The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

A 2.6 kb DNA sequence of Streptomyces coelicolor A3(2) which functions as a transposable element

Summary Streptomyces coelicolor A3(2) contains CCC DNA molecules, 2.6 kb in size, with an average copy number of less than one per ten chromosomes. Southern hybridisation revealed, in addition, two linear, integrated copies (A and B) of this mini-circle sequence per chromosome. The two integrated copies have similar (if not identical) ends and are present in the same locations in various S. coelicolor A3(2) derivatives. The mini-circle sequence is absent from S. lividans 66 and S. violaceolatus ISP5438 and from several Streptomyces species less closely related to S. coelicolor A3(2). None of a variety of Streptomyces plasmids tested contained homology to the mini-circle sequence. When a 1.8 kb fragment of the mini-circle lacking the ends of the integrated copies was inserted into KC515 (a derivative of the temperate phage C31 which is unable to lysogenise host strains by the natural route because the phage attachment site has been deleted) the resulting phage lysogenised S. coelicolor A3(2) (integrating into the genome of this host by homologous recombination with resident minicircle sequences) but not S. lividans or a variety of other C31 hosts. In contrast, a KC515 derivative (KC591) carrying the entire 2.6 kb mini-circle sequence linearised at its single BclI site (and therefore containing the integration site of the free mini-circle) lysogenised not only S. coelicolor A3(2) but also S. lividans 66 and most other strains normally lysogenised by C31. The KC591 lysogens of the eight Streptomyces species tested contained a linear, integrated prophage with termini apparently identical to those of the linear mini-cricle copies of S. coelicolor. In S. lividans, KC591 integrated preferentially at a site apparently homologous to the site occupied by mini-circle sequence A in S. coelicolor A3(2) strains, but integration into secondary sites also occurred.     

Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3^r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.     

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L^-1 to 5.5 mg L^-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.     

Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor

Summary Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent K_m values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (C31 KC515), and by larger (10–30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

The relA/spoT-Homologous Gene in Streptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.     

D-Amino acid oxidase of Streptomyces coelicolor and the effect of D-amino acids on the bacterium

The homologous gene of D-amino acid oxidase (DAO) in prokaryotic organisms is predominantly found in a group of bacteria called the Actinobacteria. We have analyzed the DAO of the model actinomycete Streptomyces coelicolor and the effect of D-amino acids on this bacterium. When expressed in Escherichia coli, the translated product of the putative dao gene of this bacterium exhibited oxidase activity against neutral and basic D-amino acids, with a higher activity toward D-valine and D-isoleucine, but not to their corresponding L-amino acids. This substrate specificity was largely different from that of the DAO of the actinobacterium Arthrobacter protophormiae. The gene message and DAO activity were constitutively detected in S. coelicolor cells, and unlike eukaryotic DAOs, the presence of a D-amino acid did not significantly induce expression. The D-amino acids that were a good substrate for S. coelicolor DAO inhibited cell growth, delayed morphological development and affected cell morphology, but they did not inhibit biofilm formation. Disruption of the dao gene had no effect on the morphology and morphological development of S. coelicolor cells, the assimilation of D-valine or the sensitivity to growth inhibition by D-valine under the experimental conditions, showing that in this bacterium DAO does not play a significant role in either morphological development or the assimilation and detoxification of D-amino acids.     

Isolation and Characterization of Novel Giant Stenotrophomonas maltophilia Phage φSMA5

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated SMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The SMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, KZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to SMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, SMA5 appears to be the first reported.     

Cell Wall Hydrolases Affect Germination,Vegetative Growth,and Sporulation in Streptomyces coelicolor

