to realize a protein function by two separate proteins.

Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here we investigated the function of imuA, imuB and dnaE2 in Myxococcus xanthus and found that imuA showed differences from imuB and dnaE2 in bacterial growth, resistance and mutation frequency. Transcriptomics analysis found that ImuA were associated with bacterial SOS response. Yeast-two-hybrid scanning revealed that ImuA interacted with RecA1 besides ImuB. Protein activity analysis proved that ImuA had no DNA binding activity, but inhibited the DNA binding and recombinase activity of RecA1. These findings highlight that ImuA not only participates in TLS by binding ImuB, but also inhibits the recombinase activity of RecA1 in M. xanthus, suggesting a role of ImuA in the two replication restart pathways.ImportanceDnaE2 is responsible for bacterial SOS mutagenesis in nearly one third of sequenced bacterial strains. However, its mechanism, especially the function of its accessory protein ImuA, is still unclear. Here we reported that M. xanthus ImuA might facilitate DnaE2 TLS by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the scientific problem of choosing one of the two restart pathways to repair the stalled replication fork.

Heterocycle modification has been widely and successfully employed in the antitumor drugs. However, the different antitumor efficacy was corelated with the heterocycle substituted, and the genetic mechanism underlying these effects has not been elucidated. In this study, the intrinsic regularity between different types of heterocycle-substituted DMEP derivative compounds and the mechanisms of their antitumor activity was preliminarily disclosed. Triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives induced more severe DNA damage and higher levels of 26S proteasomal Topo IIβ degradation, though inhibited the recruition of γH2AX to resist the DNA damage. The reduced DNA repair led to higher up-regulation of cell cycle arrest proteins, and ultimately DNA damage mediated-ATM/ATR apoptotic pathways and specifically activated DNA damage response gene TMEM133, which induced apoptosis through up-regulation of G2/M cell cycle arrest-related genes. Over-expression and knock-out of TMEM133 demonstrated that TMEM133 is essential for inhibition of the tumor cell growth during treatment with triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives.

Background Sequencing prokaryotic genomes has revolutionized our understanding of the many roles played by microorganisms. However, the cell and taxon proportions of genome-sequenced bacteria or archaea on earth remain unknown. This study aimed to explore this basic question using large-scale alignment between the sequences released by the Earth Microbiome Project and 155,810 prokaryotic genomes from public databases. Results Our results showed that the median proportions of the genome-sequenced cells and taxa (at 100% identities in the 16S-V4 region) in different biomes reached 38.1% (16.4–86.3%) and 18.8% (9.1–52.6%), respectively. The sequenced proportions of the prokaryotic genomes in biomes were significantly negatively correlated with the alpha diversity indices, and the proportions sequenced in host-associated biomes were significantly higher than those in free-living biomes. Due to a set of cosmopolitan OTUs that are found in multiple samples and preferentially sequenced, only 2.1% of the global prokaryotic taxa are represented by sequenced genomes. Most of the biomes were occupied by a few predominant taxa with a high relative abundance and much higher genome-sequenced proportions than numerous rare taxa. Conclusions These results reveal the current situation of prokaryotic genome sequencing for earth biomes, provide a more reasonable and efficient exploration of prokaryotic genomes, and promote our understanding of microbial ecological functions.

The macrolactone rapamycin (RAP) presents a broad range of bioactivities, but its clinical applications are compromised due to the poor water solubility and low bioavailability, which could probably be overcome by glycosylation. In this study, we tested a set of promiscuous glycosyltransferases (GTs) to modify rapamycin with four different sugar donors. BsGT-1 displayed the best glycosylation activity with a preference for UDP-glucose, and the glycosylation happened at C-28 or C-40 of rapamycin, producing rapamycin-40-O-β-D-glucoside (RG1), and two new compounds rapamycin-28-O-β-D-glucoside (RG2) and rapamycin-28,40-O-β-D-diglucoside (RG3). The glycosylation remarkably improved water solubility and almost completely abolished cytotoxicity but simultaneously attenuated the antifungal, antitumor, and immunosuppression bioactivities of rapamycin. We found the glycosylation at C-40 had less effect on the bioactivities than that at C-28. The molecular docking analysis revealed that the glycosylation, especially the glycosylation at C-28, weakened the hydrophobic and hydrogen bonding contacts between the rapamycin glucosides and the binding proteins: the FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain. This study highlights a succinct approach to expand the chemical diversity of the therapeutically important molecule rapamycin by using promiscuous glycosyltransferases. Moreover, the fact that glycosyl moieties at different positions of rapamycin affect bioactivity to different extents inspires further glycosylation engineering to improve properties of rapamycin. KEY POINTS: • Rapamycin was glycosylated efficiently by some promiscuous GTs. • Glycosylation improved water solubility, attenuated cytotoxicity, and bioactivities. • Glycosylation affected the interactions between ligand and binding proteins.

