- Volume 131, Issue 4, 1985

H2 production by the human protozoan parasite Trichomonas vaginalis was monitored continuously under a mobile gas phase using a membrane-inlet mass spectrometer. Simultaneous and continuous measurement of dissolved H2, O2 and CO2 indicated that H2 evolution was inhibited at levels of O2 (<0‧25 μm) undetectable by the technique, whereas CO2 production was stimulated. Respiration was not stimulated by admitting H2 to the gas phase. Metronidazole inhibited both H2 and CO2 production. Values of K i for inhibition of H2 formation in strain ATCC 30001 (metronidazole sensitive) of 0‧16 mm and in strain 85 (metronizadole resistant) of 1‧0 mm were obtained. These data suggest that metronidazole not only competes with protons as electron acceptor but that the drug itself or a product of reduction actively inhibits some hydrogenosomal enzyme or electron carrier involved in H2 production. Under these conditions metronidazole inhibition leads to irreversible loss of cell motility.

Purified LPS from a virulent cherry isolate of Pseudomonas syringae pv. morsprunorum was a mixture of smooth and rough molecular species. Mild acid hydrolysis yielded a precipitate of lipid A and a carbohydrate fraction which, by gel permeation chromatography, yielded three peaks of material. The first (high molecular weight) peak was composed almost entirely of a rhamnan, the sidechain polysaccharide. The second peak contained core oligosaccharide and comprised rhamnose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO), phosphate, glucosamine, galactosamine and alanine. The third (low molecular weight) peak contained KDO, phosphate and ethanolamine. Lipid A contained glucosamine, phosphate and the fatty acids 12:0, 3-OH 10:0, 2-OH 12:0 (all ester-linked to glucosamine), and 3-OH 12:0, which was amide-linked. The typing phage A7, which uses LPS as its binding site, was found to possess a rhamnanase which split the sidechains from smooth LPS, releasing them as oligosaccharide.

The chemotropic response of the water mould Achlya ambisexualis to nutrients was investigated. Among individual amino acids only methionine was active. Other amino acids were active only in combinations containing cysteine. Methionine was unique among the amino acids tested in its ability, when incorporated uniformly into the water agar substratum, to disrupt chemotropism towards an attractant mixture of amino acids. Among non-amino acid compounds tested for chemotropic activity only S-adenosylmethionine was active. Carboxyl methylation of protein was promoted by amino acid mixtures.

Sheared polar flagella of the type strains of monotrichate Pseudomonas aeruginosa and multitrichate P. fluorescens and P. putida were purified and the sedimentation coefficients of the corresponding flagellins determined by ultracentrifugation; from these values the approximate molecular weights of the major flagellins were calculated to be between 38000 and 43000, and other estimates based on methionine content also suggested that the range was between 38800 and 43000. Purified flagellins of these three species, and also P. syringae pv. morsprunorum and Pseudomonas sp. (‘Vibrio percolans’), were digested with trypsin, and the peptide maps obtained after two-dimensional electrophoresis were compared. Altogether 65 peptide spots were delineated and 15 peptides were found to be common to all five species. The amino acid composition of the purified flagellins of the three type species was found to accord qualitatively and quantitatively with other prokaryote flagellins. Antisera were raised against the three purified flagellins, and tested by the Ouchterlony diffusion plate method against (a) purified homologous and heterologous flagellins, (b) deflagellated cells with sheared flagella and (c) crude sheared flagella of a further 67 test strains and species (mainly Pseudomonas). With either flagella or flagellin antigens the serological reactions were virtually identical, but not all strains of a given species reacted with the relevant antiflagellin antiserum. Rapid species identification using a single antiflagellin antiserum was thus not possible. The flagellar location of the antigenic site(s) was confirmed by two serological methods, including direct immunofluorescent serology.

Proteolytic strains of Bacteroides amylophilus, Bacteroides ruminicola, Butyrivibrio fibrisolvens, Butyriribrio alactacidigens, Selenomonas ruminantium var. Ruminantium, Se. ruminantium var. lactilytica and of the genera Eubacterium, Fusobacterium and Clostridium were isolated from the rumen of sheep. The location of the proteolytic enzymens, their activity against various substrates and their sensitivity to inhibitors were compared with the same properties of mixed bacteria prepared from strained rumen fluid, in order to assess the relative importance of the different species in the hydrolysis of protein in the rumen. Bacteroides ruminicola was judged by these criteria to be the most important of these proteolytic isolates in vivo. Other isolates of higher specific activity had enzymes with properties different from rumen fluid. Species of Butyrivibrio, Selenomonas and Clostridium of lower specific activity may also be important if present in sufficiently large numbers, as their activity was of the same type as the mixed population.

