Halalkalibacterium roseum gen. nov., sp. nov., a new member of the family Balneolaceae isolated from soil.

A Gram-stain-negative, non-motile, moderately halophilic and facultatively anaerobic bacterium, designated YR4-1T, was isolated from a saline-alkali and sorghum-planting soil sample collected in Dongying, Shandong Province, PR China. Growth occurred at 28-45 °C with the presence of 4.0-20.0 % (w/v) NaCl and pH 6.0-9.0. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that YR4-1T shared the highest similarity of 92.1-92.4 % with the valid published species of Aliifodinibius. The isolate formed a separate clade at the genus level in recently described family Balneolaceae. The draft genome of strain YR4-1T is 3.83 Mbp long with 44.0 mol% G+C content. The strain possesses several genes involved in the osmotic stress response mechanism and diverse metabolic pathways, probably for the living in saline environment. This may lead to a better understanding of the underrepresented Balneolaceae lineage. The major menaquinone was MK-7. The main polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipids, aminophosphoglycolipid, one glycolipid, and four unidentified lipids. The predominant cellular fatty acids were iso-C15 : 0 (35.7 %) and anteiso-C15 : 0 (33.5 %). On the basis of its phenotypic, chemotaxonomic and phylogenetic features, strain YR4-1T represents a novel species of a new genus, for which the name Halalkalibacterium roseum gen. nov., sp. nov. is proposed. The type strain is YR4-1T (=CGMCC 1.17777T=KCTC 72795T).

A Dual-Functional Orphan Response Regulator Negatively Controls the Differential Transcription of Duplicate groELs and Plays a Global Regulatory Role in Myxococcus.

Differential transcription of functionally divergent duplicate genes is critical for bacterial cells to properly and competitively function in the environment, but the transcriptional regulation mechanisms remain in mystery. Myxococcus xanthus DK1622 possesses two duplicate groELs with divergent functions. Here, we report that MXAN_4468, an orphan gene located upstream of groEL2, encodes a response regulator (RR) and is responsible for the differential expression regulation of duplicate groELs. This RR protein realizes its negative regulatory role via a novel dual-mode functioning manner: binding to the transcription repressor HrcA to enhance its transcriptional inhibition of duplicate groELs and binding to the 3' end of the MXAN_4468 sequence to specifically decrease the transcription of the following groEL2. Phosphorylation at the conserved 61st aspartic acid is required to trigger the regulatory functions of MXAN_4468. Pull-down experiment and mutation demonstrated that two noncognate CheA proteins, respectively belonging to the Che8 and Che7 chemosensory pathways, are involved in the protein phosphorylation. A transcriptome analysis, as well as the pull-down experiment, suggested that MXAN_4468 plays a global negative regulatory role in M. xanthus. This study elucidates, for the first time, the regulatory mechanism of differential transcription of bacterial duplicate groELs and suggests a global regulatory role of a dual-functional orphan RR. IMPORTANCE Multiply copied groELs require precise regulation of transcriptions for their divergent cellular functions. Here, we reported that an orphan response regulator (RR) tunes the transcriptional discrepancy of the duplicate groELs in Myxococcus xanthus DK1622 in a dual-functional mode. This RR protein has a conserved phosphorylation site, and the phosphorylation is required for the regulatory functions. Transcriptomic analysis, as well as a pull-down experiment, suggests that the RR plays a global regulatory role in M. xanthus. This study highlights that the dual-functional orphan RR might be involved in conducting the transcriptional symphony to stabilize the complex biological functions in cells.

Two new Polyangium species, P. aurulentum sp. nov. and P. jinanense sp. nov., isolated from a soil sample.

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.

ImuA Facilitates SOS Mutagenesis by Inhibiting RecA-Mediated Activity in Myxococcus xanthus.