Vegetation probably represents the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift in pace with the colonization and succession of vegetation. In this study, we found that a Cyanobacteria monospecies genus and a Gemmatimonadetes multispecies genus are fastidiously predominant in the bare and vegetative wetlands of the Yellow River Delta, respectively. Consistently, photosynthesis genes were enriched exclusively in bare land, while genes involved in biological organic carbon metabolism and the cycling of main elements were highly expressed in vegetative wetlands, were mostly included in the MAG of Gemmatimonadetes, and were consistent with soil metabolomic results. Our results provide insight into the adaptive succession of predominant bacterial species and their ecosystem functions in response to the presence of vegetation., Vegetation represents probably the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift along with vegetation. In this study, we found that the richness and diversity of soil bacteria increased with vegetation levels and that the community composition was distinctly shifted from bare to vegetative places. The bare land displayed an extremely high abundance of Cyanobacteria as a monospecies genus, while a Gemmatimonadetes genus was predominant as multiple species in all the vegetative wetlands, suggesting their important ecosystem functions and potential mechanisms. Expression of the genes related to photosynthesis was enriched exclusively in bare land. Genes involved in biological organic carbon metabolism and the cycling of main elements (C, N, S, and P) were highly expressed in vegetative wetlands and were mostly included in the metagenome-assembled genome (MAG) of Gemmatimonadetes. Some compounds identified from soil metabolomic results also corresponded to pathways involving these key active genes. Cyanobacteria is thus responsible for the carbon sink in early infertile wetlands, and Gemmatimonadetes plays a crucial role in ecosystem functions in vegetative wetlands. Our results highlight that the soil microbial populations execute ecosystem functions for wetlands and that vegetation is the determinant for the population and functional shifts in the coastal estuarine wetland of the Yellow River Delta. IMPORTANCE Vegetation probably represents the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift in pace with the colonization and succession of vegetation. In this study, we found that a Cyanobacteria monospecies genus and a Gemmatimonadetes multispecies genus are fastidiously predominant in the bare and vegetative wetlands of the Yellow River Delta, respectively. Consistently, photosynthesis genes were enriched exclusively in bare land, while genes involved in biological organic carbon metabolism and the cycling of main elements were highly expressed in vegetative wetlands, were mostly included in the MAG of Gemmatimonadetes, and were consistent with soil metabolomic results. Our results provide insight into the adaptive succession of predominant bacterial species and their ecosystem functions in response to the presence of vegetation.

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388) with unknown differentiated functions. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation, but the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth, but significantly decreased the UV-irradiation survival, homologous recombination ability, and induction of the LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and DNA damage caused by hydrogen peroxide, but did not change the UV-radiation resistance and the SOS-gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange and LexA-autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for the DNA homologous recombination and the evolution of genomes from simple to complex.

A pale yellow bacterial strain, designated SDU1-6T, was isolated from a soil sample collected in the Jinan campus of Shandong University, Shandong Province, PR China. The strain was aerobic, motile, Gram-stain-negative and rod-shaped. Cellular growth occurred at 13–37 °C, pH 5.0–10.0 and with 0–0.1 % (w/v) NaCl, and the optimal growth was at 25 °C and pH 7.5. SDU1-6T had the highest 16S rRNA gene similarity of 95.42 % with Chryseolinea serpens DSM 24574T. The genome was 6 542 746 bp in length with 43.5 mol% DNA G+C content. The major fatty acids of SDU1-6T were C16 : 1ω5 and iso-C15 : 0, the major menaquinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. On the basis of the polyphasic evidence and the 16S rRNA gene sequence, SDU1-6T represents a novel species of the genus Chryseolinea , for which the name Chryseolinea flava sp. nov. is proposed. The type strain is SDU1-6T (=CGMCC 1.13492T=JCM 32520T).

