Pseudomonas aeruginosa and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them, pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 (polyamine utilization) encode enzymes homologous to Escherichia coli PuuABCD of the ␥-glutamylation pathway in converting Put into GABA. A series of unmarked pauA mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of pauA knockouts to abolish utilization of different polyamines and support the importance of ␥-glutamylation for polyamine catabolism in P. aeruginosa. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the pauA3B2 operon and the bauABCD operon (␤-alanine utilization). Mutation analysis led to the conclusion that pauA3B2 participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that bauABCD are essential for growth on ␤-alanine derived from DAP (or Spd) catabolism via the ␥-glutamylation pathway. Measurements of the pauA3-lacZ and bauA-lacZ expression indicated that these two promoters were differentially induced by Spd, DAP, and ␤-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the pauA3 and bauA promoters was abolished in the bauR mutant. The recombinant BauR protein was purified to demonstrate its interactions with the pauA3 and bauA regulatory regions in vitro. In summary, the present study support that the ␥-glutamylation pathway for polyamine utilization is evolutionarily conserved in E. coli and Pseudomonas spp. and is further expanded in Pseudomonas to accommodate a more diverse metabolic capacity in this group of microorganisms.Biogenic polyamines are a group of ubiquitous polycations found in all living organisms. They are essential for cell growth and participate in a variety of physiological functions (2,30,31). Depending on the specific biosynthetic pathways (12,22,26,29), different bacteria possess a preferential set of polyamines, which include the diamines diaminopropane (DAP), putrescine (Put), and cadaverine (Cad); the triamines spermidine (Spd) and norspermidine; and the tetramine spermine. It is generally believed that polyamines form complexes with nucleic acid-containing macromolecules through charge interactions in vivo (8,11,16). In vitro, excess binding of polyamines to DNA was reported to form very condensed complexes (3), which might cause difficulties in DNA unwinding during replication or transcription. Therefore, the intracellular concentrations of polyamines need to be tightly monitored to prevent adverse effects on cell growth.When released from the cells into environments, polyamines can be recycled by many bacteria or serve as sources of carbo...

The recent sequencing of the DNA region of the geldanamycin post-polyketide synthase (PKS) modification gene clusters revealed the presence of two regulatory genes: gdmRI (2,907 bp) and gdmRII (2,766 bp). The deduced products of gdmRI and gdmRII (968 and 921 amino acid residues, respectively) were identified as homologues of the LuxR transcriptional regulatory proteins. Inactivation by gene replacement of gdmRI or gdmRII in the Streptomyces hygroscopicus 17997 genome resulted in a complete loss of geldanamycin production. Complementation by a plasmid carrying gdmRI or gdmRII restored geldanamycin production, suggesting that the products of these two regulatory genes are positive regulators that are required for geldanamycin biosynthesis. The gdmRI transcript was detected in the DeltagdmRII mutant, and the gdmRII was detected in the DeltagdmRI mutant, indicating that the two genes are transcribed independently and do not regulate each other. Time course of gene expression analysis by RT-PCR of the geldanamycin biosynthetic genes showed that the transcription of gdmRI and gdmRII correlates with that of genes involved in polyketide biosynthesis, but not with the post-PKS modification gene gdmN, whose transcription is initiated earlier. gdmRI or gdmRII gene disruptants did not transcribe the polyketide biosynthetic related genes pks, gdmF, and gdnA-O-P, but did trancribe gdmN. These results demonstrated that gdmRI and gdmRII are pathway-specific positive regulators that control the polyketide biosynthetic genes in geldanamycin biosynthesis, but not the post-PKS modification gene, gdmN.

(14) and to trigger biofilm disassembly (12). Therefore, it is important to understand how bacterial cells regulate D-amino acid homeostasis.In living organisms, biosynthesis of free-form D-amino acids is catalyzed by racemases, with L-enantiomers as substrates. Peptidyl D-amino acids occur either by taking free D-amino acid as a substrate in the cell wall synthesis or by L-to-D epimerization, as in the nonribosomal peptide synthesis in microorganisms (4). In higher eukaryotic organisms, this process is infrequently catalyzed by enzyme-driven posttranslational isomerization (11). Amino acid racemization also occurs at an accelerated rate with physical and/or chemical treatments. For example, racemization of L-lysine at elevated temperatures has great potential as a commercial process of D-lysine production (20).The biochemistry of D-amino acid catabolism has not been intensively studied in comparison to those of L-amino acids. In general, D-amino acids are metabolized either directly or after conversion into the L-enantiomers. Pseudomonas aeruginosa is able to utilize many D-amino acids as nutrients and hence serves as an excellent model organism to explore novel pathways and enzymes for D-amino acid metabolism. A new type of D-to-L arginine racemization by coupled catabolic and anabolic dehydrogenases encoded by the dauBA operon was recently reported by our group (15-16). Furthermore, the molecular structure of DauA, a flavin adenine dinucleotide (FAD)-dependent D-amino acid dehydrogenase, has been determined (8).In contrast, L-alanine catabolism in Escherichia coli and other Gram-negative bacteria is mediated by DadX-dependent L-to-D racemization followed by DadA-dependent oxidative deamination of D-alanine (Fig. 1). An early report by Wasserman and coworkers established the presence of two alanine racemases, the importance of L-to-D racemization for L-Ala catabolism, and the physical proximity and coregulation of two genes encoding D-alanine dehydrogenase and catabolic alanine racemase in Salmonella enterica serovar Typhimurium (21). The same gene organization was later found in E. coli (17). The dadAX operon and its regulation by the leucine-responsive regulator Lrp and carbon catabolite repression in enteric bacteria have been characterized (23-24). DadA of E. coli has

