Production of Antibacterial Compounds from Actinomycetes

Actinomycetes are soil-dwelling Gram-positive bacteria, industrially relevant as producers of a wide range of bioactive secondary metabolites, including many antibiotics of clinical and commercial importance

The DNA cytosine methylome revealed two methylation motifs in the upstream regions of genes related to morphological and physiological differentiation in Streptomyces coelicolor A(3)2 M145

DNA methylation is an epigenetic modification detected in both prokaryotic and eukaryotic genomic DNAs. In bacteria, the importance of 5-methylcytosine (m5C) in gene expression has been less investigated than in eukaryotic systems. Through dot-blot analysis employing m5C antibodies against chromosomal DNA, we have previously demonstrated that m5C influences the differentiation of Streptomyces coelicolor A(3)2 M145 in solid sporulating and liquid non-sporulating complex media. Here, we mapped the methylated cytosines of the M145 strain growing in the defined Maltose Glutamate (MG) liquid medium. Sequencing of the M145 genome after bisulfite treatment (BS-sequencing) evidenced 3360 methylated cytosines and the two methylation motifs, GGCmCGG and GCCmCG, in the upstream regions of 321 genes. Besides, the role of cytosine methylation was investigated using the hypo-methylating agent 5'-aza-2'-deoxycytidine (5-aza-dC) in S. coelicolor cultures, demonstrating that m5C affects both growth and antibiotic biosynthesis. Finally, quantitative reverse-transcription polymerase-chain-reaction (RT-qPCR) analysis of genes containing the methylation motifs in the upstream regions showed that 5-aza-dC treatment influenced their transcriptional levels and those of the regulatory genes for two antibiotics. To the best of our knowledge, this is the first study that reports the cytosine methylome of S. coelicolor M145, supporting the crucial role ascribed to cytosine methylation in controlling bacterial gene expression

Comparison of Antibiotic Resistance Profile and Biofilm Production of Staphylococcus aureus Isolates Derived from Human Specimens and Animal-Derived Samples

Background: The diusion of antimicrobial resistance is a significant concern for public health worldwide. Staphylococcus aureus represents a paradigm microorganism for antibiotic resistance in that resistant strains appear within a decade after the introduction of new antibiotics. Methods: Fourteen S. aureus isolates from human specimens and twenty-one from samples of animal origin, were compared for their antimicrobial resistance and biofilm capability. In addition, they were characterized at the molecular level to detect the antimicrobial resistance mecA gene and genes related with enterotoxin, toxin, and biofilm production. Results: Both phenotypic and molecular analysis showed main dierences among human- and animal-derived isolates. Among the human-derived isolates, more multidrug-resistant isolates were detected and mecA gene, enterotoxin, and toxin genes were more prevalent. Dierent genes involved in biofilm production were detected with bap present only in animal-derived isolates and sasC present in both isolates, however, with a higher prevalence in the human-derived isolates. Biofilm capability was higher in human-derived isolates mainly associated to the sasC gene. Conclusions: The overall results indicate that human S. aureus isolates are more virulent and resistant than the isolates of animal origin randomly selected with no infection anamnesis. This study confirms that selection for more virulent and resistant S. aureus strains is related to the clinical practice

Correlation Between Microbial Community and Hatching Failure in Loggerhead Sea Turtle Caretta caretta

Microbial communities provide essential information about host ecology and could be helpful as a tool to improve species conservation efforts. However, microbes can also infect and compromise the host development process and viability. Caretta caretta is the most widespread marine turtle species in the Mediterranean basin and is the only species of sea turtle nesting along the Italian coasts. Little is known about the microbiota composition of the nest of sea turtles and its correlation with hatching failures. In this study, the microbial composition of two nests of C. caretta featuring different rates of hatching success from a nesting beach in Lampedusa (Italy) was analyzed and compared. The bacterial community was determined using culture-dependent methods and next-generation sequencing based on 16S rRNA gene metabarcoding analysis. Our results showed five dominant bacterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes) and indicated different bacterial families (Pseudomonadaceae and Brucellaceae) as likely causes of hatching failures. Besides, our findings demonstrated the nests' active role in modulating the sand's bacterial communities. This study suggests microbiological analysis could be a valuable tool in monitoring nests to take preventive actions and reduce hatching failures

