A large part (21%) of the wild-type Streptomyces clavuligerus genome is located in a 1.8-Mb megaplasmid that greatly influences secondary metabolites biosynthesis even if the secondary metabolites are chromosomally encoded. The megaplasmid copy number may change depending on the nutritional and environmental conditions. The S. clavuligerus oppA2::aph mutant described by Lorenzana et al. (2004) does not form aerial mycelium, spores, and clavulanic acid, but overproduces holomycin. Transcriptomic studies, polymerase chain reactions (PCR), qPCR, and RT-qPCR analysis showed that S. clavuligerus oppA2::aph has a drastically reduced number of copies (about 25,000-fold lower than the parental strain) of plasmids pSCL1 (10.5 kb), pSCL2 (149.4 kb), and the megaplasmid pSCL4 (1.8 Mb). To clarify the role of the linear plasmids and the function of OppA2 in S. clavuligerus oppA2::aph we constructed oppA2 mutants which contained: (1) a normal copy number of the linear plasmids, (2) completely lack of the linear plasmids, and (3) a parA-parB pSCL4 mutant that resulted in lack of pSCL4. In addition, a strain with a functional oppA2 gene was constructed lacking the megaplasmid pSCL4. The results confirmed that the oppA2 gene is essential for clavulanic acid production, independently of the presence or absence of linear plasmids, but oppA2 has little relevance on differentiation. We demonstrated that the lack of sporulation of S. clavuligerus oppA2::aph is due to the absence of linear plasmids (particularly pSCL4) and the holomycin overproduction is largely due to the lack of pSCL4 and is stimulated by the oppA2 mutation.
b Streptomyces clavuligerus claR::aph is a claR-defective mutant, but in addition to its claR defect it also carries fewer copies of the resident linear plasmids pSCL2 and pSCL4 (on the order of 4 ؋ 10 5 -fold lower than the wild-type strain), as shown by qPCR. To determine the function of ClaR without potential interference due to plasmid copy number, a new strain, S. clavuligerus ⌬claR:: aac, with claR deleted and carrying the wild-type level of plasmids, was constructed. Transcriptomic analyses were performed in S. clavuligerus ⌬claR::aac and S. clavuligerus ATCC 27064 as the control strain. The new ⌬claR mutant did not produce clavulanic acid (CA) and showed a partial expression of genes for the early steps of the CA biosynthesis pathway and a very poor expression (1 to 8%) of the genes for the late steps of the CA pathway. Genes for cephamycin C biosynthesis were weakly upregulated (1.7-fold at 22.5 h of culture) in the ⌬claR mutant, but genes for holomycin biosynthesis were expressed at levels from 3-to 572-fold higher than in the wild-type strain, supporting the observed overproduction of holomycin by S. clavuligerus ⌬claR:: aac. Interestingly, three secondary metabolites produced by gene clusters SMCp20, SMCp22, and SMCp24, encoding still-cryptic compounds, had partially or totally downregulated their genes in the mutant, suggesting a regulatory role for ClaR wider than previously reported. In addition, the amfR gene was downregulated, and consequently, the mutant did not produce aerial mycelium. Expression levels of about 100 genes in the genome were partially up-or downregulated in the ⌬claR mutant, many of them related to the upregulation of the sigma factor-encoding rpoE gene. The claR gene, located in the Streptomyces clavuligerus CA gene cluster, encodes a LysR-type regulator (1, 2). LysR-type transcriptional regulators (LTTRs) follow the pattern of the model regulator controlling lysA expression in Enterobacteriaceae (3). The LTTRs act on metabolic pathways but also in quorum sensing, virulence, motility, nitrogen fixation, oxidative stress responses, and other systems (3, 4, 5). These global transcriptional regulators act mostly as activators but also as repressors and control single genes or operons. The LysR proteins are tetramers, have a helix-turn-helix (HTH) motif close to the N terminus, and bind palindromic sequences identified as T-N 11 -A (6). The binding affinity is usually determined by a ligand coinducer molecule (7).The presence of LysR-type regulators in antibiotic biosynthesis gene clusters is relatively frequent (8, 9, 10). In undecylprodigiosin and actinorhodin production, the autoregulated StgR LTTR negatively controls the expression of the redD and actII-orf4 genes encoding pathway-specific activators (11).The claR gene of S. clavuligerus is expressed as a monocistronic transcript that encodes a protein of 431 amino acids (M r , 47,080). ClaR contains HTH motifs in its amino-and carboxyl-terminal regions. The HTH close to the C-terminal end of the protein contains, fully conser...
Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM , and determined to be the result of a fivefold increase in the production of the tunicamycin complex.
Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.
Background Streptomyces clavuligerus ATCC 27064, the industrial producer of the β-lactamase inhibitor clavulanic acid, carries 49 putative secondary metabolite gene clusters. These secondary metabolite gene clusters are distributed between its linear chromosome and the 1.8 Mb-plasmid pSCL4, a rich reservoir of bioactive compound gene clusters.ResultsThe transcriptome and metabolome of S. clavuligerus ATCC 27064 and the pSCL4− derived strain, S. clavuligerus pSCL4−, were analysed. Construction of the S. clavuligerus pSCL4− strain resulted in the excision of a 303 kb stretch in the right arm of the chromosome and its translocation to pSCL4, producing a 2.1 Mb plasmid named pSCL4* .The absence of pSCL4* results in changes in the transcription level of genes encoding regulatory proteins or proteins with various functions. Lack of pSCL4* results in upregulation of three chromosomal gene clusters for secondary metabolites (SMC), SMC18, for holomycin and N-propionylholothin biosynthesis, SMC11b for tunicamycin biosynthesis (located between SMC10 and SMC11), and SMC5. The SMC10, SMC11 and SMC6 gene clusters were downregulated, resulting in lower production of clavulanic acid, cephamycin C and desferrioxamine E, respectively. Clusters SMC8, SMC12, SMC13 and SMC19 were also downregulated. Production levels of bioactive compounds, such as alkylresorcinol or thiol-derived compounds, were affected in the plasmid-less strain.ConclusionsThe excision and translocation to pSCL4 of 303 kb from the right arm of the chromosome confirms that the ends of the chromosome arms are regions of high instability and supports the hypothesis that pSCL4 might have been excised from S. clavuligerus chromosomal right arm end. Cysteine and methionine metabolism in S. clavuligerus lacking pSCL4* may differ from that of the wild type strain, given the absence of sulfur metabolism genes located either in pSCL4 or at the right end of the chromosome, which led to levels of dithiolopyrrolones (holomycin, N-propionylholothin) and acetylhomocysteine thiolactone (citiolone) higher than those of the wild type strain. S. clavuligerus pSCL4− shows strong differences in its transcriptome and metabolome; however, the loss of 2.1 Mb DNA is dispensable in this strain.
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