Jiang Ye

Cloning and characterization of a novel tyrosine ammonia lyase-encoding gene involved in bagremycins biosynthesis in Streptomyces sp.

Tyrosine ammonia lyase catalyzes the deamination of l-tyrosine to trans-coumaric acid. A novel tyrosine ammonia lyase-encoding gene, bagA, was cloned and sequenced from bagremycins-producing strain Streptomyces sp. Tü 4128 whose protein product contains a Ala–Ser–Gly segment in the active site. The disruption of the bagA gene abolished trans-coumaric acid and bagremycins production. trans-coumaric acid restored the formation of bagremycin A in the mutant, but not bagremycin B. Thus, trans-coumaric acid is a precursor for biosynthesis of bagremycins and the bagA gene codes for tyrosine ammonia lyase to synthesize trans-coumaric acid. This is a novel bacterial tal gene reported in actinomycetes for the second time and for the first time in a Streptomyces sp.

Characterization of Edwardsiella tarda rpoN : roles in σ 70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN+, the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria–Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ70 class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Impact of co‐deficiency of RpoN and RpoS on stress tolerance, virulence and gene regulation in Edwardsiella tarda

Edwardsiella tarda the etiological agent for edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries was subjected to a molecular genetic study. To research into the influence when RpoN (σ54) and RpoS (σ38) were deleted simultaneously, the double deletion mutant of RpoN (σ54) and RpoS (σ38), namely rnrs, was constructed. Firstly, RpoN and RpoS are both essential for H2O2, starvation, high osmotic pressure and acid resistance, which have synergistic effect. Secondly, virulence of rnrs reduces significantly compared to E. tarda EIB 202 WT, ΔrpoN mutant and ΔrpoS mutant. Furthermore, transcriptional control of rpoS by rpoN in stationary phase was observed through qRT‐PCR, while rpoS had no influence on rpoN in the level of transcription. Meanwhile, regulation of flagellar sigma factor σF (FliA) and other flagella‐related genes including flgA, flgK, flgL, motA, and motB by rpoS, and rpoN was found. fliA and other flagella‐related genes were controlled positively by rpoN, while negatively by rpoS. At last, two differential expression genes in transcriptional level of rnrs strain were detected by DD‐RT‐PCR, namely cheY and narK. This study therefore indicated interaction between sigma factors RpoN and RpoS, which modulates stress response, virulence, motility, and provides new insights into the regulatory networks of E. tarda.

Identification of BagI as a positive transcriptional regulator of bagremycin biosynthesis in engineered Streptomyces sp. Tü 4128

Bagremycin A and B, two novel antibiotics from Streptomyces sp. Tü 4128, show a moderate activity against fungi, Gram-positive bacteria and tumor cell and have potential application values in the fields of medicine and agriculture. In this study, we obtained a bagI deletion mutant by in-frame deletion. The assays of bagI-deletion and complementation strains revealed that it was essential to bagremycin biosynthesis. Secondly, the impact of bagI mutants on mycelial growth, sporulation and pigment yields was explored throughout secondary metabolism. SEM images displayed that bagI mutation delayed the mycelium growth and sporulation and reduced pigment yields. Moreover, the yields of bagremycin A and B increased 2.5-fold and 2.6-fold in bagI-overexpressed strain compared to WT. Thirdly, we investigated genes associated with bagremycin biosynthesis by real-time fluorescent quantitative PCR (qRT-PCR). Data showed that BagI played a role of transcriptional activator in the course of bagremycin biosynthesis. This provides us new insights into the regulatory network of bagremycin biosynthesis.

Cloning and characterization of bagB and bagC, two co-transcribed genes involved in bagremycin biosynthesis in Streptomyces sp. Tü 4128

Bagremycin A and B, produced by Streptomyces sp. Tü 4128, are two antibiotics active against Gram-positive bacteria and fungi. To elucidate the biosynthetic pathway of bagremycins, two genes named bagB and bagC, were cloned and characterized from a bagremycin-producing strain. The deduced protein products of bagB and bagC show high sequence identity with aldolase and 3-dehydroquinate synthase, respectively. The bagC gene is located immediately downstream of the bagB gene and subsequent reverse transcription and polymerase chain reaction analysis revealed that bagB is co-transcribed with bagC. Inactivation of either bagB or bagC resulted in the complete abolishment of bagremycin production in fermented cultures, which suggests that these two genes are involved in the biosynthesis of bagremycins. The results from this study will allow acquisition of the entire biosynthetic cluster for bagremycins and further our understanding of the biosynthetic pathway and mechanism of action of bagremycin.

