- Volume 140, Issue 5, 1994

Immunobiological activities of chemically defined lipid A from lipopolysaccharides (LPS) of Porphyromonas gingivalis strain 381, which possesses β-(1·6)-linked glucosamine disaccharide 1-monophosphate, with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2′ -positions, respectively, were compared with those of synthetic Escherichia coli-type lipid A (compound 506) and Salmonella-type lipid A (compound 516). P. gingivalis lipid A and its LPS induced stronger or comparable production of the cytokines interleukin-1 receptor antagonist (IL-1ra), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor and interferon-γ as compared with compounds 506 and 516 in the culture supernatants of human peripheral blood monocytes or mononuclear cells. However, the P. gingivalis preparations showed low activity in inducing the production of IL-1β and tumour necrosis factor-α. Clear antagonistic effects of P. gingivalis lipid A and its LPS against IL-1β production induced by E. coli LPS or compound 506 were seen. Furthermore, P. gingivalis lipid A and its LPS had marked immunopharmacological activities, i.e. antitumour, natural killer cell and antiviral activities. Its monophosphorylation pattern and the presence and position of fatty acids possessing acyl chains of considerable length are unique to P. gingivalis lipid A, differing from enterobacterial lipid As. Its good balance between agonistic and antagonistic effects, making it a possible candidate for use as an immunomodulatory drug, may be attributable to these unique features.

Various Streptomyces spp. including S. violaceus, S. scabies, S. griseus, S. exfoliatus, S. coelicolor and S. lividans secrete indole-3-acetic acid (IAA) when fed with l-tryptophan (Trp). Production of IAA was detected in Streptomyces strains causing potato scab as well as in non-pathogenic strains. The pathways for IAA synthesis from Trp were investigated in S. violaceus and S. exfoliatus. Indole-3-acetamide (IAM), indole-3-lactic acid (ILA), indole-3-ethanol (IEt) and IAA were identified by HPLC and GC-MS. Streptomyces cells were capable of catabolizing IAM, ILA, IEt and indole-3-acetaldehyde (IAAId) into IAA. Incorporation of radioactivity into IAM, IAA and IAL but not IEt was detected when cells were fed with l-[3-14C]tryptophan. Results indicate the presence of the IAM pathway (Trp → IAM → IAA) and the possible presence of additional pathways for IAA biosynthesis in Streptomyces.

Mycobacterium abscessus and Mycobacterium chelonae, two members of the Mycobacterium fortuitum complex, contain five major glycolipids. A combination of NMR spectroscopy, fast atom bombardment mass spectrometry and chemical degradation was used to elucidate their structures. All the compounds belong to the family of glycopeptidolipids. A 6-deoxy-α-l-talosyl unit, which may bear one or two acetyl groups, invariably occupies the site of glycosylation on the threonine residue in the various compounds. A 3,4-di-O-methyl- or 2,3,4-tri-O-methyl-α-l-rhamnosyl unit modifies the alaninol end of the diglycosylated molecules. Both species also contain a multiglycosylated compound consisting of -α-l-rhamnosyl-(1 → 2)-3,4-di-O-methyl-α-l-rhamnosyl linked to alaninol, which belongs to the class of new variants of glycopeptidolipids recently described. Using an ELISA, the latter glycolipid as well as the diglycosylated ones (not previously reported to be antigenic), were shown to react with the serum raised against the whole lipid antigens of M. chelonae. A comparative serologic study of the native and chemically modified glycopeptidolipid antigens allowed the identification of their epitope as the 3,4-di-O-methyl-α-l-rhamnosyl residue. Similar experiments conducted on the glycopeptidolipids isolated from the serologically cross-reacting species M. peregrinum led to the conclusion that the epitope identified in M. chelonae and M. abscessus was involved in the cross-reactions and demonstrated the existence of a second haptenic moiety in the glycolipids of M. peregrinum, the 3-O-methyl-α-l-rhamnosyl unit. In addition to this latter non-shared epitope, the recently described sulfated glycopeptidolipid antigen of M. peregrinum did not react with the M. chelonae serum, thus further explaining the difference in the seroreactivity within the complex.