Chaperonin groEL genes are duplicated in approximately 20% of bacteria, and the duplicates are differentially transcribed due to their divergent functions. The coordinated regulation of this differential transcription is as yet undetermined. In this study, we reported that the controlling inverted repeat of chaperone expression (CIRCE) element (the HrcA-binding site located upstream of the promoter) evolved for the transcriptional regulation of duplicate groELs. CIRCE composition and locations were found to be phylogenetically conserved in bacterial taxa. Myxococcus xanthus DK1622 has two CIRCE elements (CIRCE1groESL1 and CIRCE2groESL1) in the promoter region of groESL1 and one CIRCE element (CIRCEgroEL2) before groEL2. We also found that negative HrcA and positive ?32 regulators coordinated the transcription of duplicate groELs, and that the double deletion in DK1622 eliminated transcriptional differences and reduced the heat-shock responses of groELs. In vitro binding assays showed that HrcA protein binding was biased towards CIRCE1groESL1, followed by CIRCEgroEL2, but that HrcA proteins failed to bind with CIRCE2groESL1. Mutation experiments revealed that single-nucleotide mutations in the inverted repeat regions changed the HrcA-binding abilities of CIRCEs. We constructed an in vivo transcription-regulation system in Escherichia coli to pair each of the regulators with a groEL promoter. The results indicated that the transcriptional regulation performed by HrcA and ?32 was biased towards the groEL2 and groEL1 promoters, respectively. Based on promoter-sequence characteristics, we proposed a model of the coordinated regulation of the transcription of duplicate groELs in M. xanthus DK1622.

Background Quantitative evaluation of protein structural evolution is important for our understanding of protein biological functions and their evolutionary adaptation, and is useful in guiding protein engineering. However, compared to the models for sequence evolution, the quantitative models for protein structural evolution received less attention. Ancient protein superfamilies are often considered versatile, allowing genetic and functional diversifications during long-term evolution. In this study, we investigated the quantitative impacts of sequence variations on the structural evolution of homologues in 68 ancient protein superfamilies that exist widely in sequenced eukaryotic, bacterial and archaeal genomes. Results We found that the accumulated structural variations within ancient superfamilies could be explained largely by a bilinear model that simultaneously considers amino acid substitution and insertion/deletion (indel). Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those ancient superfamilies with high bilinear multiple correlation coefficients, the influence of each unit of substitution or indel on structural variations is almost constant within each superfamily, but varies greatly among different superfamilies. The influence of each unit indel on structural variations is always larger than that of each unit substitution within each superfamily, but the accumulated contributions of indels to structural variations are lower than those of substitutions in most superfamilies. The total contributions of sequence indels and substitutions (46% and 54%, respectively) to the structural variations that result from sequence variations are slightly different in ancient superfamilies. Conclusions Structural variations within ancient protein superfamilies accumulated under the significantly bilinear influence of amino acid substitutions and indels in sequences. Both substitutions and indels are essential for explaining the structural variations within ancient superfamilies. For those structural variations resulting from sequence variations, the total contribution of indels is slightly lower than that of amino acid substitutions. The regular clock exists not only in protein sequences, but also probably in protein structures. Electronic supplementary material The online version of this article (10.1186/s12864-018-5178-8) contains supplementary material, which is available to authorized users.

Promoter optimization is an economical and effective approach to overexpress heterologous genes and improve the biosynthesis of valuable products. In this study, we swapped the original promoter of the epothilone biosynthetic gene cluster in Myxococcus xanthus with two endogenous strong promoters PpilA and PgroEL1, respectively, which, however, decreased the epothilone production ability. The transcriptional abilities by the two promoters were found to be bloomed in the growth stage but markedly decreased after the growth, whereas the original promoter Pepo functioned majorly after the exponential growth stage. Tandem repeat engineering on the original promoter Pepo remarkably increased epothilone production. The tandem promoter exerted similar expressional pattern as Pepo did in M. xanthus. We demonstrated that differential transcriptional modes markedly affected the efficiency of promoters in controlling the gene expressions for the production of the secondary metabolite epothilones. Our study provides an insight into exploiting powerful promoters to produce valuable secondary metabolites, especially in host with limited known promoters. Electronic supplementary material The online version of this article (10.1007/s00253-018-9023-4) contains supplementary material, which is available to authorized users.

