We performed molecular phylogenetic analyses of glutamine synthetase (GS) genes in order to investigate their evolutionary history. The analyses were done on 30 DNA sequences of the GS gene which included both prokaryotes and eukaryotes. Two types of GS genes are known at present: the GSI gene found so far only in prokaryotes and the GSII gene found in both prokaryotes and eukaryotes. Our study has shown that the two types of GS gene were produced by a gene duplication which preceded, perhaps by >1000 million years, the divergence of eukaryotes and prokaryotes. The results are consistent with the facts that (t0 GS is a key enzyme of nitrogen metabolism found in all extant life forms and (fi) the oldest biological fossils date back 3800 million years. Thus, we suggest that GS genes are one of the oldest existing and functioning genes in the history of gene evolution and that GSI genes should also exist in eukaryotes. Furthermore, our study may stimulate investigation on the evolution of "preprokaryotes," by which we mean the organisms that existed during the era between the origin of life and the divergence of prokaryotes and eukaryotes.Glutamine synthetase (GS) is a key enzyme in nitrogen metabolism; it has dual functions in two essential biochemical reactions, ammonia assimilation and glutamine biosynthesis (1, 2). It is also one of the few amide synthetases found in organisms. Prokaryotes and eukaryotes were once thought to synthesize different GSs: GSI for the former and GSII for the latter. It is now known, however, that GSII is also present in bacteria belonging to Rhizobiaceae (3-6), Frankiaceae (7), and Streptomycetaceae (8, 9). GSI, by contrast, has not been found in any eukaryote.Glutamine produced by GS is essential for protein synthesis, and its amide nitrogen is donated to synthesize many essential metabolites. It is thus natural to consider GS as present in, and probably indispensable to, all organisms. In view of the central roles played by GS, it is reasonable to believe that the GS gene is extremely old. From the sequence alignment of GSI from Salmonella typhimurium and GSII from alfalfa (10), we could observe that the differences in amino acids between them was 0.75 per site. This value is quite large compared with those for other proteins, suggesting also that the GSI and GSII genes share a very old comnmon ancestor.The aforementioned discovery of the GSII gene in plant symbiotic bacteria led to the suggestion that the gene had originated from host plants through lateral gene transfer (3). This was later questioned by the further findings of the GSII gene in plant nonsymbiotic actinomycetes (8, 9). Shatters and Kahn (6) have suggested that the common ancestor of the GSII genes in Rhizobiaceae and in the host plant must be older than the plant itself, and have argued against the gene transfer.In this paper we have traced the evolutionary history of the GS genes, using our own nucleotide sequence data and others' data from prokaryotic and eukaryotic species in order to estimate the age of...

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Gentamicin is a 4,6-disubstituted aminocyclitol antibiotic complex synthesised by some members of the actinomycete genus Micromonospora. In a search for the gentamicin biosynthetic gene cluster we identified, using a cosmid library approach, a region of the M. echinospora ATCC15835 chromosome that encodes homologues of aminoglycoside biosynthesis genes including gntB-a close homologue of the 2-deoxy-scyllo-inosose synthase gene (btrC) from butirosin-producing Bacillus circulans. Insertional inactivation was achieved by homologous recombination with an internal gntB fragment-containing suicide plasmid, delivered by conjugal transfer from Escherichia coli. gntB disruptants were gentamicin nonproducing mutants as assayed by an ELISA antibiotic detection system, proving the association of gntB (or a downstream region) with gentamicin biosynthesis. The function of some open reading frames within the cluster, predicted by nucleotide database homology searching, is discussed with regards to their potential roles in gentamicin biosynthesis. The discovery of this genetic region represents the first report of a gene cluster involved in the biosynthesis of a 4,6-disubstituted aminocyclitol antibiotic.

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm s ) to the Sm r S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm r ) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.

Evernimicin (EV) belongs to the orthosomycin class of antibiotics and consists of several modified L- and D-deoxysugars containing unusual orthoester and glycosyl linkages and two orsellinic acid groups, one that is halogenated. The EV biosynthetic gene cluster from Micromonospora carbonacea var. africana ATCC39149 was localized by hybridization to a dTDP-D-glucose 4,6-dehydratase probe and a 120-kb region containing the EV biosynthetic cluster and surrounding regions has been sequenced. BLAST analysis has identified a type I polyketide synthase for orsellinic acid biosynthesis as well as enzymes required for L- and D-deoxyglucose and D-deoxymannose synthesis. In addition, genes involved in glycosyltransfer and resistance were identified. Insertional mutations in several biosynthetic genes blocked EV production, indicating a role for these genes in EV biosynthesis.

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.

Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII. The GSI gene (glnA) was isolated from a cosmid library of F. alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor. The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization. The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced. In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site. F. alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E. coli lac promoter. While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F. alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life.

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