Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20 -30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

During analysis of the recently identified gene cluster for the glycopeptide antibiotic balhimycin, produced by Amycolatopsis mediterranei DSM 5908, novel genes were identified and characterized in detail. The gene products of four of the identified genes (bpsA, bpsB, bpsC and bpsD) are nonribosomal peptide synthetases (NRPSs) ; one (Orf1-protein) shows similarities to small proteins associated with several NRPSs without an assigned function. BpsA and BpsB are composed of three modules each (modules 1-6), BpsC of one module (module 7) and BpsD of a minimal module (module 8). Thus, the balhimycin gene cluster encodes eight modules, whereas its biosynthetic product is a heptapeptide. Non-producing mutants were created by a gene disruption of bpsB, an in-frame deletion of bpsC and a gene replacement of bpsD. After establishment of a gene complementation system for Amycolatopsis strains, the replacement mutant of bpsD was complemented, demonstrating for the first time that BpsD, encoding the eighth module, is indeed involved in balhimycin biosynthesis. After feeding with β-hydroxytyrosine the capability of the bpsD mutant to produce balhimycin was restored, demonstrating the participation of BpsD in the biosynthesis of this amino acid. The specificity of four of the eight adenylation domains was determined by ATP/PP i exchange assays : modules 4 and 5 activated L-4-hydroxyphenylglycine, module 6 activated β-hydroxytyrosine and module 7 activated L-3,5-dihydroxyphenylglycine, which is in accordance with the sequence of the non-proteogenic amino acids 4 to 7 of the balhimycin backbone.

Balhimycin, produced by the actinomycete Amycolatopsis balhimycina DSM5908, is a glycopeptide antibiotic highly similar to vancomycin, the antibiotic of ‘last resort’ used for the treatment of resistant Gram-positive pathogenic bacteria. Partial sequence of the balhimycin biosynthesis gene cluster was previously reported. In this work, cosmids which overlap the region of the characterized gene cluster were isolated and sequenced. At the ‘left’ end of the cluster, genes were identified which are involved in balhimycin biosynthesis, transport, resistance and regulation. The ‘right’ end border is defined by a putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (dahp) gene. The proximate gene is similar to a type I polyketide synthase gene of the rifamycin producer Amycolatopsis mediterranei indicating that another biosynthesis gene cluster might be located directly next to the balhimycin gene cluster. The newly identified StrR-like pathway-specific regulator, Bbr, was characterized to be a DNA-binding protein and may have a role in balhimycin biosynthesis. Purified N-terminally His-tagged Bbr shows specific DNA-binding to five promoter regions within the gene cluster. By in silico analysis and by comparison of the DNA sequences binding Bbr, conserved inverted repeat sequences for the Bbr-binding site are proposed. The putative Bbr consensus sequence differs from that published for StrR.

Background Salmonella enterica serovar Typhimurium is a common food-borne pathogen. S. enterica uses a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1) to invade intestinal epithelial cells. A complex network of interacting transcription factors regulates SPI-1 gene expression. In addition, SPI-1 gene expression is coupled to flagellar gene expression. Both SPI-1 and flagellar gene expression are bistable, with co-existing populations of cells expressing and not expressing these genes. Previous work demonstrated that nutrients could be used to tune the fraction of cells expressing the flagellar genes. In the present study, we tested whether nutrients could also tune the fraction of cells expressing the SPI-1 genes through transcriptional crosstalk with the flagellar genes. Results Nutrients alone were not found to induce SPI-1 gene expression. However, when the cells were also grown in the presence of acetate, the concentration of nutrients in the growth medium was able to tune the fraction of cells expressing the SPI-1 genes. During growth in nutrient-poor medium, acetate alone was unable to induce SPI-1 gene expression. These results demonstrate that acetate and nutrients synergistically activate SPI-1 gene expression. The response to acetate was governed by the BarA/SirA two-component system and the response to nutrients was governed by transcriptional crosstalk with the flagella system, specifically through the action of the flagellar regulator FliZ. Conclusions Acetate and nutrients are capable of synergistically activating SPI-1 gene expression. In addition, these signals were found to tune the fraction of cells expressing the SPI-1 genes. The governing mechanism involves transcriptional crosstalk with the flagellar gene network. Collectively, these results further our understanding of SPI-1 gene regulation and provide the basis for future studies investigating this complex regulatory mechanism. Electronic supplementary material The online version of this article (10.1186/s12866-019-1583-7) contains supplementary material, which is available to authorized users.

