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Micrococcus luteus (NCTC2665, 'Fleming strain') has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to [beta]-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome. doi: 10.1128/JB.01254-09

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific [[sigma].sup.F], suggesting that it is an antagonistic regulator of [[sigma].sup.F]. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-[[sigma].sup.F] during a subsequent global yeast two-hybrid screen, and the SCO0869- SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.

WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal 'phosphorylation pocket' were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI. The results revealed 45 genes that were affected by the deletion of whI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

Streptomyces coelicolor A3(2) does not have a canonical cell division cycle during most of its complex life cycle, yet it contains a gene (fts[K.sub.sc]) encoding a protein similar to FtsK, which couples the completion of cell division and chromosome segregation in unicellular bacteria such as Escherichia coli. Here, we show that various constructed fts[K.sub.sc] mutants all grew apparently normally and sporulated but upon restreaking gave rise to many aberrant colonies and to high frequencies of chloramphenicol-sensitive mutants, a phenotype previously associated with large terminal deletions from the linear chromosome. Indeed, most of the aberrant colonies had lost large fragments near one or both chromosomal termini, as if chromosome ends had failed to reach their prespore destination before the closure of sporulation septa. A constructed Fts[K.sub.sc]-enhanced green fluorescent protein fusion protein was particularly abundant in aerial hyphae, forming distinctive complexes before localizing to each sporulation septum, suggesting a role for Fts[K.sub.sc] in chromosome segregation during sporulation. Use of a fluorescent reporter showed that whenfts[K.sub.sc] was deleted, several spore compartments in most spore chains failed to express the late-sporulation-speeific sigma factor gene sigF, even though they contained chromosomal DNA. This suggested that sigF expression is autonomously activated in each spore compartment in response to completion of chromosome transfer, which would be a previously unknown checkpoint for late-sporulation-specific gene expression. These results provide new insight into the genetic instability prevalent among streptomycetes, including those used in the industrial production of antibiotics.

SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SC05582 as nsdA, a gene negatively affecting Streptomyces differentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster, nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SC04114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of 'current' ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.

As in other bacteria, 3'-tails are added post-transcriptionally to Streptomyces coeficolor RNA. These tails are heteropolymeric, and although there are several candidates, the enzyme responsible for their synthesis has not been definitively identified. This paper reports on three candidates for this role. First, it is confirmed that the product of S. coelicolor gene SC03896, although it bears significant sequence similarity to Escherichia coil poly(A) polymerase I, is a tRNA nucleotidyltransferase, not a poly(A) polymerase. It is further shown that SC02904 encodes an RNase PH homologue that possesses the polymerization and phosphorolysis activities expected for enzymes of that family. S. coelicolor RNase PH can add poly(A) tails to a model RNA transcript in vitro. However, disruption of the RNase PH gene has no effect on RNA 3'-tail length or composition in S. coeficolor; thus, RNase PH does not function as the RNA 3'-polyribonucleotide polymerase [poly(A) polymerase] in that organism. These results strongly suggest that the enzyme responsible for RNA 3'-tail synthesis in S. coeficolor and other streptomycetes is polynucleotide phosphorylase (PNPase). Moreover, this study shows that both PNPase and the product of SC03896 are essential. It is possible that the dual functions of PNPase in the synthesis and degradation of RNA 3'-tails make it indispensable in Streptomyces.

The Streptomyces coelicolor partitioning protein ParB binds to numerous pars sites in the oriC-proximal part of the linear chromosome. ParB binding results in the formation of large complexes, which behave differentially during the complex life cycle (D. Jakimowicz, B. Gust, J. Zakrzewska-Czerwinska, and K. F. Chater, J. Bacteriol. 187:3572-3580, 2005). Here we have analyzed the transcriptional regulation that underpins this developmentally specific behavior. Analysis of promoter mutations showed that the irregularly spaced complexes present in vegetative hyphae are dependent on the constitutive parAB[p.sub.1] promoter, while sporulation-specific induction of the promoter parAB[p.sub.2] is required for the assembly of arrays of ParB complexes in aerial hyphae and thus is necessary for efficient chromosome segregation. Expression from parAB[p.sub.2] depended absolutely on two sporulation regulatory genes, whiA and whiB, and partially on two others, whiH and whil, all four of which are needed for sporulation septation. Because of this pattern of dependence, we investigated the transcription of these four whi genes in whiA and whiB mutants, revealing significant regulatory interplay between whiA and whiB. A strain in which sporulation septation (but not vegetative septation) was blocked by mutation of a sporulation-specific promoter of ftsZ showed close to wild-type induction of parAB[p.sub.2] and formed fairly regular ParB-enhanced green fluorescent protein fuel in aerial hyphae, ruling out strong morphological coupling or checkpoint regulation between septation and DNA partitioning during sporulation. A model for developmental regulation of parAB[P.sub.2] expression is presented.