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 transcription of imuA, imuB, and dnaE2 in UV-C-irradiated Myxococcus xanthus and found that the induction of imuA occurred significantly earlier than that of the other two genes. Mutant analysis showed that unlike that of imuB or dnaE2, the deletion of imuA significantly delayed bacterial regrowth and slightly reduced the bacterial mutation frequency and UV resistance. Transcriptomic analysis revealed that the absence of ImuA released the expression of some known SOS genes, including recA1, recA2, imuB, and dnaE2. Yeast two-hybrid and pulldown analyses proved that ImuA interacts physically with RecA1 besides ImuB. Protein activity analysis indicated that ImuA had no DNA-binding activity but inhibited the DNA-binding and recombinase activity of RecA1. These findings indicate the new role of ImuA in SOS mutagenesis; that is, ImuA inhibits the recombinase activity of RecA1, thereby facilitating SOS mutagenesis in M. xanthus. IMPORTANCE DnaE2 is responsible for bacterial SOS mutagenesis in nearly one-third of sequenced bacterial strains. However, its mechanism, especially the function of one of its accessory proteins, ImuA, is still unclear. Here, we report that M. xanthus ImuA could affect SOS mutagenesis by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the selection of the two restart pathways to repair the stalled replication fork.

The Regulation of LexA on UV-Induced SOS Response in Myxococcus xanthus Based on Transcriptome Analysis.

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.

Two PAAR Proteins with Different C-Terminal Extended Domains Have Distinct Ecological Functions in Myxococcus xanthus.

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 xanthusMXAN_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, but not 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. xanthusIMPORTANCE 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 kin discrimination 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.

Psychroflexus aurantiacus sp. nov., isolated from soil in the Yellow River Delta wetlands.

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).

Deinococcus terrestris sp. nov., a gamma ray- and ultraviolet-resistant bacterium isolated from soil.

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).

Functional Division Between the RecA1 and RecA2 Proteins in Myxococcus xanthus.

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388), with unknown functional differentiation. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation but that the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth but significantly decreased the UV-radiation survival, homologous recombination (HR) ability, and induction of LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and increased the sensitivity to DNA damage caused by hydrogen peroxide but did not change the UV-radiation resistance or SOS gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange (DSE) and LexA autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved mainly to regulate gene expression 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 DNA HR and genome sophistication.

A Hierarchical Network of Four Regulatory Genes Controlling Production of the Polyene Antibiotic Candicidin in Streptomyces sp. Strain FR-008.

The four regulatory genes fscR1 to fscR4 in Streptomyces sp. strain FR-008 form a genetic arrangement that is widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the roles of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1 to fscR4, although differentially expressed throughout the growth phase, displayed similar temporal expression patterns, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1 to fscR4 have various degrees of control over structural genes in the biosynthetic cluster under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with fscR1 and fscR4 functioning at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3 or -4 and fscR4 complementing loss of fscR2 Our study provides more insights into the roles of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound.IMPORTANCE The regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, high-performance liquid chromatography (HPLC) analysis, and genetic cross-complementation, we showed that FscR1 to FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.

Insights into the persistence and phenotypic effects of the endogenous and cryptic plasmid pMF1 in its host strain Myxococcus fulvus 124B02.

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.

ParC, a New Partitioning Protein, Is Necessary for the Active Form of ParA From Myxococcus pMF1 Plasmid.

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.

Whole transcriptome analysis and gene deletion to understand the chloramphenicol resistance mechanism and develop a screening method for homologous recombination in Myxococcus xanthus.