Agaricus bisporus has the enzymic potential to assimilate ammonia by the activities of glutamine synthetase (EC 6·3.1·2), NADp-dependent glutamate dehydrogenase (EC 1·4.1·2) and NADP-dependent glutamate dehydrogenase (EC 1·4.1·4). It also contains glutamate synthase (EC 1·4.7·1) and a number of transaminating activities like glutamate-oxaloacetate transaminase (EC 2·6.1·1), glutamate-pyruvate transaminase (EC 2·6.1·2) and alanine-glyoxylate transaminase (EC 2·6.1·44). A. bisporus showed good growth in a defined buffered medium on glucose as a carbon source and a number of organic nitrogen compounds or ammonia as a nitrogen source. No growth was observed using nitrate as a nitrogen source. A. bisporus was not able to use organic nitrogen containing substances as a sole nitrogen and carbon source. Specific activities of the ammonia assimilating enzymes showed some variation when mycelia were cultivated on different nitrogen sources. Highest specific activities for glutamine synthetase, NAD-dependent glutamate dehydrogenase and NADP-dependent glutamate dehydrogenase were found when mycelia were grown on glutamate as a nitrogen source. Lowest values were found when the mycelia were grown on ammonia or glutamine. The specific activities of the ammonia assimilating enzymes showed no variation during maturation of the sporophores.

We have isolated an active 719 bp fragment from the 4·3 kb region of the genome of Streptomyces antibioticus that activates a silent phenoxazinone synthase (PHS) gene in Streptomyces lividans. Sequencing of the 719 bp fragment revealed several potential open reading frames (ORFs); however, the distribution of G + C in these putative ORFs was uncharacteristic of streptomycete genes. No RNA products transcribed from the active sequence were detected by dot-blot hybridization and no proteins corresponding in size to the predicted products from the ORFs were observed when appropriate plasmids were used as templates in a streptomycete coupled transcription-translation system. Fragments of 249 and 243 bp, respectively, were obtained from the 719 bp fragment from S. antibioticus and from the S. lividans genome by PCR cloning. Both fragments activated phs in S. lividans when cloned on a high copy number plasmid.

In glycerol-grown, but not in glucose-grown cultures, expression in Streptomyces lividans TK24 of the cloned α-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.

By differential hybridization we have identified cDNA clones that are derived from iron-regulated genes in Saccharomyces cerevisiae. Sequencing of seven cDNA clones revealed that five clones correspond to TPI1 encoding triosephosphate isomerase (Tpi1p) and one corresponds to TDH3 encoding glyceraldehyde-3-phosphate dehydrogenase (Tdh3p). During iron-limited growth mRNA levels for Tpi1p and Tdh3p were at least 3-fold lower than during iron-saturated growth; as shown with a hem1 mutant strain this regulation does not require haem synthesis. mRNA half-lives of TPI1 (TDH3) were 11·5 min (18 min) in low-iron medium and 30 min (32·5 min) in Nigh-iron medium, indicating iron-regulation of transcript half-lives; the stabilities of the ACT1 and PDC1 transcripts were not influenced by iron. Increased glycerol production during growth in low-iron, as compared to high-iron medium, is consistent with a modification of the glycolytic flux during iron-limited growth in S. cerevisiae.

The 23S rDNA sequences of Mycobacterium paratuberculosis, M. avium and M. phlei and the sequences of the spacer regions between the 16S and 23S rRNA genes were determined. The overall 23S rDNA sequence identity between M. paratuberculosis and M. avium was 99·7% (nine mismatches), showing the very close relatedness of these mycobacteria. Evolutionary distances between the five known mycobacterial 23S rRNA/rDNA sequences and those of other Gram-positive G + C-rich bacteria were determined. The 23S rDNA sequences of mycobacteria showed two inserted regions compared to the other bacteria. A mycobacterial unique region contained one mismatch between M. paratuberculosis and M. avium. An Actinomycetales-specific insertion, consisting of 111 nucleotides, was completely identical for M. paratuberculosis and M. avium. The sequence of the intergenic spacer region between 16S and 23S rDNA had a length of 278 bp for M. paratuberculosis and M. avium with only two mismatches. The spacer region of the fast-growing M. phlei was 85 bp longer. No tRNA-encoding region was found in the spacer region.

The gene for proline iminopeptidase from Lactobacillus delbrueckii subsp. lactis DSM 7290 coding for an enzyme that hydrolyses the synthetic substrate l-prolyl-β-naphthylamide (Pro-βNA) was cloned in Escherichia coli. An enzymic plate assay was used to screen for positive clones. The gene, designated pepI, was subcloned into vector pUC18 and sequenced. The nucleotide sequence revealed an 882 bp open reading frame encoding 294 amino acids, coding for an enzyme with a calculated molecular mass of 32883 Da. By cloning under control of the lac promoter the peptidase was highly expressed. Sequence analysis showed that pepI is of a new sequence type, distinct from all peptidases so far sequenced. Amino acid homology to the active site of a Pseudomonas putida esterase and inhibitor studies of the enzyme imply involvement of a serine residue in catalysis.

