Cutting-edge technology. I. Global gene expression profiling using DNA microarrays

Having the complete human genomic sequence poses a new challenge: to use genomic structural information to display and analyze biological processes on a genome-wide scale to assign gene function. DNA microarrays are a miniaturized, ordered arrangement of nucleic acid fragments from individual genes located at defined positions on a solid support, enabling the analysis of thousands of genes in parallel by specific hybridization. This review describes technical aspects, discusses relevant applications, and suggests factors affecting the use of this technology and how it fits in the grand scheme of meeting the needs of the postgenomic era.     

Bacteriophages of Saccharopolyspora erythraea

Five bacteriophages infecting only Saccharopolyspora erythraea (formerly Streptomyces erythreus) among 43 Streptomyces spp. tested were classified into two groups by phage-host relationships, restriction enzyme mapping, cohesive-end determinations, and Southern hybridizations. phi SE6, the most frequently isolated phage, produced clear plaques on all hosts tested, while phi SE45, phi SE57, phi SE60, and phi SE69 produced turbid plaques. phi SE6 DNA was linear, had a molecular weight of (27.6 +/- 1) X 10(6) and, like the DNAs of phi SE45, phi SE57, and phi SE69, lacked cohesive ends. The characteristic patterns of of ClaI and HindIII restriction digests of phi SE6 DNA and the results of Southern hybridizations with three different ClaI fragments of phi SE6 DNA as probes indicated that phi SE6 DNA was partially circularly permuted and terminally redundant, suggesting that it was packaged by a headful packaging mechanism. Southern hybridization data also showed that phi SE45, phi SE57, and phi SE69 were closely related to phi SE6. phi SE60 DNA, in contrast, had cohesive ends, and restriction mapping plus Southern hybridization data showed that phi SE60 was unrelated to the other four phages.     

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

Saccharopolyspora erythraea is used for the industrial-scale production of the antibiotic erythromycin A, derivatives of which play a vital role in medicine. The sequenced chromosome of this soil bacterium comprises 8,212,805 base pairs, predicted to encode 7,264 genes. It is circular, like those of the pathogenic actinomycetes Mycobacterium tuberculosis and Corynebacterium diphtheriae, but unlike the linear chromosomes of the model actinomycete Streptomyces coelicolor A3(2) and the closely related Streptomyces avermitilis. The S. erythraea genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, at least 72 genes predicted to confer resistance to a range of common antibiotic classes and many sets of duplicated genes to support its saprophytic lifestyle. The availability of the genome sequence of S. erythraea will improve insight into its biology and facilitate rational development of strains to generate high-titer producers of clinically important antibiotics.     

New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster

Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis.     

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

Improving the scaling normalization for high-density oligonucleotide GeneChip expression microarrays

Background

Normalization is an important step for microarray data analysis to minimize biological and technical variations. Choosing a suitable approach can be critical. The default method in GeneChip expression microarray uses a constant factor, the scaling factor (SF), for every gene on an array. The SF is obtained from a trimmed average signal of the array after excluding the 2% of the probe sets with the highest and the lowest values.

Results

Among the 76 U34A GeneChip experiments, the total signals on each array showed 25.8% variations in terms of the coefficient of variation, although all microarrays were hybridized with the same amount of biotin-labeled cRNA. The 2% of the probe sets with the highest signals that were normally excluded from SF calculation accounted for 34% to 54% of the total signals (40.7% ± 4.4%, mean ± sd). In comparison with normalization factors obtained from the median signal or from the mean of the log transformed signal, SF showed the greatest variation. The normalization factors obtained from log transformed signals showed least variation.

Conclusions

Eliminating 40% of the signal data during SF calculation failed to show any benefit. Normalization factors obtained with log transformed signals performed the best. Thus, it is suggested to use the mean of the logarithm transformed data for normalization, rather than the arithmetic mean of signals in GeneChip gene expression microarrays.

     

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

Background  

There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.     

Disruption of a rhodaneselike gene results in cysteine auxotrophy in Saccharopolyspora erythraea.

A 3,373-base-pair DNA segment from a clone fortuitously isolated from Saccharopolyspora erythraea by hybridization to an oligodeoxynucleotide probe was sequenced. Computer-assisted analysis of the nucleotide sequence reveals three closely linked Streptomyces open reading frames plus a fourth converging on the others. The deduced product of one of them, ORF2, shows considerable similarity to bovine liver rhodanese. orf2, and the closely linked orf3 located just downstream of it, were disrupted by insertion of an apramycin resistance cassette into the orf2 coding sequence along with inversion of the fragment carrying most of orf2 and orf3 via two successive recombinational events in the wild-type strain. The mutant strain thus created contains wild-type levels of rhodanese activity but cannot grow on minimal medium. It is a cysteine auxotroph, capable of utilizing efficiently only thiosulfate among the inorganic sulfur sources tested. orf2 has been designated cysA. The possible role of the rhodaneselike cysA gene product in thiosulfate formation is discussed.