The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2- cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.

Background The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. Results In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. Conclusions Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites. Electronic supplementary material The online version of this article (10.1186/s12934-018-0867-1) contains supplementary material, which is available to authorized users.

RecA is a ubiquitous multifunctional protein for bacterial homologous recombination and SOS response activation.Myxococcus xanthusDK1622 possesses tworecAgenes, and their functions and mechanisms are almost unclear. Here, we showed that the transcription ofrecA1(MXAN_1441) was less than one-tenth ofrecA2(MXAN_1388). Expressions of the tworecAgenes were both induced by ultraviolet (UV) irradiation, but in different periods. Deletion ofrecA1did not affect the growth, but significantly decreased the UV-irradiation survival, the homologous recombination ability, and the induction of the LexA-dependent SOS genes. Comparably, the deletion ofrecA2markedly prolonged the lag phase for cellular growth and antioxidation of hydrogen peroxide, but did not change the UV-irradiation resistance and the SOS-gene inducibility. The two RecA proteins are both DNA-dependent ATPase enzymes. We demonstrated that RecA1, but not RecA2, hadin vitroDNA recombination capacity and LexA-autolysis promotion activity. Transcriptomic analysis indicated that the duplicate RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. We discuss the potential mechanisms for the functional divergence. This is the first time to clearly determine the divergent functions of duplicatedrecAgenes in bacterial cells. The present results highlight that the functional divergence of RecA duplicates facilitates the exertion of multiple RecA functions.Author summaryMyxobacteria has a large-size genome, contains many DNA repeats that are rare in the prokaryotic genome. It encodes bacterial RecA that could promote recombination between homologous DNA sequences. How myxobacteria avoid the undesired recombination between DNA repeats in its genome is an interesting question.M. xanthusencodes two RecA proteins, RecA1 (MXAN_1441) and RecA2 (MXAN_1388). Both RecA1 and RecA2 shows more than 60% sequence consistency withE. coliRecA (EcRecA) and can partly restore the UV resistance ofE. coli recAmutant. Here, our results proved their divergent functions of the two RecAs. RecA1 retains the ability to catalyze DNA recombination, but its basal expression level is very low. RecA2 basal expression level is high, but no recombination activity is detected in vitro. This may be a strategy forM. xanthusto adapt to more repetitive sequences in its genome and avoid incorrect recombination.HighlightsM. xanthushas tworecAs, which are expressed with significantly different levels. BothrecAs are inducible by UV irradiation, but in different stages.The absence ofrecA1reduces bacterial UV-irradiation resistance, while the absence ofrecA2delays bacterial growth and antioxidant capacity.RecA1 retains the DNA recombination and SOS induction abilities, while RecA2 has evolved to regulate the expression of genes for cellular transport and antioxidation.

Myxococcus xanthus kills susceptible bacteria using myxovirescin A (TA) during predation. However, whether prey cells in nature can escape M. xanthus by developing resistance to TA is unknown. We observed that many field-isolated Bacillus licheniformis strains could survive encounters with M. xanthus, which was correlated to their TA resistance. A TA glycoside was identified in the broth of predation-resistant B. licheniformis J32 co-cultured with M. xanthus, and a glycosyltransferase gene (yjiC) was up-regulated in J32 after the addition of TA. Hetero-expressed YjiC-modified TA to a TA glucoside (TA-Gluc) by conjugating a glucose moiety to the C-21 hydroxyl group, and the resulting compound was identical to the TA glycoside present in the co-culture broth. TA-Gluc exhibited diminished bactericidal activity due to its weaker binding with LspA, as suggested by in silico docking data. Heterologous expression of the yjiC gene conferred both TA and M. xanthus-predation resistance to the host Escherichia coli cells. Furthermore, under predatory pressure, B. licheniformis Y071 rapidly developed predation resistance by acquiring TA resistance through the overexpression of yjiC and lspA genes. These results suggest that M. xanthus predation resistance in B. licheniformis is due to the TA deactivation by glucosylation, which is induced in a predator-mediated manner.