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the S. coelicolor life cycle.Peptidoglycan (PG) is a primary constituent of the gram-positive bacterial cell wall and, despite its rigid structure, is a remarkably dynamic macromolecule. It functions in maintaining cell shape and cytoplasmic turgor pressure and serves as the scaffolding to which cell wall-associated components, such as proteins and teichoic acids, are anchored (16). PG comprises alternating N-acetylglucosamine and N-acetylmuramic acid residues, which make up the glycan backbone, and peptide side chains that link the glycan strands together (49). PG biosynthesis is a complex process involving the concerted efforts of many enzymes, beginning with precursor synthesis in the cytoplasm and concluding with polymerization outside the cytoplasmic membrane (3, 8, 48). During bacterial growth, PG is actively remodeled to allow incorporation of new PG and to accommodate changes in cell shape. The enzymes responsible for this remodeling are collectively termed cell wall hydrolases, and they act by cleaving covalent bonds within either the glycan strands or the peptide side chains. The essential nature of PG requires that synthesis and cleavage be tightly regulated, with the activities of biosynthetic and hydrolytic enzymes coordinated in both space and time.Cell wall hydrolases are diverse enzymes that are typically grouped on the basis of substrate specificity and the resulting cleavage products. The major groups include the lysozymes and lytic transglycosylases, which hydrolyze the β-(1,4)-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine; the endopeptidases, which cleave the peptide bonds in the amino acid side chains connecting the parallel glycan strands; the carboxypeptidases, which cleave the C-terminal amino acids of peptide chains; and the amidases, which cleave between N-acetylmuramic acid and the first residue (l-Ala) of the peptide side chain (55).In addition to remodeling the PG, cell wall hydrolases also contribute to a multitude of specialized cellular processes, from the assembly of secretion systems, flagella, and pili (55) to the resuscitation of dormant cells by a recently discovered class of hydrolases known as the resuscitation-promoting factors (Rpfs) (37). The Rpfs are secreted proteins that are structurally related to lysozymes (12, 13) and are found in a subset of the actinomycetes, including Micrococcus, Mycobacterium, Corynebacterium, and Streptomyces (44). The sole Micrococcus luteus Rpf is essential for viability (36), while in Mycobacterium tuberculosis, which encodes five Rpf proteins, the enzymes are required for virulence and resumption of active growth during emergence from a latent state (26). The sporulating actinomycete Streptomyces coelicolor is predicted to encode seven Rpf proteins, along with a plethora of other cell wall hydrolases. Surprisingly little is known about cell wall remodeling in the streptomycetes, despite the fact that significant remodeling must accompany the filamentous growth and morphological changes associated with the different stages of the Streptomyces life cycle. The S. coelicolor life cycle initiates with spore germination; this process likely depends on cell wall hydrolase activity, as spore germination in Bacillus subtilis requires the activity of at least two hydrolases (50). Following spore germination in S. coelicolor, germ tubes elongate and branch in a filamentous manner, forming a network of cells termed the vegetative mycelium. A second type of filamentous (but nonbranching) cells, the aerial hyphae, then emerge from the vegetative mycelium, and it is within these cells that chains of spores develop. Cell wall hydrolase activity and the associated cell wall remodeling are thought to be essential for vegetative hyphal branch formation, vegetative and aerial hyphal tip extension, spore chain formation, and spore dispersal. In this work, we describe the first investigation of cell wall hydrolase activity and function in Streptomyces. We identify a subset of hydrolases whose genes share a conserved 5′ untranslated region (UTR), demonstrate enzymatic activity for four of these proteins, and reveal that these enzymes function at multiple stages in the S. coelicolor life cycle.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

Polyhydroxyalkanoate accumulation in Streptomyces coelicolor affected by SCO7613 gene region

Polyhydroxyalkanoate (PHA) is stored as an important carbon and energy source in bacterial cells. For biomedical applications, gram-positive bacteria can be better sources of PHAs, since they lack outer membrane lipopolysaccharide. Although gram-positive Streptomyces coelicolor A3(2) has been indicated as a high potential PHA producer, pha C gene that encodes the key enzyme PHA synthase in the metabolic pathway is not determined in its genome. BLAST search results of the GenBank database argued that SCO7613 could specify a putative polyhydroxyalkanoate synthase (PhaC) responsible for PHA biosynthesis. Deduced amino acid sequence of SCO7613 showed the presence of conserved lipase box like sequence, ⁵⁵⁵GASAG⁵⁵⁹, in which serine residue was present as the active nucleophile. Present study describes deletion of putative S. coelicolor pha C gene via PCR dependent method. We showed that SCO7613 is not an essential gene in S. coelicolor and its deletion affected PHA accumulation negatively although it is not ceased. Transcomplementation abolished the mutant phenotype, demonstrating that the decrease in PHA resulted from the deletion of SCO7613.     