Streptococcus bovis had a low proteolytic activity as determined by the conversion of 14C-labelled casein to TCA-soluble products, but was able to grow on casein, probably by virtue of its exceptionally high leucine aminopeptidase activity.

The generalized transducing phage Pf 16h2 has been used to confirm linkage relationships of chromosomal markers of Pseudomonas putida previously determined from their time-of-entry in Hfr crosses, and to map new auxotrophic mutations. By means of spot matings using Hfr donors of known origin of transfer, catabolic markers forming part of a closely linked group of operons referred to as a superoperonic cluster have been shown to be chromosomally located and their map positions determined. R-prime-mediated interspecific complementation has been used to equate functionally 21 auxotrophic loci in P. putida and P. aeruginosa, and the distribution of these loci on the two genetic maps has been compared. While both maps reveal that auxotrophic markers are largely restricted to about 40% of the chromosome and that auxotrophic markers of similar phenotype are not clustered, there is evidence of at least seven chromosomal rearrangements since divergence from a presumed common ancestor.

The Pseudomonas gene coding for carboxypeptidase G2 was introduced into Saccharomyces cereυisiae on an Escherichia coli/ yeast shuttle vector pROG5. The level of enzyme activity obtained was independent of the orientation of the gene within the pBR322-derived tetracycline resistance gene of the vector, indicating that expression can occur from a Pseudomonas promoter in yeast.

The transport of the neutral amino acid l-glutamine by Neurospora crassa occurred by means of two permeases : the neutral-specific permease and the general permease. For both transport systems, accumulation of l-glutamine was a saturable process that occurred against a concentration gradient and was dependent upon metabolic energy, suggesting that accumulation is a carrier-mediated active transport process. The transported glutamine was incorporated into cellular protein. The kinetic values K m and V max were determined for glutamine transport by both systems. The glutamine analogues methionine sulphoximine and γ-glutamyl hydroxamate appear to be transported by the same permeases that transport glutamine.

In addition to determining the physiological properties of glutamine transport, we examined whether the pmn;pmb;pmg;nit-2 strain could transport this amino acid. This strain is defective for constitutive amino acid transport and for the ability to utilize amino acids as sole nitrogen sources, with the exception of glutamine. No glutamine transport was detected, suggesting that glutamine utilization by this strain is not due to its ability to transport this amino acid.

In vitro growth conditions of Bordetella bronchiseptica led to an enrichment of phase variants. The frequency of phase variation was about 10‒6 per cell per generation. Therefore phase variation in Bordetella may result from a random mutation in a controlling region, followed by selection.

We have attempted to undertake genetic analysis in Bacillus megaterium using the technique of protoplast fusion that has been successfully applied in Staphylococcus and Streptomyces. Efficient production of protoplasts, fusion and regeneration techniques have been established. However, variability in numbers and types of recombinants using two-, three-, and four-factor crosses was observed throughout these studies. No linkages were detected, even between loci known to be linked by cotransduction with bacteriophage MP 13. These results were similar to those reported by Alföldi and coworkers using B. megaterium strain 216, even though the experimental design was significantly changed. During initial subculturing, segregants were observed in a 1 : 2 : 2 ratio of noncomplementing diploids : parental-l:parental-2. The ratio changed dramatically after seven subcultures. Double recombinants appeared after nine subcultures. These results corroborate those reported in B. subtilis and suggest that there is a locus-inactivation phenomenon present in Bacillus which is not evident in Streptomyces or Staphylococcus. Until the mechanism is elucidated, protoplast fusion should not be used for chromosomal mapping in B. megaterium. However, it can be used to transfer plasmids among the bacilli at a frequency of 10‒5‒10‒6 per regenerated protoplast.