BACKGROUND: Myxococcus xanthus DK1622 is a model system for studying multicellular development, predation, cellular differentiation, and evolution. Furthermore, it is a rich source of novel secondary metabolites and is widely used as heterologous expression host of exogenous biosynthetic gene clusters. For decades, genetic modification of M. xanthus DK1622 has mainly relied on kanamycin and tetracycline selection systems. RESULTS: Here, we introduce an alternative selection system based on chloramphenicol (Cm) to broaden the spectrum of available molecular tools. A chloramphenicol-resistant growth phase and a chloramphenicol-susceptible growth phase before and after chloramphenicol-induction were prepared, and later sequenced to identify specific genes related to chloramphenicol-repercussion and drug-resistance. A total of 481 differentially expressed genes were revealed in chloramphenicol-resistant Cm5_36h and 1920 differentially expressed genes in chloramphenicol-dormant Cm_8h. Moreover, the gene expression profile in the chloramphenicol-dormant strain Cm_8h was quite different from that of Cm5_36 which had completely adapted to Cm, and 1513 differentially expression genes were identified between these two phenotypes. Besides upregulated acetyltransferases, several transporter encoding genes, including ABC transporters, major facilitator superfamily transporters (MFS), resistance-nodulation-cell division (RND) super family transporters and multidrug and toxic compound extrusion family transporters (MATE) were found to be involved in Cm resistance. After the knockout of the most highly upregulated MXAN_2566 MFS family gene, mutant strain DK-2566 was proved to be sensitive to Cm by measuring the growth curve in the Cm-added condition. A plasmid with a Cm resistance marker was constructed and integrated into chromosomes via homologous recombination and Cm screening. The integration efficiency was about 20% at different concentrations of Cm. CONCLUSIONS: This study provides a new antibiotic-based selection system, and will help to understand antibiotic resistance mechanisms in M. xanthus DK1622.

Phylogeny-guided characterization of glycosyltransferases for epothilone glycosylation.

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.

The developmental regulator MtrA binds GlnR boxes and represses nitrogen metabolism genes in Streptomyces coelicolor.

In Streptomyces, GlnR is an activator protein that activates nitrogen-assimilation genes under nitrogen-limiting conditions. However, less is known regarding the regulation of these genes under nitrogen-rich conditions. We determined that the developmental regulator MtrA represses nitrogen-assimilation genes in nitrogen-rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP-qPCR analysis. Transcriptional analysis indicated that, on nutrient-rich medium, MtrA profoundly repressed expression of nitrogen-associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.

Genome Editing in Model Strain Myxococcus xanthus DK1622 by a Site-Specific Cre/loxP Recombination System.

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.

CIRCE element evolved for the coordinated transcriptional regulation of bacterial duplicate groELs.

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.

SCO5351 is a pleiotropic factor that impacts secondary metabolism and morphological development in Streptomyces coelicolor.

The genome of Streptomyces coelicolor encodes hundreds of putative regulatory proteins, most of which are of unknown function, including SCO5351. In this study, we determined that deletion of sco5351 largely abrogates production of actinorhodin (ACT) and reduces production of the calcium-dependent antibiotic (CDA). Comprehensive transcriptional analyses indicated that transcription of genes of the ACT pathway, including the pathway-specific regulator actII-orf4 and those involved in the building of the chemical compound, was markedly lower in Δsco5351 in the late growth phase. However, transcription of genes in the CDA cluster was notably reduced in Δsco5351 only in the early growth phase, suggesting that SCO5351 has a regulatory role throughout growth. Similar to the observations with Δsco5351, ACT production was blocked by mutagenesis of three conserved amino acids potentially involved in dimerization of SCO5351, indicating that protein dimerization is critical to the function of SCO5351. In addition, disruption of sco5351 delayed the formation of aerial mycelium and spores under the conditions tested and, consistent with this, transcription of developmental genes associated with spore formation was reduced in Δsco5351, implying that SCO5351 is involved in developmental control. Our findings reveal SCO5351 as a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor.

O-Phenylenediamine: a privileged pharmacophore of ferrostatins for radical-trapping reactivity in blocking ferroptosis.

Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by the accumulation of lipid hydroperoxides. It has drawn considerable attention owing to its putative involvement in diverse neurodegenerative diseases. Ferrostatins are the first identified inhibitors of ferroptosis and they inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers. However, their further medicinal application has been limited due to the deficient knowledge of the lipid peroxyl radical-trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid hydroperoxide inhibition mechanism of ferrostatins. The results show that an ortho-amine (-NH) moiety from ferrostatins can simultaneously interact with lipid radicals, and then form a planar seven-membered ring in the transition state, and finally present greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin delocalization in the transition state. Additionally, a classical H-bond type interaction was discovered between a radical and an o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel and has not been found in diphenylamine or traditional polyphenol antioxidants. It can be said that o-phenylenediamine is a privileged pharmacophore for the design and development of ferroptosis inhibitors.