Streptomyces youssoufiensis sp. nov., isolated from a Moroccan phosphate mine.

A novel actinomycete, strain X4(T), was isolated from a phosphate mine in Youssoufia, 100 km north of Marrakesh, Morocco. The taxonomic status of this strain was evaluated by a polyphasic approach. Strain X4(T) had white aerial mycelium with Rectiflexibiles spore chains bearing smooth-surfaced spores and did not produce diffusible pigments. Chemotaxonomic analysis showed that the cell wall of strain X4(T) contained LL-diaminopimelic acid and glycine. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence indicated that strain X4(T) belongs to the Group I streptomycetes, branching off next to Streptomyces ramulosus NRRL B-2714(T) and Streptomyces kasugaensis M338-M1(T). DNA-DNA relatedness and phenotypic data enabled strain X4(T) to be distinguished from the phylogenetically most closely related type strains. It is therefore proposed that strain X4(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces youssoufiensis sp. nov. is proposed; the type strain is X4(T) ( = CCMM B709(T)  = DSM 41920(T)).

Phosphoinositides are involved in control of the glucose-dependent growth resumption that follows the transition phase in Streptomyces lividans.

The interruption of the sblA gene of Streptomyces lividans was previously shown to lead to relief of glucose repression of the normally strongly glucose-repressed alpha-amylase gene. In addition to this relief, an early entry into stationary phase was observed when cells were grown in a minimal medium containing glucose as the main carbon source. In this study, we established that this mutant does not resume growth after the transition phase when cultured in the complex glucose-rich liquid medium R2YE and sporulates much earlier than the wild-type strain when plated on solid R2YE. These phenotypic differences, which were abolished when glucose was omitted from the R2YE medium, correlated with a reduced glucose uptake ability of the sblA mutant strain. sblA was shown to encode a bifunctional enzyme possessing phospholipase C-like and phosphoinositide phosphatase activities. The cleavage of phosphoinositides by SblA seems necessary to trigger the glucose-dependent renewed growth that follows the transition phase. The transient expression of sblA that takes place just before the transition phase is consistent with a regulatory role for this gene during the late stages of growth. The tight temporal control of sblA expression was shown to depend on two operator sites. One, located just upstream of the -35 promoter region, likely constitutes a repressor binding site. The other, located 170 bp downstream of the GTG sblA translational start codon, may be involved in the regulation of the degradation of the sblA transcript. This study suggests that phosphoinositides constitute important regulatory molecules in Streptomyces, as they do in eukaryotes.

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3.

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

Regulation of the expression of amy TO1 encoding a thermostable alpha-amylase from Streptomyces sp. TO1, in its original host and in Streptomyces lividans TK24.

In its original host, the thermophilic Streptomyces strain sp. TO1, the amy TO1 gene was expressed during growth but only in the presence of starch in the growth medium. When cloned in Streptomyces lividans, on a low copy number replicative plasmid, amy TO1 expression was detectable in fructose-, mannitol- and galactose-grown cultures but not in glucose- or glycerol-grown cultures. This basal expression could be further induced by maltotriose. In a mutant strain of S. lividans disrupted for the LacI-like negative transcriptional regulator (NTR) Reg1, and when the symmetry of the dyadic symmetry element located in the promoter region of amy TO1 was altered, the basal levels of amy TO1 expression were significantly higher than those of the wild-type strain, and the maltotriose inducibility was abolished. These results suggest that, in S. lividans, amy TO1 expression is under the control of the NTR Reg1 due to its interaction with the dyadic symmetry element.

amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24.

The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

alpha-Amylase gene of thermophilic Streptomyces sp. TO1: nucleotide sequence, transcriptional and amino acid sequence analysis.

The nucleotide sequence of a 1860-bp region encoding a thermostable alpha-amylase of Streptomyces sp. TO1 was determined. Frame analysis revealed the presence of a 1359-bp long open reading frame (amy TO1) encoding a 453 amino acid protein with a deduced M(r) of 49 kDa. Northern blot analysis revealed that amy TO1 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp. TO1 strains. Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of -35 (TTGCTG) and -10 (TACGCG) eubacterial-like promoter sequences. Amy TO1 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S. thermoviolaceus alpha-amylase. Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces alpha-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy TO1 might account for the thermostable properties of Amy TO1.

Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.

Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene.

A regulatory gene, reg1, was identified in Streptomyces lividans. It encodes a 345-amino-acid protein (Reg1) which contains a helix-turn-helix DNA-binding motif in the N-terminal region. Reg1 exhibits similarity with the LacI/GalR family members over the entire sequence. It displays 95% identity with MalR (the repressor of malE in S. coelicolor), 65% identity with ORF-Sl (a putative regulatory gene of alpha-amylase of S. limosus), and 31% identity with CcpA (the carbon catabolite repressor in Bacillus subtilis). In S. lividans, the chromosomal disruption of reg1 affected the expression of several genes. The production of alpha-amylases of S. lividans and that of the alpha-amylase of S. limosus in S. lividans were enhanced in the reg1 mutant strains and relieved of carbon catabolite repression. As a result, the transcription level of the alpha-amylase of S. limosus was noticeably increased in the reg1 mutant strain. Moreover, the induction of chitinase production in S. lividans was relieved of carbon catabolite repression by glucose in the reg1 mutant strain, while the induction by chitin was lost. Therefore, reg1 can be regarded as a pleiotropic regulatory gene in S. lividans.