In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b 6 -f complex and Photosystem I. In these organisms plastocyanin is the electron carrier under copper-replete conditions, and cytochrome c-553 is the electron carrier during copper deprivation. In this paper we report the cloning, sequencing and transcriptional analysis of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. The gene for cytochrome c-553 encodes a preprotein containing 111 amino acids with a predicted n-terminal transit peptide sequence of 25 amino acids. The gene for plastocyanin encodes a preprotein containing 139 amino acids with a n-terminal transit peptide sequence of 34 amino acids. RNA transcript analyses indicate that the expression of the genes for cytochrome c-553 (petJ) and plastocyanin (petE) are regulated in reciprocal ways in response to copper concentration. In copper-replete conditions, petJ is expressed at very low levels, but is transcribed at high levels under copper deprivation; petE is down-regulated in the absence of copper, but is rapidly up-regulated when copper is added back to the medium.

Anucleate primary sterigmata (aps) mutants of Aspergillus nidulans are partially blocked in conidiation (asexual sporulation) due to failure of the organized migration of nuclei into the conidiophore metulae. The mutants also have a slightly reduced hyphal growth rate and irregular distribution of nuclei in vegetative hyphae; the hyphal phenotype appears somewhat more variable than the conidiation defect. The mutants fall into two complementation groups, apsA and apsB, mapping on chromosomes IV and VI, respectively. apsB mutants are also partially defective in sexual reproduction.

Clostripain-specific antibodies were used to analyse the maturation of clostripain prepro-enzyme and core protein heterologously synthesized in Escherichia coli and Bacillus subtilis. Core protein purified from E. coli cells harbouring plasmid pHM3-23 underwent calcium-dependent, self-triggered maturation. Concomitantly, the inactive form of the enzyme was converted into an active form, demonstrating the self-activation capacity of the clostripain core protein. As judged from Western blot analysis, the major portion of the protein in E. coli was degraded, presumably by the activated clostripain. The enzyme was not exported to the E. coli periplasm, either by use of the putative Clostridium histolyticum signal peptide or by use of the E. coli OmpA signal peptide. Therefore, the Gram-positive micro-organism B. subtilis was chosen as an alternative host for the expression of the prepro-enzyme and the core protein. BR 151 cells harbouring pHM7-10B secreted clostripain precursor to the growth medium and matured subsequently to the active enzyme. As only a small amount of activity was detected intracellularly, the putative C. histolyticum signal peptide was efficiently recognized by the B.subtilis secretion apparatus. Under optimized conditions, a level of 4500 UI-1 could be obtained in batch cultures.

The Acetobacter xylinum gene (celB) encoding phosphoglucomutase (EC 5·4.2·2) has previously been cloned by complementation of cellulose-negative mutants. In the present report the nucleotide sequence of a 2·0 kb DNA fragment containing celB is described. Expression analysis using the bacteriophage T7 RNA polymerase promoter ø10 resulted in identification of a probable translational start codon of celB, and this conclusion was confirmed by N-terminal amino acid sequencing of the recombinant protein. From the nucleotide sequence data it was deduced that celB encodes a protein with a calculated molecular mass of 59·6 kDa. A protein of similar size was visualized after in vitro transcription and translation, using the cloned 2·0 kb fragment as template. The results of an amino acid sequence comparison and a biochemical analysis indicated that the CelB protein is structurally and functionally related to the previously characterized human and rabbit phosphoglucomutases.

Genes encoding superoxide dismutase (SOD: EC 1·15.1·1) were cloned from Campylobacter jejuni NCTC 11351 and Campylobacter coli UA585 by heterologous complementation of a SOD-deficient Escherichia coli mutant. Deletion analysis of the cloned C. jejuni DNA assigned the sod gene to a 1·2 kb insert and this contained an open reading frame of 660 bp. The deduced gene product of 220 amino acids was 71% identical to the E. coli iron-containing SOD and 60% identical to the E. coli manganese-containing SOD. The recombinant SOD was expressed at high levels in E. coli and protected a sodA sodB double mutant from the toxic effects of methyl viologen. Nucleotide sequence analysis of the corresponding gene from C. coli showed it to be 92% identical to that from C. jejuni.