Global regulator BldA regulates morphological differentiation and lincomycin production in Streptomyces lincolnensis

Global regulator BldA, the only tRNA for a rare leucine codon UUA, is best known for its ability to affect morphological differentiation and secondary metabolism in the genus Streptomyces. In this study, we confirmed the regulatory function of the bldA gene (Genbank accession no. EU124663.1) in Streptomyces lincolnensis. Disruption of bldA hinders the sporulation and lincomycin production, that can recur when complemented with a functional bldA gene. Western blotting assays demonstrate that translation of the lmbB2 gene which encodes a L-tyrosine hydroxylase is absolutely dependent on BldA; however, mistranslation of the lmbU gene which encodes a cluster-situated regulator (CSR) is observed in a bldA mutant. Intriguingly, when the preferential cognate codon CTG was used, the expression level of LmbU was not the highest compared to the usage of rare codon TTA or CTA, indicating the rare codon in this position is significant for the regulation of lmbU expression. Moreover, replacement of TTA codons in both genes with another leucin codon in the bldA mutant did not restore lincomycin production. Thus, we believe that the bldA gene regulates lincomycin production via controlling the translation of not only lmbB2 and lmbU, but also the other TTA-containing genes. In conclusion, the present study demonstrated the importance of the bldA gene in morphological differentiation and lincomycin production in S. lincolnensis.

EsrE-A yigP Locus-Encoded Transcript-Is a 3′ UTR sRNA Involved in the Respiratory Chain of E. coli

The yigP locus is widely conserved among γ-proteobacteria. Mutation of the yigP locus impacts aerobic growth of Gram-negative bacteria. However, the underlying mechanism of how the yigP locus influences aerobic growth remains largely unknown. Here, we demonstrated that the yigP locus in Escherichia coli encodes two transcripts; the mRNA of ubiquinone biosynthesis protein, UbiJ, and the 3′ untranslated region small regulatory RNA (sRNA), EsrE. EsrE is an independent transcript that is transcribed using an internal promoter of the yigP locus. Surprisingly, we found that both the EsrE sRNA and UbiJ protein were required for Q8 biosynthesis, and were sufficient to rescue the growth defect ascribed to deletion of the yigP locus. Moreover, our data showed that EsrE targeted multiple mRNAs involved in several cellular processes including murein biosynthesis and the tricarboxylic acid cycle. Among these targets, sdhD mRNA that encodes one subunit of succinate dehydrogenase (SDH), was significantly activated. Our findings provided an insight into the important function of EsrE in bacterial adaptation to various environments, as well as coordinating different aspects of bacterial physiology.

A yigP mutant strain is a small colony variant of E. coli , and shows pleiotropic antibiotic resistance

Small colony variants (SCVs) are a commonly observed subpopulation of bacteria that have a small colony size and distinctive biochemical characteristics. SCVs are more resistant than the wild type to some antibiotics and usually cause persistent infections in the clinic. SCV studies have been very active during the past 2 decades, especially Staphylococcus aureus SCVs. However, fewer studies on Escherichia coli SCVs exist, so we studied an E. coli SCV during an experiment involving the deletion of the yigP locus. PCR and DNA sequencing revealed that the SCV was attributable to a defect in the yigP function. Furthermore, we investigated the antibiotic resistance profile of the E. coli SCV and it showed increased erythromycin, kanamycin, and d-cycloserine resistance, but collateral sensitivity to ampicillin, polymyxin, chloramphenicol, tetracycline, rifampin, and nalidixic acid. We tried to determine the association between yigP and the pleiotropic antibiotic resistance of the SCV by analyzing biofilm formation, cellular morphology, and coenzyme Q (Q8) production. Our results indicated that impaired Q8 biosynthesis was the primary factor that contributed to the increased resistance and collateral sensitivity of the SCV. This study offers a novel genetic basis for E. coli SCVs and an insight into the development of alternative antimicrobial strategies for clinical therapy.