The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of L-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A.

The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of L-DOPA, but not of L-tyrosine. The purified LmbB1 protein showed a Km for L-DOPA of 258.3 microM. The L-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an L-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an L-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on L-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian L-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from L-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis, cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11.

The lincomycin (LM)-production gene cluster of the overproducing strain Streptomyces lincolnensis 78-11 was cloned, analysed by hybridization, as well as by DNA sequencing, and compared with the respective genome segments of other lincomycin producers. The lmb/lmr gene cluster is composed of 27 open reading frames with putative biosynthetic or regulatory functions (lmb genes) and three resistance (lmr) genes, two of which, lmrA and lmrC, flank the cluster. A very similar overall organization of the lmb/lmr cluster seems to be conserved in four other LM producers, although the clusters are embedded in non-homologous genomic surroundings. In the wild-type strain (S. lincolnensis NRRL2936), the lmb/lmr-cluster apparently is present only in single copy. However, in the industrial strain S. lincolnensis 78-11 the non-adjacent gene clusters for the production of LM and melanin (melC) both are duplicated on a large (0.45-0.5 Mb) fragment, accompanied by deletion events. This indicates that enhanced gene dosage is one of the factors for the overproduction of LM and demonstrates that large-scale genome rearrangements can be a result of classical strain improvement by mutagenesis. Only a minority of the putative Lmb proteins belong to known protein families. These include members of the gamma-glutamyl transferases (LmbA), amino acid acylases (LmbC), aromatic amino acid aminotransferases (LmbF), imidazoleglycerolphosphate dehydratases (LmbK), dTDP-glucose synthases (LmbO), dTDP-glucose 4,6-dehydratases (LmbM) and (NDP-) ketohexose (or ketocyclitol) aminotransferases (LmbS). In contrast to earlier proposals on the biosynthetic pathway of the C-8 sugar moiety (methylthiolincosaminide), this branch of the LM pathway actually seems to be based on nucleotide-activated sugars as precursors.

Dependence on reporter gene of apparent activity in gene fusions of a Streptomyces griseus streptomycin biosynthesis promoter.

The adjacent genes strR-strA-strB1 lie within the large cluster of genes of streptomycin biosynthesis and resistance in Streptomyces griseus. strR encodes a pathway-specific activator StrR, suggested by previous work to be either an antiterminator or a conventional activator, binding to its DNA target via a helix-turn-helix motif. strB1 is transcribed in an StrR-dependent fashion from a promoter (PstrB1) that lies downstream from strA; between PstrB1 and strB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modulatable transcription termination sites. Hybrid plasmids were constructed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region ("long" fragments) or a small part of it (the "short" fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3'-phosphotransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insertion of an extraneous fragment into PstrB1 in the short fragment construct led to loss of APH activity, demonstrating that no adventitious promoter had been formed in the short construct. In vitro deletion of part of the leader region in a long fragment construct led to high APH expression with or without StrR present. Although these results are consistent with the target of StrR being within the leader region, and thus with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expression of catechol oxygenase (the product of xylE) unless strR was also present on a compatible plasmid. Transfer of PstrB1-xylE fragments to the high copy number vector did not increase catechol oxygenase expression. We interpret these results in terms of an effect, in the hybrid constructs, of one of the reporter genes on promoter function, possibly by affecting local DNA topology.

Heterotopic ossification after spinal cord injury. Epidemiology and risk factors.

From 1981 to 1986 we treated 413 patients for acute spinal-cord injuries. We reviewed 356 patients followed for a minimum of two years of whom 71 (20%) developed heterotopic ossification around one or more joints. Heterotopic ossification occurred more often in male patients (23%) than in female (10%), and was most frequent in the 20- to 30-year age group. It was also more common after injuries of the lower cervical or thoracic spine than after those of the lumbar spine. Patients with severe neurological deficits (Frankel grades A and B) showed significantly more heterotopic ossification but there was no correlation with the number or severity of associated head and limb injuries. Serum calcium levels did not change significantly in either group for 30 weeks after injury, but the erythrocyte sedimentation rate and the alkaline phosphatase level were significantly increased at six weeks in patients with heterotopic ossification.

Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis.

Using purified sigma 55 RNA polymerase from Bacillus subtilis in an in vitro transcription system, we have shown that both promoters and terminators of Gram negative origin are recognized by this enzyme. Furthermore, when B. subtilis is transformed with a shuttle vector containing certain of these promoters, synthesis of the Staphylococcus aureus CAT protein is achieved, and levels up to 25% of the total cellular protein can be obtained. These findings indicate a closer evolutionary relationship of the expression machinery of these two bacterial species than has been assumed so far. On the basis of these results, the construction of new expression vectors for B. subtilis is likely to be facilitated, since a variety of well-characterized signal elements from Escherichia coli are available.