To clone and study the geldanamycin biosynthetic gene cluster in Streptomyces hygroscopicus 17997, we designed degenerate primers based on the conserved sequence of the ansamycin 3-amino-5-hydroxybenzoic acid (AHBA) synthase gene. A 755-bp polymerase chain reaction product was obtained from S. hygroscopicus 17997 genomic DNA, which showed high similarity to ansamycin AHBA synthase genes. Through screening the cosmid library of S. hygroscopicus 17997, two loci of separated AHBA biosynthetic gene clusters were discovered. Comparisons of sequence homology and gene organization indicated that the two AHBA biosynthetic gene clusters could be divided into a benzenic and a naphthalenic subgroup. Gene disruption demonstrated that the benzenic AHBA gene cluster is involved in the biosynthesis of geldanamycin. However, the naphthalenic AHBA genes in the genome of Streptomyces hygroscopicus 17997 could not complement the deficiency of the benzenic AHBA genes. This is the first report on the AHBA biosynthetic gene cluster in a geldanamycin-producing strain.

Background Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells. Methods We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway. Results ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells. Conclusions We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

COVID-19 pandemic caused by SARS-CoV-2 infection severely threatens global health and economic development. No effective antiviral drug is currently available to treat COVID-19 and any other human coronavirus infections. We report herein that a CFDA-approved macrolide antibiotic, carrimycin, potently inhibited the cytopathic effects (CPE) and reduced the levels of viral protein and RNA in multiple cell types infected by human coronavirus 229E, OC43, and SARS-CoV-2. Time-of-addition and pseudotype virus infection studies indicated that carrimycin inhibited one or multiple post-entry replication events of human coronavirus infection. In support of this notion, metabolic labelling studies showed that carrimycin significantly inhibited the synthesis of viral RNA. Our studies thus strongly suggest that carrimycin is an antiviral agent against a broad-spectrum of human coronaviruses and its therapeutic efficacy to COVID-19 is currently under clinical investigation.

To investigate the distribution of dTDP-glucose-4,6-dehydratase (dTGD) gene and diversity of the potential 6-deoxyhexose (6DOH) glycosylated compounds in marine microorganisms, a total of 91 marine sediment-derived bacteria, representing 48 operational taxonomic units and belonging to 25 genera, were screened by polymerase chain reaction. In total, 84% of the strains were dTGD gene positive, suggesting 6DOH biosynthetic pathway is widespread in these marine sediment-derived bacteria. BLASTp results of dTGD gene fragments indicate a high chemical diversity of the potential 6DOH glycosylated compounds. Close phylogenetic relationship occurred between dTGDs involved in the production of same or similar 6DOH glycosylated compounds, suggesting dTGD can be used to predict the structure of potential 6DOH glycosylated compounds produced by new strains. In two cases, where dTGD shared ≥85% amino acid identity and close phylogenetic relationship with their counterparts, 6DOH glycosylated compounds were accurately predicted. Our results demonstrate that phylogenetic analysis of dTGD gene is useful for structure prediction of glycosylated compounds from newly isolated strains and can therefore guide the chemical purification and structure identification process. The rapid identification of strains that possess dTGD gene provides a bioinformatics assessment of the greatest potential to produce glycosylated compounds despite the absence of fully biosynthetic pathways or genome sequences.

Background Bitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene ( ist ). Exogenous l -Leucine ( l -Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and l -Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus . Results SSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23 , bsm42 and acyB2 , in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the Δ SSP_Lrp -SP strain containing an SSP_Lrp deficient of putative l -Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 μg/L versus 313.3 ± 25.2 μg/L, respectively), especially SP III increased remarkably. The yield of BT in Δ SSP_Lrp- BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the Δ SSP_Lrp -SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the Δ SSP_Lrp -SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine. Conclusion SSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative l -Leu binding domain was advantageous for production of BT and SP, especially their III components. Electronic supplementary material The online version of this article (10.1186/s12934-019-1086-0) contains supplementary material, which is available to authorized users.