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SC04263. Evidence for the control of a cluster of function-unknown genes by the SC04263 regulator revealed a new aspect of the pleiotropic bldA phenotype.

In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright loci accompanied sporulation-associated chromosome condensation and septation.

Paraquat (methyl viologen)-resistant mutants of Streptomyces coelicolor A3(2) that grew and sporulated normally in the presence of paraquat were isolated. Based on the positions of the mutant loci in the genetic map, we isolated the pqr (paraquat resistance) gene whose mutation (pqr501) caused a dominant paraquat-resistant phenotype. The pqr locus consists of two genes (pqrA and pqrB) that form a transcription unit. The pqrA gene encodes a protein with a TetR-like DNA-binding motif, and the pqrB gene encodes a putative efflux pump of the major facilitator superfamily. The pqr501 mutation was a base substitution changing arginine-18 to glutamine (R18Q) near the helix-turn-helix motif in PqrA. A pqrA null mutant exhibited similar paraquat resistance, and an increase in the amount of pqrA promoter-driven transcripts of about eightfold was observed for the pqrA501 mutant. These results suggest that PqrA is a negative regulator of its own operon. Deletion of the pqrAB operon caused cells to be very sensitive to paraquat, consistent with the prediction that PqrB may function as a paraquat-efllux pump. Purified PqrA protein specifically bound to the pqrA promoter region, whereas mutant R18Q protein did not, indicating that PqrA is a direct autoregulator of its own operon.

The bldG gene is not involved in the regulation of antibiotic production and sporulation in Streptomyces coelicolor. A DNA fragment that can induce antibiotic production in a bldG mutant codes for a protein similar to the anti-anti-sigma factors of Bacillus and Staphylococcus.

Research demonstrates that the linear chromosomal partitioning genes parA and parB are located near the replication origin and together they form an operon. Data also point out that the translation start points of the two genes overlap suggesting a coregulation.

Early sporulation genes and association with suggested developmental decision points in Streptomyces coelicolor A3(2) are discussed. New information about the role of whiF (ital) has been found through cytological analysis of a series of mutants. Knowledge about whiG, whiA and whiB (all ital) has been confirmed and extended. A model explains the mutant phenotypes proposing two early developmental decision points related to commitment to sporulation septation.

Research concerning identification, characterization and cloning of five whi loci in Streptomyces coelicolor is analyzed. Results describe phenotypes that result from the mutations at each of the loci.

A study was conducted to examine the functions of Streptomyces coelicolor genes in the master control systems for differentiation. Reverse genetics is carried out to produce mutants. The sequences of the early whi genes were not correlated with phosphorelay production in S. coelicolor. Results indicated genes having cell biology and metabolism functions. Findings also showed that the FtsZ protein supports a critical role in bacterial cell division.

Spore pigment in Streptomyces coelicolor is determined by the whiE locus which codes for the polyketide pigment. This locus is a gene cluster containing two promoters, whiEP1 and whiEP2. Transcripts from these promoters are detected on the emergence of the sporulation septum. The promoter whiEP2 regulates the transcription of of an open reading frame coding for a flavin adenine dinucleotide-dependent hydroxylase in the pigment biosynthetic pathway.

Streptomyces coelicolor A3(2) offers a good model system for the study of secondary metabolism, due to its well developed genetics and ability to produce different antibiotics. BldA mutants in Streptomyces coelicolor A3(2), lacking tRNA for the rare leucine codon UUA, cannot make the red undecylprodigiosin antibiotic complex. A cloning strategy was used to localize the red gene cluster, and a PWb mutation derivative of a bldA mutant, was located between red structural genes in the sector 1-4 kb from the pathway-specific regulatory gene redD.

Research was conducted to examine the cloning of a glgC gene from Streptomyces coelicolor A3(2) using a polymerase chain reaction approach. The DNA fragments were transferred from agarose gels to nylon membranes while DNA was extracted from Streptomyces and from C31 phage derivatives. Results showed that a disruption of glgC eliminated only one of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation.

The denaturation of the double-stranded circular or linear DNA of Streptomyces coelicolor A3(2) stimulated insert-directed integrative transformation. The stimulatory effects of plasmid DNA denaturation on Streptomyces coelicolor A3(2) integrative transformation also depressed transformation by an autonomously replicating plasmid vector. Application of the technique in linearized cloned DNA was also effective in the transformation of the microbe. Furthermore, the technique can applied to any streptomycete that is capable of accepting DNA from Escherichia coli.

A gene product of schC gene which is similar to hydroxylase is involved in the preparation of a polyketide spore pigment in Streptomyces halstedii. The green spores of S. halstedii turn lilac when schC is ruptured. Flavin adenine nucleotide is present in hydroxylases which play an important role in the catabolic pathways of aromatic compounds and the biosynthesis of polycyclic aromatic polyketide antibiotics.