The fast progression of nanotechnology has led to novel therapeutic interventions. Antimicrobial activities of silver nanoparticles (Ag NPs) were tested against standard ATCC strains of Staphylococcus aureus (ATCC 9144), Escherichia coli (O157:H7), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 90028) in addition to 60 clinical isolates collected from cancer patients. Antimicrobial activity was tested by disk diffusion method and MIC values for Ag NPs alone and in combination with N-acetylcysteine (NAC) against tested pathogens were determined by broth microdilution method. Ag NPs showed a robust antimicrobial activity against all tested pathogens and NAC substantially enhanced the antimicrobial activity of Ag NPs against all tested pathogens. Synergism between Ag NPs and NAC has been confirmed by checkerboard assay. The effect of Ag NPs on tested pathogens was further scrutinized by Transmission Electron Microscope (TEM) which showed disruption of cell wall in both bacteria and fungi. Ag NPs abrogated the activity of respiratory chain dehydrogenase of all tested pathogens and released muramic acid content from S. aureus in culture. The cytotoxic effect of Ag NPs alone and in combination with NAC was examined using human HepG2 cells and this revealed no cytotoxicity at MIC values of Ag NPs and interestingly, NAC reduced the cytotoxic effect of Ag NPs at concentrations higher than their MIC values. Taken together, Ag NPs have robust antimicrobial activity and NAC substantially enhances their antimicrobial activities against MDR pathogens which would provide a novel safe, effective, and inexpensive therapeutic approach to control the prevalence of MDR pathogens.

A group of 11 medicinal plants, including Lavandula pubescens, Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Silene succulenta, Silene villosa, Bogonvillea glabra, Cakile maritime, Gomphrene celesoids, Mirabilis jalaba, and Silene nocturna growing in Egypt, were extracted and examined for their immunomodulatory and antioxidant activities. RAW 264.7 cells were recruited to investigate the immunomodulatory effect through multiple parameters analysis. First, the proliferation index of macrophages cells was evaluated revealing that Trigonella foenugricium, Silene succulenta and Silene villosa have a significant cytotoxic effect on RAW cells. Interestingly, we observed enhancement of macrophages phagocytic function of by all extracts except Cakile maritime, Gomphrena celosioides and Silene nocturna. Afterwards, macrophages were challenged by incubation with LPS and the effect of various extracts on inflammatory responses was investigated; the generation of NO from activated macrophage was substantially suppressed by 7 extracts namely, Trigonella foenugricium, Calligonum comosum, Silene succulenta, Bougainvillea glabra, Mirabilis jalaba, Gomphrena celosioides and Silene nocturna. TNF-α was decreased by percentage range from 3.8 to 85.8% and Trigonella foenugricium extract showed the highest inhibition of TNF-α release. All extracts except Trigonella foenugricium, Salsola schweinforthi, Silene succulenta and Mirabilis jalaba significantly inhibited COX-2 production from stimulated macrophage. Moreover, evaluating the potential antioxidant activity of these extracts showed that Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba exhibited some antioxidant activities. Taken together, our results suggest that some of these extracts may have a considerable antinflammatory and antioxidant effects and may be a potential therapeutic choice in the treatment of inflammatory diseases.

Background Salmonella enterica serovar Typhimurium is an intestinal pathogen capable of infecting a wide range of animals. It initiates infection by invading intestinal epithelial cells using a type III secretion system encoded within Salmonella pathogenicity island 1 (SPI-1). The SPI-1 genes are regulated by multiple interacting transcription factors. The master regulator is HilD. HilE represses SPI-1 gene expression by binding HilD and preventing it from activating its target promoters. Previous work found that acetate and nutrients synergistically induce SPI-1 gene expression. In the present study, we investigated the role of HilE, nominally a repressor of SPI-1 gene expression, in mediating this response to acetate and nutrients. Results HilE is necessary for activation of SPI-1 gene expression by acetate and nutrients. In mutants lacking hilE, acetate and nutrients no longer increase SPI-1 gene expression but rather repress it. This puzzling response is not due to the BarA/SirA two component system, which governs the response to acetate. To identify the mechanism, we profiled gene expression using RNAseq in the wild type and a ΔhilE mutant under different growth conditions. Analysis of these data suggested that the Rcs system, which regulates gene expression in response to envelope stress, is involved. Consistent with this hypothesis, acetate and nutrients were able to induce SPI-1 gene expression in mutants lacking hilE and the Rcs system. Conclusions While the exact mechanism is unknown, these results demonstrate the HilE, nominally a repressor of SPI-1 gene expression, can also function as an activator under the growth conditions investigated. Collectively, these results provide new insights regarding SPI-1 gene regulation and demonstrate that HilE is more complex than initially envisioned.

Quorum sensing has been shown to play a crucial role in Pseudomonas aeruginosa pathogenesis where it activates expression of myriad genes that regulate the production of important virulence factors such as biofilm formation. Antagonism of quorum sensing is an excellent target for antimicrobial therapy and represents a novel approach to combat drug resistance. In this study, Chromobacterium violaceum biosensor strain was employed as a fast, sensitive, reliable, and easy to use tool for rapid screening of soil samples for Quorum Sensing Inhibitors (QSI) and the optimal conditions for maximal QSI production were scrutinized. Screening of 127 soil isolates showed that 43 isolates were able to breakdown the HHL signal. Out of the 43 isolates, 38 isolates were able to inhibit the violet color of the biosensor and to form easily detectable zones of color inhibition around their growth. A confirmatory bioassay was carried out after concentrating the putative positive cell-free lysates. Three different isolates that belonged to Bacillus cereus group were shown to have QSI activities and their QSI activities were optimized by changing their culture conditions. Further experiments revealed that the cell-free lysates of these isolates were able to inhibit biofilm formation by P. aeruginosa clinical isolates.