Two unrecognizable strains of the same bacterial species form a distinct colony boundary. During growth as colonies, Myxococcus xanthus uses multiple factors to establish cooperation between recognized strains and prevent interactions with unrecognized strains of the same species. Here, &Delta, MXAN_0049 is a mutant strain deficient in immunity for the paired nuclease gene, MXAN_0050, that has a function in the colony-merger incompatibility of Myxococcus xanthus DK1622. With the aim to investigate the factors involved in boundary formation, a proteome and metabolome study was employed. Visualization of the boundary between DK1622 and &Delta, MXAN_0049 was done scanning electron microscope (SEM), which displayed the presence of many damaged cells in the boundary. Proteome analysis of the DK1622- boundary disclosed many possible proteins, such as cold shock proteins, cell shape-determining protein MreC, along with a few pathways, such as RNA degradation, phenylalanine, tyrosine and tryptophan biosynthesis, and Type VI secretion system (T6SS), which may play major roles in the boundary formation. Metabolomics studies revealed various secondary metabolites that were significantly produced during boundary formation. Overall, the results concluded that multiple factors participated in the boundary formation in M. xanthus, leading to cellular damage that is helpful in solving the mystery of the boundary formation mechanism.

Many endogenous plasmids carry no noticeable benefits for their bacterial hosts, and the persistence of these ‘cryptic plasmids’ and their functional impacts are mostly unclear. In this study, we investigated these uncertainties using the social bacterium Myxococcus fulvus 124B02 and its endogenous plasmid pMF1. pMF1 possesses diverse genes that originated from myxobacteria, suggesting a longstanding co-existence of the plasmid with various myxobacterial species. The curing of pMF1 from 124B02 had almost no phenotypic effects on the host. Laboratory evolution experiments showed that the 124B02 strain retained pMF1 when subcultured on dead Escherichia coli cells but lost pMF1 when subcultured on living E. coli cells or on casitone medium; these results indicated that the persistence of pMF1 in 124B02 was environment-dependent. Curing pMF1 caused the mutant to lose the ability to predate and develop fruiting bodies more quickly than the pMF1-containing strain after they were subcultured on dead E. coli cells, which indicated that the presence of pMF1 in M. fulvus 124B02 has some long-term effects on its host. The results provide some new insights into the persistence and impacts of cryptic plasmids in their natural bacterial cells.

Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.

A strictly anaerobic, Gram-stain-positive bacterium, designated FW431T, was isolated from a mud cellar used for producing strong aromatic Chinese liquors. The strain was able to produce butanoic acid, an important component of the aroma style of Chinese liquors. Cells of strain FW431T were straight or slightly curved rods with a polar endospore and peritrichous flagella. The major cellular fatty acids (>10 % of the total) were C16 : 0, C18 : 1ω9c and C18 : 0. Biolog assays indicated that the strain preferably metabolizes palatinose, l-fucose, β-hydroxybutyric acid, l-rhamnose and α-ketobutyric acid among 95 carbon sources tested. FW431T was related most closely to Clostridium ljungdahlii DSM 13528T and Clostridium kluyveri DSM 555T based on 16S rRNA gene sequence similarities of 95.0 and 94.2 %, respectively. The DNA G+C content of the genomic DNA was 44.4 mol%. Based on the evidence presented here, FW431T ( = CGMCC 1.5201T = KCTC 15519T) is proposed as the type strain of a novel species, Clostridium luticellarii sp. nov.

