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Background Streptomycete bacteria are prolific producers of specialized metabolites, many of which have clinically relevant bioactivity. A striking feature of their genomes is the expansion of gene families that encode the same enzymatic function. Genes that undergo expansion events, either by horizontal gene transfer or duplication, can have a range of fates: genes can be lost, or they can undergo neo-functionalization or sub-functionalization. To test whether expanded gene families in Streptomyces exhibit differential expression, an RNA-Seq approach was used to examine cultures of wild-type Streptomyces coelicolor grown with either glucose or tween as the sole carbon source. Results RNA-Seq analysis showed that two-thirds of genes within expanded gene families show transcriptional differences when strains were grown on tween compared to glucose. In addition, expression of specialized metabolite gene clusters (actinorhodin, isorenieratane, coelichelin and a cryptic NRPS) was also influenced by carbon source. Conclusions Expression of genes encoding the same enzymatic function had transcriptional differences when grown on different carbon sources. This transcriptional divergence enables partitioning to function under different physiological conditions. These approaches can inform metabolic engineering of industrial Streptomyces strains and may help develop cultivation conditions to activate the so-called silent biosynthetic gene clusters.

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

The fate of Streptomyces lividans lysogens was studied in sterile and nonsterile soil microcosms. It was found that in sterile soil lysogens grew as well as the parental strain. However, in nonsterile soil, numbers of the lysogen decreased rapidly, indicating a decreased fitness when compared to the original organism. In addition, the release of this phage from a lysogen and its subsequent infection and lysogenisation of a recipient strain was demonstrated in sterile soil.

Ecosystems, including engineered ones, are complex systems in which microorganisms occur in heterogenous communities. Their behaviour in the environment is often unknown due to the lack of proper detection and identification techniques. Molecular ecology is a new field in which microbes can be recognized and their function can be understood at the DNA/RNA level without unreliable steps of cultivation of microbes. During the last few years genetically modified microbes have been constructed by recombinant DNA techniques for putative use in the environment. The slow progress in this field is due to the lack of integration of microbial ecology and molecular biology. In the present review, examples will be given of the use of DNA probes and marker genes in our study on the ecology of genetically modified microbes and wild-type recalcitrant microorganisms that are difficult to cultivate or even `non-culturable'. Emphasis is given to the development and use of oligonucleotide probes directed towards 16S rRNA, to detect microbes in various engineered ecosystems: (i) Frankia in root nodules, and (li) propionate-oxidizing sulfate-reducing bacteria in anaerobic granular sludge. Expression of genes is demonstrated by studies on the localization of nifH transcripts in root nodules of Coriaria and Alnus. In addition we will describe examples of the use of marker genes (gusA gene and aphV gene) to study competition and genetic stability of released engineered Rhizobium and Streptomyces strains.

The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.

The ecology of bacteriophages in soil is poorly understood with respect to the distribution, activity and life cycle of their hosts [29]. Perhaps the main reason for this is simply one of enumeration. Whilst it is relatively easy to detect a given phage if one knows its propagative host, it is not possible to observe those phages for which hosts are not known. It is estimated that at present only a small fraction of bacteria in soil can be cultivated and therefore the absolute numbers of bacteriophages within a soil sample remain hidden. Recent studies in aquatic systems have shown using direct counting procedures that the traditional phage assay may well underestimate phage numbers by as much as two orders of magnitude [15]. Studies on phage ecology are further complicated by the fact that their activity is dependent on the resources accessible to them, which, in the case of obligate parasites, are the availability of active hosts. In soil, microbial activity and abundance is discontinuous in space and time. Phage activity is dependent on this and must therefore mirror this by rapid replication in restricted growth periods and sites in order to maintain sufficient numbers of infective phages in the soil environment for the survival of the population. The concentration of active hosts in microsites may well increase the likelihood of phage-host encounters, at least in the short term. Unfortunately no information is available on the spatial distribution of bacteriophages in soil and it is likely that counts derived from a given mass of soil underestimate the concentration and significance in such sites. Phages may be involved in either lysogenic or virulent associations with their hosts in soil, however current detection procedures provide no indication of total numbers in the environment as these methods rely on the virulent reaction of the virus with its host. The importance of this point was illustrated by Reanney [18], who estimated that free phages of Bacillus stearothermophilus in soil, when detected by phage extraction and assay techniques, comprised only 20% of the population, the remainder being present as lysogens. Thus, even when working with a known host, the numbers of free phages infecting that host in a soil sample can only be taken as an indication of the total number of bacteriophages present. If it is true that 80% of temperate phages are present in soil as prophages, it perhaps serves as an indicator of how active bacteriophages actually occur in soil. Due to the hostile conditions that can prevail in the soil environment, the ability of a bacteriophage to lysogenise its host obviously confers a greater capacity to survive environmental stress such as: proteolytic enzymes, abiotic stress etc. If one considers the evolution of bacteriophages through the reshuffling of gene modules [16,21,22,23], then the lysogenic state must be one of the most important states for the interaction of different phage genomes and their subsequent recombination into new genomes in the environment.

A new method for the isolation and enumeration of streptomycete spores from soil was developed. This method makes use of a cation-exchange resin to disperse soil particles. It allowed the detection of 10 spores in 100 g of sterile soil, while ca. 10 3 could be accurately enumerated in 100 g. This method was applied to studying the fate of a marked actinophage in soil. In sterile amended and nonsterile soil, relatively high numbers of actinophages were only found during the first few days of the experiment when the host streptomycete was in the mycelial form. Later, after sporulation, lysogens could be detected in sterile amended soil and could still be found 60 days after inoculation. Although no lysogens were found in nonsterile soil, the introduced phage could still be detected in the free state after 60 days, albeit at a low titer.