Identification of Tail Genes in the Temperate Phage 16-3 of Sinorhizobium meliloti 41

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.The Sinorhizobium meliloti-Medicago symbiosis is an important model for endosymbiotic nitrogen fixation. The genome sequence of S. meliloti (strain 1021) has been established (14), and the Medicago truncatula genome is under intensive investigation (3). Phage 16-3 is a temperate, double-stranded DNA phage of S. meliloti strain 41. It is by far the best-studied rhizobiophage and serves as a tool in analyses of rhizobium genetics, in the isolation of some symbiotic mutants, and in the construction of special vectors. Genetic determinants and molecular mechanisms of many aspects of the 16-3 life cycle, such as phage integration and excision (8, 26, 38), regulation of the lytic/lysogenic switch (5, 6, 9, 24, 28), immunity to superinfection (4), phage DNA packaging (15), and the role of gene h in the host range (32), have been examined in detail. Moreover, the complete 60-kb phage genome sequence (accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ500118","term_id":"111054886","term_text":"DQ500118"}}DQ500118) has been determined recently (P. P. Papp et al., unpublished results). However, little is known about the genes and structural elements involved in the interaction between the phage and its host, and furthermore, only one study of the 16-3 virion proteins has been reported (11).The initial interaction between a tailed phage and its bacterial host cell is mediated by the distal part of the phage tail, which specifically binds to the phage receptor located on the host surface. Earlier results demonstrated that phage 16-3 adsorption is connected to the strain-specific capsular polysaccharide of S. meliloti 41, the K_R5 antigen. So far, three bacterial gene clusters involved in K_R5 antigen production, including the rkp-1, rkp-2, and rkp-3 regions, have been described. rkp mutants are defective in the invasion of the host plant for symbiosis. In addition, they cannot adsorb phage 16-3, suggesting that the K_R5 antigen is required for both functions (19, 20, 30).In order to elucidate the molecular mechanism of phage 16-3 and S. meliloti 41 recognition, bacterial mutants carrying an altered phage receptor and host range phage mutants able to overcome the adsorption block have been characterized previously (32). It was shown that the RkpM protein, together with other yet uncharacterized elements, is a component of the phage receptor. With the use of rkpM mutants, host range mutations in phage gene h, which probably encodes the tail fiber protein, were identified. Interestingly, some mutations influencing phage-host recognition could not be localized in the rkpM and h genes, indicating that on both sides, additional components are important for bacteriophage-host recognition.The aim of this study was to identify additional genetic determinants involved in S. meliloti 41 and phage 16-3 recognition by characterizing new host range and receptor mutants. Furthermore, by using insertional mutagenesis, we examined a region of the phage chromosome supposed to be responsible for tail formation and identified six new genes essential for phage assembly.     

The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA

Summary Glycerol-3-phosphate dehydrogenase (gylB) mutations (which cause glycerol sensitivity), and presumed gylcerol kinase (gylA) and/or regulatory mutations eliminating both glycerol-3-phosphate dehydrogenase and glcerol kinase activities, map close to the argA locus of Streptomyces coelicolor A3(2). Using the plasmid vector pIJ702 and restriction enzymes Bg/II and SstI, extensively overlapping S. coelicolor DNA fragments of 2.74 kb and 2.84 kb were isolated, either of which could restore the wild-type phenotype to gylB and some gylA mutants. Genetic and biochemical analyses of mutants carrying the cloned gylDNA suggested that a functional gyl promoter had not been cloned, and that restoration of the Gyl⁺ phenotype was achieved by recombination between the cloned and chromosomal gyl DNA sequences. After subcloning parts of this DNA into the phage vector C31 KC400, gene disruption analysis was carried out, which confirmed the absence of the gyl promoter, and indicated that a polycistronic mRNA traverses gylA and then gylB.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Linear Plasmid SLP2 Is Maintained by Partitioning,Intrahyphal Spread,and Conjugal Transfer in Streptomyces

Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces lividans, which contains a typical parAB partitioning operon. In S. lividans and Streptomyces coelicolor, a parAB deletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-containing hyphae to plasmidless hyphae. Deletion of the transfer (traB) gene eliminated conjugal transfer, lessened the growth retardation of colonies, and increased plasmid loss through sporulation cycles. The additional deletion of an intrahyphal spread gene (spd1) caused almost complete plasmid loss in a sporulation cycle and eliminated all growth retardation. Moreover, deletion of spd1 alone severely reduced conjugal transfer and stability of SLP2 in S. coelicolor M145 but had no effect on S. lividans TK64. These results revealed the following three systems for SLP2 maintenance: partitioning and spread for moving the plasmid DNA along the hyphae and into spores and conjugal transfer for rescuing plasmidless hyphae. In S. lividans, both spread and partitioning appear to overlap functionally, but in S. coelicolor, spread appears to play the main role.Soil bacteria of the genus Streptomyces possess terminal protein (TP)-capped linear chromosomes (23) and often harbor one or more linear plasmids with similar structures (29). Like their circular counterparts, many of these linear plasmids mediate conjugation and transfer of themselves and the chromosomes (reviewed in references 12 and 13).Conjugation in Streptomyces often produces a unique transfer-related phenotype known as “pocking” or “lethal zygosis” (4, 5). On certain solid media, transfer of a conjugative plasmid from donor to recipient leads to a growth inhibition zone (“pock”), reflecting retarded growth and development of recipient mycelia. Different plasmids display distinct pock morphology.Streptomyces plasmids often contain only a single transfer gene (tra or kil) for mobilization of the plasmid from donor to recipient hyphae. The Streptomyces TraB proteins resemble the SpoIIIE protein of Bacillus subtilis, which translocates double-stranded chromosomal DNA during prespore formation (35). The genetic study of Possoz et al. (27) suggests that pSAM2, a circular Streptomyces plasmid, is transferred in double-stranded form. In support of this, Reuther et al. (28) showed that the TraB protein of the circular Streptomyces plasmid pSVH1 binds specifically to a 14-bp direct repeat downstream of the traB gene without any detectable nicking activity, which is required for conjugal transfer of plasmid DNA in single-stranded form via rolling circle replication.Overexpression of the Streptomyces traB genes is lethal to the host (hence the name kil in some cases), and the presence of kil override (kor) genes is necessary for viability of the host (17). Mutations in the tra or kil gene completely abolish conjugal transfer and pocking. The mechanism of killing by traB or kil is unknown.After the entry into the recipient mycelium, spreading of the incoming plasmid along the recipient hyphae has been proposed to rely on the plasmid-carried spread (spd) genes. The conjugative plasmids of Streptomyces usually contain two or more spd genes, which encode small hydrophobic proteins that often do not show extensive sequence similarity to one another or to other proteins in the database (9). Inactivation of a single spd gene is sufficient to cause a reduction in pock sizes (16, 19), and it was therefore postulated that the spd genes promote migration of plasmid copies inside the recipient mycelium (19).Recently, Tiffert et al. (33) showed that overexpression of Spd2, like that of TraB, was also lethal to the host. They also demonstrated that in vitro, a spread gene product, SpdB2, formed oligomers and interacted with the TraB protein. Those authors proposed that TraB, SpdB2, and other spread proteins formed a channel at the septal cross walls for intrahyphal spread of the plasmid DNA. Involvement of TraB in spreading has also been suggested by Kataoka et al. (16), Kosono et al. (21), and Pettis and Cohen (26). This postulated involvement of Spd proteins and TraB in intrahyphal spread has, however, not yet been experimentally demonstrated.During vegetative growth, distribution of low-copy-number plasmids in dividing bacterial cells usually involves a functional partitioning system. In Streptomyces, proper postreplicational partitioning of low-copy-number plasmids is important during the development of haploid spores from aerial hyphae to avoid plasmid loss in the subcultures, and this task is generally assumed to rely on a parAB operon, which is also used in the partitioning of many other bacterial chromosomes and plasmids (reviewed in reference 8). ParB is a DNA binding protein, which binds specifically to one or more centromere-like parS sites. ParA, a membrane-associated ATPase, is recruited by ParB to the parS site and forms a cytoskeletal structure required for the symmetric movement of the ParB-parS complex during partitioning. The involvement of conjugal transfer, spread, and partitioning in the movement of plasmids in a Streptomyces colony is depicted in Fig. ​Fig.1A1A.Open in a separate windowFIG. 