Myxococcus xanthus DK1622 is a rich source of novel secondary metabolites, and it is often used as an expression host of exogenous biosynthetic gene clusters. However, the frequency of obtaining large genome-deletion variants by using traditional strategies is low, and progenies generated by homologous recombination contain irregular deletions. The present study aims to develop an efficient genome-engineering system for this bacterium based on the Cre/loxP system. We first verified the functionality of the native cre system that was integrated into the chromosome with an inducible promoter PcuoA. Then we assayed the deletion frequency of 8-bp-spacer-sequence mutants in loxP by Cre recombinase which was expressed by suicide vector pBJ113 or self-replicative vector pZJY41. It was found that higher guanine content in a spacer sequence had higher deletion frequency, and the self-replicative vector was more suitable for the Cre/loxP system, probably due to the leaky expression of inducible promoter PcuoA. We also inspected the effects of different antibiotics and the native or synthetic cre gene. Polymerase chain reaction (PCR) and sequencing of new genome joints confirmed that the Cre/loxP system was able to delete a 466 kb fragment in M. xanthus. This Cre/loxP-mediated recombination could serve as an alternative genetic manipulation method.

Although plasmids provide additional functions for cellular adaptation to the environment, they also create a metabolic burden, which causes the host cells to be less competitive with their siblings. Low-copy-number plasmids have thus evolved several mechanisms for their long-term maintenance in host cells. pMF1, discovered in Myxococcus fulvus 124B02, is the only endogenous autonomously replicated plasmid yet found in myxobacteria. Here we report that a post-segregational killing system, encoded by a co-transcriptional gene pair of pMF1.19 and pMF1.20, is involved in maintaining the pMF1 plasmid in its host cells. We demonstrate that the protein encoded by pMF1.20 is a new kind of nuclease, which is able to cleave DNA in vitro. The nuclease activity can be neutralized by the protein encoded by pMF1.19 through protein–protein interaction, suggesting that the protein is an immune protein for nuclease cleavage. We propose that the post-segregational killing mechanism of the nuclease toxin and immune protein pair encoded by pMF1.20 and pMF1.19 is helpful for the stable maintenance of pMF1 in M. fulvus cells.

Due to the high similarity in their requirements for space and food, close bacterial relatives may be each other's strongest competitors. Close bacterial relatives often form visible boundaries to separate their swarming colonies, a phenomenon termed colony-merger incompatibility. While bacterial species are known to have many incompatible strains, it is largely unclear which traits lead to multiple incompatibilities and the interactions between multiple incompatible siblings. To investigate the competitive interactions of closely related incompatible strains, we mutated Myxococcus xanthus DK1622, a predatory bacterium with complex social behavior. From 3392 random transposon mutations, we obtained 11 self-identification (SI) deficient mutants that formed unmerged colony boundaries with the ancestral strain. The mutations were at nine loci with unknown functions and formed nine independent SI mutants. Compared with their ancestral strain, most of the SI mutants showed reduced growth, swarming and development abilities, but some remained unchanged from their monocultures. When pairwise mixed with their ancestral strain for co-cultivation, these mutants exhibited improved, reduced or unchanged competitive abilities compared with the ancestral strain. The sporulation efficiencies were affected by the DK1622 partner, ranging from almost complete inhibition to 360% stimulation. The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies. However, the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures. We propose that the colony-merger incompatibility in M. xanthus is associated with multiple independent genetic loci, and the incompatible strains hold competitive interaction abilities, which probably determine the complex relationships between multiple incompatible M. xanthus strains and their co-existence strategies.

Significance Natural products biosynthesized by cryptic gene clusters represent a largely untapped source for drug discovery. However, mining of these products by promoter engineering is restricted by the lack of streamlined genetic tools, especially in nonmodel biosynthetic gene cluster (BGC)-rich bacteria. Here, we describe the discovery of a pair of bacteriophage recombinases and application of recombinase-assisted promoter engineering to rapidly identify and activate several cryptic biosynthetic gene clusters in two Burkholderiales strains that currently lack effective genetic tools. Construction of an efficient genome engineering platform in a natural product producer expedites mining of cryptic BGCs in their native backgrounds, and host melioration for yield or structure optimization. This strategy enables potentially scalable discovery of novel metabolites with intriguing bioactivities from many other bacteria., Bacterial genomes encode numerous cryptic biosynthetic gene clusters (BGCs) that represent a largely untapped source of drugs or pesticides. Mining of the cryptic products is limited by the unavailability of streamlined genetic tools in native producers. Precise genome engineering using bacteriophage recombinases is particularly useful for genome mining. However, recombinases are usually host-specific. The genome-guided discovery of novel recombinases and their transient expression could boost cryptic BGC mining. Herein, we reported a genetic system employing Red recombinases from Burkholderiales strain DSM 7029 for efficient genome engineering in several Burkholderiales species that currently lack effective genetic tools. Using specialized recombinases-assisted in situ insertion of functional promoters, we successfully mined five cryptic nonribosomal peptide synthetase/polyketide synthase BGCs, two of which were silent. Two classes of lipopeptides, glidopeptins and rhizomides, were identified through extensive spectroscopic characterization. This recombinase expression strategy offers utility within other bacteria species, allowing bioprospecting for potentially scalable discovery of novel metabolites with attractive bioactivities.

Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and Orf3, were modulated in the epothilone production strain Burkholderia DSM7029::Tn5-km-epo (named G32) via Red/ET engineering to increase heterologous polyketide epothilone yields. Compared with the prior G32 strain, the total production of several epothilones in the G32::orf14-orf3 mutant was meaningfully doubled according to high-performance liquid chromatography-mass spectrometer analysis. Typically for epothilone B, in simple and clear liquid medium CYMG, the overall productivity in the engineered high-yield producer G32::orf14-orf3 was improved for almost 3-fold, from 2.7 to about 8.1 μg/l. Additionally, the ratio of extracellular to intracellular accumulation of epothilone B was raised from 9.3:1 to 13.7:1 in response to expression of two putative transport genes orf14 and orf3. Hence, we strongly recommend that the Orf14 and Orf3 transporters export epothilone, thus promotes the forward reaction of biosynthesis on epothilone manufacture inside the cells. Our results afford a practical stage for yield improvement of other heterologous natural products in broad chassis cells.

A Glycoside hydrolase (GH) typically contains one catalytic module and varied non-catalytic regions (NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules (CBMs). AgaG4 is a GH16 endo-β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the truncated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

The use of toxin to attack neighbours and immunity proteins to protect against toxin has been observed in bacterial conflicts, including kin discrimination. Here, we report a novel nuclease-toxin and its immunity protein function in the colony-merger incompatibility, a kind of bacterial kin discrimination, in Myxococcus xanthus DK1622. The MXAN_0049 gene was determined to be a genetic determinant for colony-merger incompatibility, and the incompatibility could be eliminated by deletion of the upstream co-transcribed MXAN_0050 gene. We demonstrated that the MXAN_0050 protein was a nuclease, and MXAN_0049 protein was able to bind to MXAN_0050 to block nuclease activity in vitro. Expression of MXAN_0050 in Escherichia coli inhibited cellular growth, and the inhibition effect could be recovered by co-expression of MXAN_0049. We found that deletion of the PAAR-encoding gene (MXAN_0044) or the type VI secretion system led to the colony-merger and co-existence with the ΔMXAN_0049 mutant, suggesting that they were associated with colony-merger incompatibility. Homologues of the nuclease-toxin and cognate immunity pair are widely distributed in bacteria. We propose a simplified model to explain the kin discrimination mechanism mediated by the nuclease-toxin and immunity protein.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. Results In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. Conclusions By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0758-x) contains supplementary material, which is available to authorized users.

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L

Cervus albirostris (white-lipped deer) is an endemic species in China. As the name implies, C. albirostris has a characteristic pure white marking around their mouth and on the underside of the throat. The animal is a typical alpine species normally living at the height of 3500–4300 m. In this study, by pyrosequencing the 16S rRNA gene sequences, we for the first time analyzed the gut bacterial community composition in eight feces samples of wild C. albirostris. From a total of 243,634 high-quality sequences, we identified 186 genera, included in 17 prokaryotic phyla in the feces. The relative proportions of Firmicutes and Bacteroidetes were highly consistent in each individual sample. The most frequently detected genus was Ruminococcaceae UCG-005, ranging from 6.70 to 21.00%, displaying positively connections with the Rikenellaceae RC9 gut group. The bacterial communities associated with C. albirostris provide the basic knowledge for further microbiological studies and facilitates the conservation efforts of this vulnerable deer species.