- Volume 134, Issue 7, 1988

Resistance to the low M r fungal epipolythiodiketopiperazine toxin, gliotoxin, varied threefold between the phytopathogens Pythium ultimum and Rhizoctonia solani. Uptake of radiolabelled gliotoxin was rapid and concentration dependent. Uptake by P. ultimum was largely complete within 1 min while the rate of uptake peaked within 10 min for R. solani (anastomosis groups 2-2 and 4). Uptake of gliotoxin by P. ultimum was twice that shown by the more resistant R. solani. A deep rough mutant of Salmonella typhimurium, deficient in outer-membrane polysaccharide synthesis, was hypersensitive to gliotoxin, indicating that diffusion barriers play a role in relative sensitivity to gliotoxin. Fungal glutathione levels (reduced and oxidized) did not differ appreciably before or after gliotoxin exposure, indicating that this cytoplasm-based detoxification mechanism was not important in the relative fungal sensitivity to gliotoxin. Binding of the radiolabelled thiol reagents N-ethylmaleimide (NEM) and iodoacetic acid to fungal thiol groups was inhibited by gliotoxin. Conversely, the thiol reagents NEM and p-chloromercuribenzoic acid inhibited the uptake of radiolabelled gliotoxin. Uptake of radiolabelled amino acids and glucose was reduced by up to 85% by gliotoxin (8 μg ml−1). It is suggested that the primary mechanism of action of gliotoxin involves selective binding to cytoplasmic membrane thiol groups.

SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer. The host range of the phage is narrow, limited to some strains of this species. Another strain of Sac. erythraea, and a strain of Sac. hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication. SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping. The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.

A number of expression vectors have been constructed to allow over-production of selected gene products in Klebsiella pneumoniae and other enteric bacteria. The plasmids use the strong hybrid trp-lac (tac) promoter for gene expression, which is regulated by the lacI Q allele of the lac repressor carried on the vector. This provides very tight regulation of gene expression, which is important for over-production of proteins which may be detrimental to cell growth. The vectors carry the standard mpl8 cloning nest in which all the restriction sites are unique to the plasmid (with the exception of EcoRI in pDK7). Derivatives were constructed carrying kanamycin, chloramphenicol or ampillicin resistance as selectable markers, the first two of which are advantageous in K. pneumoniae due to the high inherent -lactamase activity of this organism.

The DNA of an isolate of Borrelia duttonii, an agent of relapsing fever is present as seven major species ranging in size from 10 kb to greater than 150 kb. Additionally, this isolate contains low copy number species, both smaller and larger than these seven major elements. No one of these individual DNA species obviously corresponds to the bacterial chromosome, unlike the situation in Borrelia hermsii, another relapsing fever Borrelia. Thus it appears that B. duttonii has a unique segmented arrangement of its genetic material. Cloned DNA fragments containing coding sequences specific for variant surface antigens of B. duttonii hybridize to a closely migrating, high copy number subset of these genetic elements.

The gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promoter sequence (TTTTCC for the –35 region and TATTGT for the – 10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upsteam of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.

A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (M r 60000, pI 5·0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64kDa polypeptide of pI 8·2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.

The mixed-ligand complexes of platinum(II) [Pt(Gly)(uracil)Cl2].H2O and [Pt(His)(adenine)]-Cl.2H2O eliminated multicopy plasmids with the ColE1 origin of replication in Escherichia coli with 100% frequency. However, plasmids of the IncF1, H1 and X groups were totally refractory to these agents under similar conditions.

Four highly amplified DNA sequences (ADS) ranging from 5·8 to 24·8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24·8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).

A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neoμm. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the trans-formant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.

During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative (‘cured’) strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWWO. Experiments with two plasmids identical to pWWO but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the ‘benzoate caring’ occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWWO-174, a derivative of pWWO which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWWO, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the ‘cured’ phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously ‘cured’ derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10−2 and 10−3. We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the ‘cured’ segregants grow faster than the wild-type using the chromosomally determined β-ketoadipate pathway.

The rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host. Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors. Genetically, the regions of DNA responsible for phase variation have not been identified. We have sought to determine whether the plasmid identified in acute disease isolates, QpHl, which represents approximately 5% of the coding capacity of this organism is involved in phase variation. Plasmids from phase 1 and phase 2 variants (designated QpHl and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells. Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage. The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type. Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E. coli extract was also identical. Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical. These data indicate that within the limits of our analysis, the plasmid DNA from C. burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation.

Haemophilus somnus expresses two types of receptors that bind to the Fc region of bovine IgG, IgA and IgM. In this study, the relationship between these two types of Fc receptors is characterized. The high molecular mass receptors (350, 270 and 120 kDa) were secreted into the culture medium and were also in the insoluble protein fraction of the culture medium. The 41 kDa Fc receptor, which is a major outer-membrane protein, was only present in the insoluble protein fraction. Peptide mapping of the two types of Fc receptors suggests that the 41 kDa receptor is related to the high molecular mass receptor complex. Disulphide linkage is unlikely to be the mechanism of association of the 41 kDa receptor with the high molecular mass receptors since reducing agents had no effect on separating the individual receptors. Although the 41 kDa receptor is a major protein in the outer membrane of H. somnus, it does not react with convalescent bovine sera in Western blots. In contrast, convalescent bovine sera reacts intensely with the high molecular mass receptors in Western blots.

Clinical isolates of Shigella spp. were examined for their susceptibility to human serum. The susceptibility of the strains to immune and nonimmune human serum was dependent upon the size of the bacterial inoculum and the concentration of serum. There were differences among Shigella spp. in susceptibility to human serum: S. sonnei strains were the least susceptible, strains of S. boydii and S. flexneri serotype 6 were intermediate, and those of S. flexneri other than serotype 6 and S. dysenteriae were the most susceptible. Experiments in which heat-treated (56 °C for 30 min, or 50 °C for 20 min) serum was used, and analysis of activation of complement by lipopolysaccharides (LPS) from each Shigella sp., suggested that LPS composition, especially the O antigen polysaccharide chains, contributes to the differences among Shigella spp. in susceptibility to human serum.

An in vitro model of the regenerative phase of the human endometrial cycle was developed in order to study the growth of Chlamydia trachomatis during the period following menses. Glandular epithelial fragments were prepared from curettings of endometria and explanted onto coated substrata. Epithelial cells migrated rapidly from the explant in a fashion which closely mimicked the regeneration of the surface epithelium after menses. The cultures were then experimentally infected with C. trachomatis serotype E at various times during formation of the outgrowth. Chlamydial inclusions developed both within the explants and in the outgrowing epithelial sheets. They were also found in isolated epithelial and non-epithelial cells. However, the most striking feature of chlamydial inclusion development within these cultures was the tendency for inclusions to be located in cells at the periphery of the epithelial sheets. This was partly due to the failure of the cells within the sheets to bind chlamydiae after centrifugation of the organisms onto the culture and partly due to a phenomenon similar to phagokinesis. During this process infectious chlamydial particles were cleared from the substratum by migrating cells with free motile edges, which occasionally led to internalization and inclusion development within these cells.