Antibiotics from Gram-negative bacteria: a comprehensive overview and selected biosynthetic highlights.

Covering: up to 2017The overwhelming majority of antibiotics in clinical use originate from Gram-positive Actinobacteria. In recent years, however, Gram-negative bacteria have become increasingly recognised as a rich yet underexplored source of novel antimicrobials, with the potential to combat the looming health threat posed by antibiotic resistance. In this article, we have compiled a comprehensive list of natural products with antimicrobial activity from Gram-negative bacteria, including information on their biosynthetic origin(s) and molecular target(s), where known. We also provide a detailed discussion of several unusual pathways for antibiotic biosynthesis in Gram-negative bacteria, serving to highlight the exceptional biocatalytic repertoire of this group of microorganisms.

Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis.

Actinobacteria produce a variety of polyketide alkaloids with unusual structures. Recently, it was shown that a type I modular polyketide synthase (PKS) is involved in the assembly of coelimycin P1, a polyketide alkaloid produced by Streptomyces coelicolor M145. However, the mechanisms for converting the product of the PKS to coelimycin P1 remain to be elucidated. Here we show that the C-terminal thioester reductase (TR) domain of the PKS and an ω-transaminase are responsible for release of the polyketide chain as an aldehyde and its subsequent reductive amination. Bioinformatics analyses identified numerous gene clusters in actinobacterial genomes that encode modular PKSs with a C-terminal TR domain and a homolog of the ω-transaminase. These are predicted to direct the biosynthesis of both known and novel polyketide alkaloids, suggesting that reductive chain release and transamination constitutes a conserved mechanism for the biosynthesis of such metabolites.

Structural changes in freshwater fish and chironomids exposed to bacterial exotoxins.

Mass fish mortalities have been reported in the past decade from British waters, often coinciding with blooms of filamentous actinobacteria, particularly strains of Streptomyces griseus. The present study has shown that some fractions of the exudate of S. griseus, prepared after a series of high pressure liquid chromatography (HPLC) separations, and analysed with liquid chromatography mass spectrometry (LC-MS), induced pathological changes to the gills of carp and/or tench fry following exposure under laboratory conditions up to 96 h Similar changes were induced by streptomycin, a secondary metabolite of S. griseus, and these included loss of microridging and fusion of secondary lamellae, with carp fry in the case of the exudate of S. griseus being more sensitive than tench fry, especially with exposure to fraction 9 and selected sub-fractions of 9. Some deformities using a severity index were also observed in the head capsule of larvae of the non-biting midge Chironomus riparius, including loss and splitting of teeth on the mentum. The results are discussed in relation to further identification of metabolites derived from samples of the organic filtrate of S. griseus and implications for the functioning of freshwater ecosystems.

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Analysis of the prodiginine biosynthesis gene cluster of Streptomyces coelicolor A3(2): new mechanisms for chain initiation and termination in modular multienzymes.

BACKGROUND: Prodiginines are a large family of pigmented oligopyrrole antibiotics with medicinal potential as immunosuppressants and antitumour agents that are produced by several actinomycetes and other eubacteria. Recently, a gene cluster in Streptomyces coelicolor encoding the biosynthesis of undecylprodiginine and butyl-meta-cycloheptylprodiginine has been sequenced. RESULTS: Using sequence comparisons, functions have been assigned to the majority of the genes in the cluster, several of which encode homologues of enzymes involved in polyketide, non-ribosomal peptide, and fatty acid biosynthesis. Based on these assignments, a complete pathway for undecylprodiginine and butyl-meta-cycloheptylprodiginine biosynthesis in S. coelicolor has been deduced. Gene knockout experiments have confirmed the deduced roles of some of the genes in the cluster. CONCLUSIONS: The analysis presented provides a framework for a general understanding of the genetics and biochemistry of prodiginine biosynthesis, which should stimulate rational approaches to the engineered biosynthesis of novel prodiginines with improved immunosuppressant or antitumour activities. In addition, new mechanisms for chain initiation and termination catalysed by hitherto unobserved domains in modular multienzyme systems have been deduced.

Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure prediction from the sequence of its non-ribosomal peptide synthetase.

A gene cluster for the non-ribosomal synthesis of a peptide of unknown structure has been identified in the partial genome sequence of Streptomyces coelicolor. Using molecular and computational analyses, the total structure of a tripeptide siderophore synthesized by the non-ribosomal peptide synthetase within the cluster has been deduced from the translated sequence of its encoding gene. This represents a novel method for the structural assignment of natural products from genome sequence data.

Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains.

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular proteins that selectively bind, activate and condense amino acids in an ordered manner. Substrate recognition and activation occurs by reaction with ATP within the adenylation (A) domain of each module. Recently, the crystal structure of the A domain from the gramicidin synthetase (GrsA) with L-phenylalanine and adenosine monophosphate bound has been determined. RESULTS: Critical residues in all known NRPS A domains have been identified that align with eight binding-pocket residues in the GrsA A domain and define sets of remarkably conserved recognition templates. Phylogenetic relationships among these sets and the likely specificity determinants for polar and nonpolar amino acids were determined in light of extensive published biochemical data for these enzymes. The binding specificity of greater than 80% of the known NRPS A domains has been correlated with more than 30 amino acid substrates. CONCLUSIONS: The analysis presented allows the specificity of A domains of unknown function (e.g. from polymerase chain reaction amplification or genome sequencing) to be predicted. Furthermore, it provides a rational framework for altering of A domain specificity by site-directed mutagenesis, which has significant potential for engineering the biosynthesis of novel natural products.