Epigenetic control of Streptomyces coelicolor differentiation

DNA cytosine methylation is one of the most important epigenetic modifications in eukaryotes regulating chromatin organization, genome maintenance and gene expression. The role of DNA cytosine methylation in prokaryotes has not been deeply investigated. In Escherichia coli cytosine methylation regulates gene expression during the stationary phase and cytosine hypermethylation leads to chromosomal DNA cleavage and cell death. Streptomyces coelicolor is a mycelial soil microorganism, which exhibits a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate and aerial mycelium, MII). The importance of DNA methylation was already described in Streptomycetes, but its biological role remained unknown. The main objectives of this study were to analyze the pattern of cytosine methylation in Streptomyces coelicolor and to investigate the relationship between DNA cytosine methylation and morphological/physiological differentiation. Dot-blot analysis of genomic DNA using antibody anti-5-methylcytosine revealed that DNA methylation is modulated during hyphae differentiation. Specifically DNA cytosine methylation is higher at the MI stage than in the MII or spores. Cytosine methylome was investigated by bisulphite DNA sequencing showing that 30% of S. coelicolor genes contain a methylated motif in their upstream region. The biological effect of cytosine methylation was studied using 5-aza-2’-deoxycytidine (aza-dC), a hypomethylating agent. Phenotypic analyses of cultures treated with aza-dC demonstrated that they were impaired in germination, aerial mycelium formation and sporulation. In addition, they showed a strong reduction in antibiotic production. Overall, our results suggest a role for DNA cytosine methylation in morphological and physiological differentiation of S. coelicolor. Further experiments are ongoing to characterize the molecular mechanisms and pathways behind the observed phenotypes

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

<p>Abstract</p> <p>Background</p> <p>A bacterial artificial chromosomal library of <it>Planobispora rosea</it>, a genetically intractable actinomycete strain, was constructed using <it>Escherichia coli</it>-<it>Streptomyces </it>artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.</p> <p>Results</p> <p>One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These <it>orfs </it>encode two proteins with high similarity to a putative membrane protein of <it>Streptomyces coelicolor </it>and to the nogalamycin resistance protein SnorO of <it>Streptomyces nogalater</it>, respectively. The role of these two Orfs is unknown in <it>Planobispora. </it>Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker.</p> <p>Conclusion</p> <p>Two <it>Planobispora rosea orfs </it>are responsible for increasing intracellular amounts and storage of thiostrepton in <it>Streptomyces lividans</it>.</p

Initial pH Conditions Shape the Microbial Community Structure of Sewage Sludge in Batch Fermentations for the Improvement of Volatile Fatty Acid Production

Conversion of wastewater treatment plants into biorefineries is a sustainable alternative for obtaining valuable compounds, thus reducing pollutants and costs and protecting the environment and human health. Under specific operating conditions, microbial fermentative products of sewage sludge are volatile fatty acids (VFA) that can be precursors of polyhydroxyalkanoate thermoplastic polyesters. The role of various operating parameters in VFA production has yet to be elucidated. This study aimed to correlate the levels of VFA yields with prokaryotic microbiota structures of sewage sludge in two sets of batch fermentations with an initial pH of 8 and 10. The sewage sludge used to inoculate the batch fermentations was collected from a Sicilian WWTP located in Marineo (Italy) as a case study. Gas chromatography analysis revealed that initial pH 10 stimulated chemical oxygen demands (sCOD) and VFA yields (2020 mg COD/L) in comparison with initial pH 8. Characterization of the sewage sludge prokaryotic community structures-analyzed by next-generation sequencing of 16S rRNA gene amplicons-demonstrated that the improved yield of VFA paralleled the increased abundance of fermenting bacteria belonging to Proteobacteria, Bacteroidetes, Chloroflexi, and Firmicutes phyla and, conversely, the reduced abundance of VFA-degrading strains, such as archaeal methanogens

Antibiotic Resistance Profiling, Analysis of Virulence Aspects and Molecular Genotyping of Staphylococcus aureus Isolated in Sicily, Italy

Staphylococcus aureus is the major cause of foodborne diseases worldwide. In this retrospective study, 84 S. aureus strains were characterized. The collection comprises 78 strains isolated during 1998 and 2014 from dairy products and tissue samples from livestock bred for dairy production in Sicily. One isolate was obtained from a pet (dog), one from an exotic animal (a circus elephant), and four human isolates were obtained during a severe food poisoning outbreak that occurred in Sicily in 2015. All the strains were characterized by pulsed-field gel electrophoresis (PFGE), for antibiotic resistance and presence of toxin genes. PFGE results showed 10 different pulsotypes, with three relatively frequent and three unique. The antibiotic resistance profiling showed that penicillin G (35.7%) and tetracycline (20.2%) resistance is largely spread. Most isolates contained at least one toxin gene making them a potential threat for public health. Enterotoxin sec gene was observed in 28.6% and seg in 23.8% of the strains, respectively; the human isolates were the only ones to concurrently harbor both seg and sei genes. In addition, 24 isolates were randomly selected and analyzed by multilocus sequence typing. Interestingly, the analysis showed the presence of 12 sequence types (STs), of which 6 were novel. One of them, ST700, was detected in 29% of the isolates and was found to be spread throughout Sicily. ST700 has been present in the island for almost 16 years (1998-2014) and it shows no host preference since it was isolated from different ruminant species. Four human isolates shared both the pulsotype (PT10) and the sequence type (ST9), as well as the virulence genes (seg-sei); this observation suggests that the isolates originated from a single clone, although they were obtained from two different individuals

Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

Background Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. Results Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38+/-0.02 and 0.33+/-0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2D reference-map (http://www.unipa.it/ampuglia/Abal-proteome-maps) matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). Conclusion In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement