Modified xylE and xylTE reporter genes for use in Streptomyces: analysis of the effect of xylT.

The reporter gene xylE (encoding catechol 2,3-dioxygenase) has been modified for a more rational use in Streptomyces. Two reporter fragments, one containing xylE, and the other containing also the upstream gene xylT (which encodes a soluble ferredoxin), have been constructed to allow precise fusion of regulatory regions to the reporter genes. Identical fusions of these xylE and xylTE reporter fragments to the Streptomyces dagA and tipA promoters, in low and high copy number plasmids, show that the levels of xylE mRNA and catechol 2,3-dioxygenase activities are significantly higher when xylT is present.

Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution.

BACKGROUND: Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS: The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS: The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator.

Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA.

Identification and properties of a novel clt locus in the Streptomyces phaeochromogenes plasmid pJV1.

A novel clt locus required for efficient transfer of the Streptomyces phaeochromogenes plasmid pJV1 was identified and mapped. The clt region was functional in both orientations, and its absence caused a severe reduction in plasmid transfer. Chromosome mobilization, on the other hand, was not affected by absence of the clt locus. The clt region showed structural, but not sequence, similarity to transfer origins of gram-negative plasmids.

Sequence and functional analysis of the Streptomyces phaeochromogenes plasmid pJV1 reveals a modular organization of Streptomyces plasmids that replicate by rolling circle.

pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.

Transcriptional regulation of the four promoters of the agarase gene (dagA) of Streptomyces coelicolor A3(2).

The agarase gene (dagA) of Streptomyces coelicolor A3(2) is transcribed from four promoters that are recognized by at least three, and probably four, different RNA polymerase holoenzymes, each containing a different sigma factor. S1 nuclease protection studies revealed that transcription from all four promoters is induced by the products of agar hydrolysis and strongly repressed by glucose. Mutants deficient in glucose kinase activity were defective in glucose repression of all four promoters. Mutants were isolated or identified in which transcription from all four promoters had become inducer-independent (i.e. constitutive), establishing the existence of a repressor gene for dagA that does not appear to be located within 9 kb of the structural gene. The cloned dagA gene was also constitutively expressed in the closely related strain Streptomyces lividans, which does not normally make agarase and which presumably lacks the repressor gene. Glucose was still able to repress dagA transcription even under conditions of constitutive expression, suggesting that glucose kinase does not mediate its effect via inducer exclusion. Relative differences in the use of the four promoters were not detected during different stages of growth of surface-grown cultures, although dagA transcription appeared to peak during the production of aerial mycelium.

Sequence of the Streptomyces albus G lipase-encoding gene reveals the presence of a prokaryotic lipase family.

An extracellular lipase (Lip)-encoding gene from Streptomyces albus G has been cloned and sequenced. It encodes a Lip with 82% sequence identity to another previously cloned Lip from a Streptomyces species not closely related. These two sequences can be aligned with 33% identity to the sequence of Lip1 from the antarctic psychrotroph Moraxella TA144 [G. Feller et al., Nucleic Acids Res. 18 (1990) 6431]. An alignment of the three sequences revealed amino-acid substitutions which might be responsible for the greater thermal stability of the Streptomyces lipases. The presence of this lip gene family in several members of the Streptomyces genus was also shown.

Relationship between the replication functions of Streptomyces plasmids pJV1 and pIJ101.

The essential replication region of the Streptomyces phaeochromogenes plasmid pJV1 was sequenced and compared to the equivalent region of the well-characterized Streptomyces plasmid pIJ101. The sequence revealed a similar organization in both plasmids, including a conserved region within both plasmid origins, which is just upstream of a gene encoding the replication protein (Rep). The Rep proteins of pJV1 and pIJ101 are very similar, and both have a conserved motif, also present in Rep proteins of single-stranded DNA plasmids of the low G + C gram+ bacteria that includes a tyrosine residue presumably involved in creating the single-stranded nick that initiates the replication event. A co-integration experiment between these two replicons led to the formation of a novel replicon with a hybrid origin and allowed the identification of the nick site where replication starts, also showing that the replication functions of both plasmids are related.

Cloning, characterization, and expression in Streptomyces lividans 66 of an extracellular lipase-encoding gene from Streptomyces sp. M11.

A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.

Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation.

Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.

glnA mutations conferring resistance to methylammonium in Escherichia coli K12.

Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.

Nitrogen regulation of synthesis of the high affinity methylammonium transport system of Escherichia coli.

Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4.

cis-Dominant, glutamine synthetase constitutive mutations of Escherichia coli independent of activation by the glnG and glnF products.

Mutants resistant to 80 microM L-methionine-DL-sulfoximine (MS) were isolated on glucose-minimal 15 mM NH4+ medium plates from Escherichia coli cells which were hypersensitive to this concentration of the analogue by virtue of their harboring glnG mutations. MS-resistant mutants derived from strain MX902 carried, in addition to its glnG74 ::Tn5 allele, mutations tightly linked to glnA, as shown by P1-mediated transduction experiments. One particular allele, gln-76, which suppressed the MS-sensitivity conferred by glnG74 ::Tn5 but not its Ntr- phenotype (inability to transport and utilize compounds such as arginine or proline as the only nitrogen sources), was shown to allow constitutive expression of glutamine synthetase in the absence not only of a functional glnG product but also of a functional glnF product. This behavior was found to be cis-dominant in complementation experiments with F'14 merogenotes . In an otherwise wild-type genetic background as in MX929 (gln-76 glnA+ glnL+ glnG+ glnF +), however, normal activation, mediated by the glnG and glnF products was preferred over that mediated by gln-76.