- Volume 135, Issue 12, 1989

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fevl. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36·5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.

Streptomyces lividans TK21 possesses inducible ribosomal RNA methylase activity that confers high-level resistance to lincomycin and lower levels of resistance to certain macrolides. The methylase gene (designated lrm) is inducible by erythromycin and other macrolides and also by celesticetin (a lincosamide) but not by lincomycin. The lrm enzyme monomethylates the N 6-amino group of adenosine at position 2058 within 23S-like ribosomal RNA.

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The M r of the native enzyme was determined to be 200000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of M r 49000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and l-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9·2 for oxidative deamination of glutamate and 8·4 for reductive amination of 2-oxoglutarate. The Michaelis constants (K m) were 28·6 mm for l-glutamate and 0·12 mm for NADP. K m values for reductive amination were 1·54 mm for 2-oxoglutarate, 0·07 mm for NADPH and 30·8 mm for NH4 +. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.

A Ca2+- and calmodulin-dependent protein kinase was purified from baker’s yeast to near homogeneity. At pH 7·5 and 0·1 m-NaCl it has a native molecular mass close to 100 kDa, and is a dimer of apparently identical 56 kDa autophosphorylatable subunits. At 60 μm-CaCl2 and with mixed histones as substrate, half-maximal activation required concentrations of beef calmodulin above 1 μm. At 0·14 μm-beef calmodulin the enzyme showed apparent negative cooperativity towards ATP, with limiting apparent K m values of 4 μm and 60 μm ATP. The enzyme has a broad substrate specificity in vitro, including two yeast proteins that yield, respectively, 50 kDa and 200 kDa phosphopolypeptides.

Six Gram-negative bacteria (Klebsiella pneumoniae, Erwinia chrysanthemi, Proteus vulgaris, Serratia marescens, Salmonella typhimurium, and Pseudomonas aeruginosa) were shown to contain an FA-type protein capable of activating aponitrate reductase, apotrimethylamine N-oxide reductase and apoformate dehydrogenase of Escherichia coli. Protein FA activity was highest in Erwinia chrysanthemi and lowest in Pseudomonas aeruginosa. All the species also contained the low-M r (≤ 1500) heat-resistant material previously reported to be necessary for the protein-FA-dependent activation of E. coli chlB nitrate reductase.

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a ~ 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of ~ 34·5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence database was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.

The gene from Thermomonospora curvata CCM 3312 coding for thermostable a-amylase (tarn) has been cloned in Streptomyces lividans TK 24 and localized to a 2·6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tarn gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.

A recombinant plasmid, pSM2513, containing an 8·5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2·7 kb BglII-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.

Comparisons of 23 virulent phages of Streptococcus salivarius subsp. thermophilus were made based on morphology, host-range, structural protein analysis. DNA restriction patterns and DNA homology. All the phages had isometric heads (diameters 55–58 nm) and striated tails (lengths 221–275 nm). The genome sizes ranged from 37 to 43 kb. The electrophoretic profiles of the structural proteins were related, with at least two major bands of about 23 and 29 kDa in common for all the phages except three, 𝜙80, 𝜙96 and 𝜙99. These latter phages, which had a particular protein composition (main proteins of 29 and 40 kDa) and high DNA homology between each other, showed only a low DNA homology with the other phages. Extensive DNA homologies were demonstrated by Southern hybridization for all the other phages. All these results suggest a close evolutionary relationship between the phages.

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013 - a narrow-host-range plasmid with the broad-hosfrange IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1O11, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.

The restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support he current taxonomic classification of C. neoformans into two varieties.

Summary: A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsl. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnl. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1·8 kb fragment of DNA which complemented ger-97::Tn9/7and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential σEand σApromoter regions and encoding a predicted polypeptide of 21117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 171 was cloned in Escherichia coli. The cloned CGTase gene consisted of a single open reading frame which would encode a polypeptide of 713 amino acids, and the first 27 amino acid residues comprised a signal peptide. The nucleotide sequence and the amino acid sequence of this CGTase (CGTase 17-1) gene had strong homology with those of the CGTase (CGTase 38-2) gene previously cloned in our laboratory from the alkalophilic Bacillus sp. strain no. 38-2, although the enzymic properties of the CGTase 17-1 were distinct from those of the CGTase 38-2. To analyse those enzymic properties further, we constructed 12 chimeric CGTases using three restriction nuclease sites and compared the enzymic properties of the chimeric CGTases. The N-terminal part of the enzyme was important for heat stability, and the pH-activity profile was influenced by both the N- and the C-terminal parts. A third segment was less important for enzymic properties.

Two freshly isolated strains, Xc and Yc, of Streptococcus mutans serotype c from human dental plaque were subcultured 100 times in Brain Heart Infusion broth. The cell-surface hydrophobicity of strain Xc markedly decreased after subculturing 60 times, but that of strain Yc remained unaltered. Radioimmunoassay showed a close correlation between surface hydrophobicity and the amount of a cell-surface protein antigen (PAc) of M r 190000. One hydrophilic variant (strain Xc100L), one relatively hydrophobic variant (strain Xc100H), and two hydrophobic variants (strains Yc100H1 and Ycl00H2) were isolated from the 100-fold subcultures of hydrophobic strains Xc and Yc, respectively. SDS-PAGE showed that the amount of cell-associated and cell-free PAc of strain Xc100L was smaller than that of strains Xc and Xc100H. Strain Yc100H2 produced larger amounts of cell-associated PAc than strains Yc and Yc100H1. Resting cells of hydrophilic strain Xc100L attached in smaller numbers to saliva-coated hydroxyapatite than did other hydrophobic strains. RNA dot-blot analysis demonstrated a significant decrease in PAc-specific mRNA in strain Xc100L, as compared with strains Xc and Xc100H. Neither rearrangement nor deletion in the structural gene (pac) for PAc of these strains was observed by Southern blot analysis. These findings suggest that a mechanism which regulates the transcription of the pac gene participates in the quantitative variation of PAc after repeated subculturing.

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determine by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product o a larger, heat-modifiable polypeptide. Based on the results o SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a ‘rough’ lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology o the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath an core structure and polypeptide composition characteristic o that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure o antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detaile molecular analysis of the ultrastructure an antigenicity of T. denticola.

The surface exposure of computer-predicted, linear B-cell epitopes on the major outer-membrane protein (MOMP) of Chlamydia trachomatis serovar B was assessed using antibodies raised against synthetic peptides in conjunction with immunogold transmission electron microscopy. Several of the chosen peptides elicited antibodies which reacted with both denatured and native MOMP. The majority of the exposed epitopes were found within the variable segments of MOMP. For each of the epitopes identified, the extent of their surface accesssibility varied both among individual organisms and different developmental forms. Evidence for two distinct subspecies-specific epitopes within VS4 is presented.