1.Models for partitioning, transferring, and spreading of SLP2 and their interactions in Streptomyces. (A) During vegetative growth of the mycelium, the spread of the low-copy-number plasmid SLP2 (dumbbells) along the substrate and into the aerial hyphae is aided by both the parAB partitioning system (Par) and the spread system (Spd). The proper partitioning of the plasmids into the haploid spores also involves the partitioning system. Gray blotches represent the chromosomes. (B) Involvement of conjugal transfer (Tra) and Spd systems in compensation of SLP2 loss and growth retardation caused by the ΔparAB mutation. Partitioning defect during formation of spores from aerial hyphae (top) results in the absence of SLP2tsrΔpar plasmids in some spores (path a). Colonies developed from the plasmidless spores would be of normal size and produce Thio^s spores. If a plasmid-containing spore suffers severe plasmid loss during colony development (path b), extensive Tra-mediated interhyphal transfer and Spd-mediated intrahyphal transfer of the plasmid (arrows) would result in acute growth retardation, and the spores produced would contain SLP2tsrΔpar and be Thio^r. If the plasmid suffers no or little loss during colony formation (path c), conjugal transfer and growth retardation would be minimal, and most of the spores produced would be Thio^r.One or more parAB operons are found on low-copy-number plasmids in Streptomyces, the linear SLP2 plasmid in Streptomyces lividans (15), and the linear SCP1 plasmid (2) and the circular plasmid SCP2 (11) in Streptomyces coelicolor, all of which are stably maintained in their hosts. Of these, the parAB system of SCP2 has been demonstrated to be important for stable maintenance of SCP2 through sporulation cycles in S. coelicolor (11). Supposedly, the parAB system in the linear plasmids plays the same functional role.The linear chromosome of Streptomyces also possesses a parAB operon. In S. coelicolor, about 20 parS sequences are clustered within 520 kb of oriC (3, 20). Disruption of the parAB operon on the S. coelicolor chromosome resulted in 13% of anucleate spores (20). Interestingly, this defect can be suppressed by the presence of an integrated form of the linear plasmid SCP1 (the NF state), which contains two parAB homologs (2). Other than these studies, little is known about the mechanisms of stable maintenance of low-copy-number plasmids in Streptomyces, especially the linear ones.In this study, we initiated a study of the genetic controls of SLP2 stability. SLP2, a 50-kb linear plasmid originally identified in S. lividans 1326 (6, 15), may be conjugally transferred to and stably maintained in various other species (14). It contains 43 putative genes, including 3 involved in conjugation, spd1 (SLP2.18), traB (SLP2.19), and spd2 (SLP2.26), and a putative par operon consisting of parA (SLP2.30c) and parB (SLP2.29c) (15). We discovered that deletion of parAB (ΔparAB) causes only a mild defect in maintenance of SLP2 and surprisingly resulted in growth retardation of SLP2-containing colonies. We present evidence showing that the mild effect of the ΔparAB mutation was due to recovery of the plasmid through traB-mediated conjugal transfer from plasmid-containing to plasmidless hyphae during growth, and the growth retardation was caused by the interhyphal conjugal transfer and spd1-mediated intrahyphal spread. In addition, the intrahyphal spread function is also important for SLP2 maintenance during vegetative growth. It appears to overlap fully with the partitioning function in S. lividans (the original host) but dominates over the partitioning function in S. coelicolor, such that deletion of spd1 caused little effect on S. lividans but severely reduced the stability and conjugal transfer efficiency of SLP2 in S. coelicolor.In contrast to unicellular bacteria, the involvement of multiple mechanisms for plasmid maintenance reflects the demand for efficient movement of plasmid DNA along the substrate and aerial hyphae before partitioning into the spores during the relatively complex life cycle of Streptomyces.