Sequences involved in growth-phase-dependent expression and glucose repression of a Streptomyces alpha-amylase gene.

In glycerol-grown, but not in glucose-grown cultures, expression in Streptomyces lividans TK24 of the cloned alpha-amylase gene (aml) of Streptomyces limosus is switched on toward the end of exponential growth. During this period, aml expression is further inducible by maltotriose. We showed that a 378 bp fragment, extending from position -204 through to +174 (relative to the transcriptional start site), included cis-acting sequences involved in aml regulation. When this fragment was present on a multicopy plasmid, the growth-phase-dependent aml expression conferred by a DNA fragment cloned on a compatible low-copy-number plasmid was greatly enhanced, as if negative regulators were being titrated. A study of the regulation of aml expression in variants with deletions in the aml promoter region indicated that a direct repeat (DR) between positions -124 and -106 (relative to the transcriptional start site) and an inverted repeat (IR) between positions +9 and +24 were good candidates for secondary and primary operator sites, respectively. Deletion of a 29 bp fragment containing the IR rendered aml expression partly growth-phase-independent, resistant to glucose repression, and insensitive to maltotriose induction.

Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces venezuelae.

The alpha-amylase gene (aml) of Streptomyces venezuelae ATCC15068 was cloned in Streptomyces lividans TK24 using the plasmid vector pIJ702. Sub-cloning and exonuclease III deletion experiments localised the sequences required for alpha-amylase production to a segment of 2.05 kb. Low-resolution nuclease S1 mapping revealed a aml transcript of approx. 1.7 kb, and the extracellular form of alpha-amylase was estimated by SDS-polyacrylamide gel electrophoresis to be 59 kDa, suggesting that aml mRNA is monocistronic. The nucleotide sequence of aml was determined and high-resolution nuclease S1 mapping experiments identified transcripts that appeared to initiate at a promoter identical to that of the alpha-amylase gene of Streptomyces limosus [Long et al., J. Bacteriol. 169 (1987) 5745-5754]. Transcription of aml in S. venezuelae, and of the cloned gene in Streptomyces coelicolor A3(2), was induced by maltose and repressed by glucose. Glucose repression in S. coelicolor A3(2) depended on a functional glucose kinase gene. The predicted amino acid sequence of the extracellular enzyme was very similar (75% identity) to the alpha-amylase of S. limosus and shared with this enzyme a strong susceptibility to tendamistat, a potent inhibitor of mammalian alpha-amylases. Sequence inspection revealed a putative signal sequence of 28 amino acids that preceded the probable signal peptidase cleavage site.

Cloning, characterization and regulation of an alpha-amylase gene from Streptomyces limosus.

An alpha-amylase gene (aml) of Streptomyces limosus ATCC 19778 was cloned in Streptomyces lividans 66. S1 mapping experiments identified an aml transcript 1.8 kb in length and the extracellular enzyme was estimated to be 59 kD in size, suggesting that aml was transcribed as a monocistronic mRNA species. Expression of the gene was induced by maltose (or maltodextrins) in S. limosus and when aml was cloned in S. lividans or Streptomyces coelicolor A3(2). In S. limosus, mannitol repressed aml expression while glucose had little or no effect; in S. lividans and S. coelicolor the relative effects of the two sugars were reversed. Both induction and carbon-source repression of aml expression appeared to occur at the level of transcriptional initiation. Glucose repression in S. coelicolor was dependent upon a functional glucose kinase gene.

alpha-Amylase gene of Streptomyces limosus: nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases.

The nucleotide sequence of the coding and regulatory regions of the alpha-amylase gene (aml) of Streptomyces limosus was determined. High-resolution S1 mapping was used to locate the 5' end of the transcript and demonstrated that the gene is transcribed from a unique promoter. The predicted amino acid sequence has considerable identity to mammalian and invertebrate alpha-amylases, but not to those of plant, fungal, or eubacterial origin. Consistent with this is the susceptibility of the enzyme to an inhibitor of mammalian alpha-amylases. The amino-terminal sequence of the extracellular enzyme was determined, revealing the presence of a typical signal peptide preceding the mature form of the alpha-amylase.

Origin of Escherichia coli K-12 Hfr B7.

Several F' plasmids encoding resistance to tetracycline have been derived from a trg::Tn10 Hfr B7 strain of Escherichia coli K-12. One of these plasmids, JGF312, was analyzed by restriction endonuclease digestion and Southern blot hybridization to cloned chromosomal fragments. This analysis revealed that JGF312 was formed by Tn10-promoted deletion from the Tn10 insertion (31.4 min) to within the prophage rac at 30.1 min. Hfr B7 was shown to result from recombination between IS2 of F delta (33-43) and a chromosomal IS2 located within the rac-man region at 30.9 min on the genetic map.