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Streptomyces coelicolor A3(2) has three additional glnA-type genes besides the glutamine synthetase genes glnA (encoding GSI) and glnII (encoding GSII). The aim of this work was to characterize their functional properties and regulation. Sequence analyses revealed that GlnA2, GlnA3, and GlnA4 are dissimilar to S. coelicolor GSI and lack highly conserved amino acid residues involved in catalysis. In heterologous expression experiments, glnA2, glnA3, and glnA4, in contrast to glnA and glnII, were not capable of complementing the L-glutamine auxotrophy of an Escherichia coli glnA mutant. The lack of a conserved sequence motif reflecting adenylylation control of enzyme activity suggests that GlnA2, GlnA3, and GlnA4 are not regulated via adenylyltransferase-mediated modification. In DNA-binding assays, the OmpR-like regulator of nitrogen metabolism GlnRII, which interacts with the glnA and glnII promoters, did not bind to the upstream regions of glnA2, glnA3, and glnA4. These findings support the conclusion that glnA2, glnA3, and glnA4 are not directly involved in L-glutamine synthesis and nitrogen assimilation and are not subject to nitrogen control in S. coelicolor. The glnA3 gene product is similar to FluG, which is required for asexual sporulation in Aspergillus nidulans. However, inactivation of glnA3 does not block morphological differentiation in S. coelicolor.