James E. M. Stach

Statistical Approaches for Estimating Actinobacterial Diversity in Marine Sediments

ABSTRACT Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with ≥99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpsons index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. FST and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.

Diversity of cultivable actinobacteria in geographically widespread marine sediments

Reports describing actinobacteria isolated from marine environments have been dominated by Micromonospora, Rhodococcus and Streptomyces species. Recent culture-independent studies have shown that marine environments contain a high diversity of actinobacterial species that are rarely, if at all, recovered by cultivation-based methods. In this study, it is shown that cultivation-independent methods can be used to guide the application of selective isolation methods. The detection of marine-derived actinobacterial species that have previously only been reported from terrestrial habitats is highlighted. This study provides good evidence that the previously described low diversity of actinobacterial species isolated from marine environments does not reflect an actual low species diversity, and that the use of informed selective isolation procedures can aid in the isolation of members of novel taxa.

Marine actinobacteria: perspectives, challenges, future directions

In this paper we evaluate the current state of research on the biology and biotechnology of marine actinobacteria. The topics covered include the abundance, diversity, novelty and biogeographic distribution of marine actinobacteria, ecosystem function, bioprospecting, and a new approach to the exploration of actinobacterial taxonomic space. An agenda for future marine actinobacterial research is suggested based upon consideration of the above issues.

Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain Streptomyces sp. NTK 937*

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

The Catalytic Mechanism of a Natural Diels–Alderase Revealed in Molecular Detail

The Diels-Alder reaction, a [4 + 2] cycloaddition of a conjugated diene to a dienophile, is one of the most powerful reactions in synthetic chemistry. Biocatalysts capable of unlocking new and efficient Diels-Alder reactions would have major impact. Here we present a molecular-level description of the reaction mechanism of the spirotetronate cyclase AbyU, an enzyme shown here to be a bona fide natural Diels-Alderase. Using enzyme assays, X-ray crystal structures, and simulations of the reaction in the enzyme, we reveal how linear substrate chains are contorted within the AbyU active site to facilitate a transannular pericyclic reaction. This study provides compelling evidence for the existence of a natural enzyme evolved to catalyze a Diels-Alder reaction and shows how catalysis is achieved.

Abyssomicin Biosynthesis: Formation of an Unusual Polyketide, Antibiotic-Feeding Studies and Genetic Analysis

Abyssomicin C, produced by the marine actinomycete Verrucosispora maris AB-18-032, is active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and inhibits p-aminobenzoate formation during tetrahydrofolate synthesis; it is the first natural product active against this therapeutic target. To investigate the biosynthesis of this small but structurally complex secondary metabolite, we carried out feeding studies using 13C labelled polyketide building blocks. Formation of abyssomicin C requires two propionates, five acetates and one glucose-derived metabolite. Identification and sequencing of the abyssomicin biosynthetic gene cluster revealed a 57 kb segment of Verrucosispora maris AB-18-032 DNA that contained all of the genes necessary for abyssomicin biosynthesis. The identity of the biosynthetic gene cluster was confirmed by gene inactivation and complementation experiments (the first genetic manipulation of a member of this genus) and a model for abyssomicin C biosynthesis is proposed.

Synthetic RNA Silencing in Bacteria – Antimicrobial Discovery and Resistance Breaking

The increasing incidence and prevalence of antibiotic resistance in bacteria threatens the “antibiotic miracle.” Conventional antimicrobial drug development has failed to replace the armamentarium needed to combat this problem, and novel solutions are urgently required. Here we review both natural and synthetic RNA silencing and its potential to provide new antibacterials through improved target selection, evaluation, and screening. Furthermore, we focus on synthetic RNA silencers as a novel class of antibacterials and review their unique properties.

Computer-assisted numerical analysis of colour-group data for dereplication of streptomycetes for bioprospecting and ecological purposes

Large numbers of alkaliphilic streptomycetes isolated from a beach and dune sand system were dereplicated manually based on aerial spore mass, colony reverse and diffusible pigment colours formed on oatmeal agar, and on their capacity to produce melanin pigments on peptone-yeast extract-iron agar. The resultant data were converted to their respective red, blue and green shade intensities. The Euclidean distances between each of the colours were calculated by considering red, green and blue shade intensity values as X, Y and Z coordinates in three dimensional space. The clusters of isolates delineated in the dendrogram generated using the distances were found to match those obtained by manual colour-grouping of the isolates. A reasonable linear correlation was found between the colour-group and corresponding rep-PCR data. The implications of the computer-assisted colour-grouping method for bioprospecting and ecological studies are discussed.

Characterisation of micromonosporae from aquatic environments using molecular taxonomic methods

Large numbers of strains assigned to the genus Micromonospora on the basis of typical colonial and pigmentation features were isolated from diverse aquatic sediments using a standard selective isolation procedure. Two hundred and six isolates and eight representatives of the genus Micromonospora were assigned to 24 multimembered groups based on a numerical analysis of banding patterns generated using BOX and ERIC primers. Representatives of multimembered groups encompassing isolated micromonosporae were the subject of 16S rRNA gene sequencing analyses. Good congruence was found between the molecular fingerprinting and 16S rRNA sequence data indicating that the groups based upon the former are taxonomically meaningful. Nearly all of the isolates that were chosen for the 16S rRNA gene sequencing analyses showed that the ecosystems studied are a rich source of novel micromonosporae. These findings have implications for high throughput screening for novel micromonosporae as BOX and ERIC fingerprinting, which is rapid and reproducible, can be applied as a robust dereplication procedure to indicate which environmental isolates have been cultured previously.

Species-Selective Killing of Bacteria by Antimicrobial Peptide-PNAs

Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.