- Volume 132, Issue 4, 1986

Transfer of Neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic NAD-specific glutamate dehydrogenase (NAD-GDH), within 30 min of the shift. Immunoprecipitation of in vivo pulse-labelled NAD-GDH demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. Derepression was shown to be under transcriptional control by using the RNA synthesis inhibitor, picolinic acid, and by immunoprecipitation of the in vitro translation products of poly(A)-containing mRNA from repressed and derepressed cells. A brief (5 min) shift to derepression medium followed by a return to 1% (w/v) sucrose medium was sufficient to trigger synthesis of abundant NAD-GDH transcripts and low levels of the active enzyme. A secondary level of translational control is proposed to account for the discrepancy between the detectable levels of NAD-GDH transcripts and protein, following transient derepression.

Nitrate assimilation by suspensions of Azotobacter chroococcum, as determined by the disappearance of the ion from the external medium, displayed saturation kinetics, was inhibited by nitrite, and exhibited an affinity for nitrate higher than that of nitrate reductase. This suggests that the entry of nitrate into the cell is mediated by a specific transporter. Nitrate assimilation required a readily utilizable carbon source and aerobic conditions and was blocked by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) but not by N, N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of ATPases. The inhibition of nitrate assimilation in the absence of an appropriate carbon source was overcome by the non-physiological energy source ascorbate plus N-methylphenazinium methylsulphate (PMS), a substrate combination that allowed respiration. Though an ATP-dependent nitrate uptake mechanism cannot be ruled out, these data suggest that transport of nitrate into the cell is directly dependent on the proton electrochemical gradient across the cytoplasmic membrane.

Cells of an industrial strain of the l-glutamate producer Corynebacterium glutamicum grown in biotin-starvation conditions lost 30% of their membrane phospholipids. This was accompanied by a progressive decrease in the rate of accumulation of glutamate and of the intracellular glutamate accumulated at the plateau. Addition of an acylated surfactant to growing cultures of the same strain also induced a progressive loss of glutamate uptake, without changing the K T of the process. The surfactant-treated cells almost completely excreted the labelled glutamate loaded during a preincubation. These results, together with those obtained previously, allow us to propose that glutamate excretion is mediated by a glutamate permease, after uncoupling of this uptake system resulting from a marked loss of membrane phospholipids. This loss of phospholipids can be induced either by biotin starvation or by addition of an acylated surfactant to growing cells.

Sodium dodecyltriethoxy sulphate (SDTES), either pure or as a component of commercial surfactant mixtures, underwent rapid primary biodegradation by mixed bacterial cultures in OECD screen and river-water die-away tests. Inoculation of [35S]SDTES-containing solutions with OECD screen test media acclimatized to surfactants or their degradation products led to production of various 35S-labelled glycol sulphates and their oxidation products, all known to occur during degradation of [35S]SDTES by pure bacterial isolates. Triethylene glycol monosulphate was the major catabolite together with smaller amounts of di- and monoethylene glycol monosulphates implying, by analogy with pure cultures, that ether-cleavage was the major primary biodegradation step. The oxidation product (carboxylate derivative) of each glycol sulphate was also detected together with metabolites tentatively identified as Ω-/β-oxidation products of the dodecyl chain. Relatively little SO4 2- was liberated directly from SDTES but mixed cultures derived from sewage could metabolize the sulphated glycols to SO4 2-. The environmental relevance of these degradation routes was established by following metabolite production from [35S]SDTES in full-scale river-water die-away tests. Triethylene glycol sulphate was formed first, then rapidly oxidized to acetic acid 2-(diethoxy sulphate) which persisted as the major metabolite for 2–3 weeks. Small amounts of sulphated derivatives of di- and monoethylene glycols were also detected during the same period. Very little SO4 2- was formed directly from SDTES but large amounts accompanied the eventual disappearance of glycol sulphate derivatives. None of the 35S-labelled organic metabolites was persistent and, whenever [35S]SDTES was a component of a commercial mixture, all ester sulphate was completely mineralized to 35SO4 2- within 28 d.

A detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325. Inserts carrying the 1·2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance. One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell. A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure. This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host. These results suggest that the inverted repeat sequence of the X1 structure serves as a ‘hot-spot’ for generalized recombination. Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.

A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 μm-thallium salt and susceptibility to the nocardia-specific bacteriophage ϕBl. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 x 10-4 with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, ϕBls) and 2 x 10-3 with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, ϕBlr) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 μm-thallium acetate). The frequency of thalliuM-resistant transconjugants was 10-1 to 10-2 per donor; all thalliuM-resistant transconjugants contained the donor plasmid.

We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.

The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.

We have characterized a copy number mutant of the pBR322-based plasmid pWT111. A single nucleotide transversion in loop II’ of RNAI results in an eightfold increase in plasmid copy number. Removal of the rom coding region from pWT111cop results in a further sixfold increase in copy number. We present evidence that ROM is involved in the strong incompatibility effect seen between pMB1 and ColE1 type plasmids.

Transmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains habouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum is one of a group of developmentally regulated proteins which enable cells to aggregate by chemotaxis during the early stages of development. We report the identification and DNA sequence of a cDNA clone encoding the amino-terminal region of the phosphodiesterase. The clone, pPD-3, was selected from a cDNA library created by priming first strand synthesis using a set of oligonucleotides with sequences predicted from the amino-terminal amino acid sequence of purified phosphodiesterase. The DNA sequence of pPD-3 encodes perfectly the available phosphodiesterase amino acid sequence, and pPD-3 selects an mRNA which can be translated into material recognized by phosphodiesterase antisera. The nucleotide sequence of pPD-3 indicates there are 49 amino acids, which contain a segment possessing the characteristics of a signal peptide, that separate the amino-terminal residue identified in the purified protein from the methionine codon at which translation originates. DNA blot analysis demonstrates that the phosphodiesterase gene exists as a single copy in the nuclear genome. Analysis of RNA indicates that the phosphodiesterase transcript is 2·1 kb long, which is approximately 0·8 kb more than the minimum required to encode this protein.

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100° respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A7 organisms was purified and characterized by chemical analysis and by 1H and 13C NMR spectroscopy using one- and two-dimensional methods. The polymer has the repeating unit → 3)-β-2-acetamido-2-deoxygalactopyranose-(1 → 3)-α-2-acetamido-2-deoxy-6-O-acetylglucopyranose-(1-phosphate →. It was immunogenic (capable of eliciting antibodies) for sheep. Chemical removal of O-acetyl groups destroyed both the ability of the polymer to adhere to sheep erythrocytes at neutral pH and the ability to form immune precipitates with specific antisera. Studies using the protein A-gold technique in the electron microscope showed the polysaccharide to be peripherally localized on the bacterial surface.

A protein toxin apparently composed of one polypeptide with an estimated M r of 155000 was purified from sonicated cells of a type D strain of Pasteurella multocida (LFB3) by preparative polyacrylamide gel electrophoresis (PAGE) and DEAE-Sephadex A50 chromatography. Its specific activity was 150-fold greater than that of the crude extract. The partially purified protein was cytotoxic for embryonic bovine lung cells, lethal for mice and caused turbinate atrophy in gnotobiotic pigs; a single intraperitoneal injection of approximately 360 ng kg-1 caused 50% turbinate atrophy. Reversal of the two-step purification procedure using DEAE-Sephacel chromatography followed by preparative PAGE increased the yield of toxin 30-fold; the specific activity of the partially purified toxin was 1970-fold greater than that of the crude extract.