Examination of a series of isolates of Providencia stuartii collected over an 18 month period from a chronic-care patient at Bristol Royal Infirmary revealed the emergence of resistance to carbenicillin. Resistence was mediated by a 47 kb plasmid which transferred by conjugation to a plasmid-free strain of P. stuartii but not to Escherichia coli. Carbenicillin-sensitive isolates were either plasmid free or contained a 36 kb cryptic plasmid. Restriction endonuclease mapping of this plasmid showed it to be closely related to 32 kb and 34 kb cryptic plasmids reported previously in P. stuartii from Bristol. Mapping of the R plasmid showed it to be derived from the 34 kb cryptic plasmid by transposition of two copies of Tn1.

Two independently-isolated thymine-requiring mutant strains of Escherichia coli were found to possess an unusual phenotype: in the presence of CO2, growth was independent of thymine. The two strains showed different profiles of temperature sensitivity. Glycine, serine and methionine were unable to relieve the thymine-dependence. Both strains were susceptible to trimethoprim under conditions where they were thymine-independent. The results are consistent with the occurrence of partial defects in thymidylate synthase.

The copper resistance in Escherichia coli determined by plasmid pRJ1004 is inducible. The level of resistance is proportional to the inducing dose of copper. The level of copper resistance in induced and uninduced cells changes with the growth phase of the culture. Induced resistant cells accumulate less copper than uninduced cells, so that reduced accumulation may be the mechanism of resistance. We propose that the inducible plasmid-coded copper resistance interacts with the normal metabolism of the cell to protect against toxic levels of copper while allowing continued operation of copper-dependent functions.

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.

Three site-specific endonucleases, AflI, AflII and AflIII, have been partially purified from the cyanobacterium Anabaena flos-aquae CCAP 1403/13f. Their recognition and cleavage specificities have been determined to be:

AflII and AflIII are new specificities and may be useful in molecular cloning, as well as in the analysis of DNA. The distribution of type II restriction endonucleases in the cyanobacteria is briefly discussed.

We have determined the changes in DNA sequence corresponding to three mutations in the promoter-proximal open reading frame of spoIIA, a locus that regulates sporulation in Bacillus subtilis. All three mutations prevent the synthesis of two sporulation-associated enzymes, but they differ in their effects on spore incidence. We now find that mutation spo-42, which allows spores to be produced at a low incidence, is a transition that changes Gly95 to Asp in the protein encoded by the open reading frame. Mutation spo-69, which blocks sporulation entirely, consists of two transitions: these change Gly62 to Asp and Ala116 to Thr. Mutation sas-1, which partially suppresses spo-69, is also a transition : this changes residue 62 (which had become Asp as a result of the spo-69 mutation) to Asn.

Legionella pneumophila attached to, penetrated and replicated within the three eukaryotic cell lines, MRC-5, HEp-2 and Vero. Multiplication occurred rapidly in these cells for an initial 48 h after inoculation and declined thereafter. Infected MRC-5 cell monolayers developed lytic-type cytopathic changes, with organisms being readily released. HEp-2 cells showed a more chronic infection, with slowly developing granular changes in the monolayers, and slow release of intracellular bacteria. In Vero cells, organisms were released rapidly along with a more progressively developing granular cytopathic effect in the monolayers. L. pneumophila was unable to grow in cell-free culture fluids. Uptake and intracellular development was similar for each cell type, and was initiated by ‘bacteriopexis’, a process in which the organisms bound via receptors and were surrounded by cellular microvilli which eventually fused, leading to bacterial engulfment. Replication of organisms in vacuoles within the cytoplasm of infected cells was confirmed by thorium labelling. These vacuoles were lined with ribosomes and, at the early stages of intracellular development, were found in close proximity to mitochondria, cytoplasmic filaments and banded enclosures. Ruthenium red staining showed that acid mucopolysaccharide capsular material was not present on these organisms during the attachment process or intracellular phase. Organism release was by lysis of the infected cells.

Corynebacterium ulcerans strain 378 produces a bacteriocin (ulceracin 378) and a toxin when grown on semi-solid medium. Ulceracin 378 was purified 360-fold by dialysis and chromatography on DEAE-cellulose and Sephadex G-200. On the basis of Ultrogel AcA22 gel filtration its molecular weight was about 900000. It could be dissociated by 2-mercaptoethanol and sodium dodecyl sulphate into smaller subunits of 25000. The bactericidal activity was associated with this subunit which contained no carbohydrate or lipid. Ulceracin 378 was thermostable and stable over a wide pH range. Purified ulceracin 378 did not have a toxic effect (lethal) on guinea-pigs and rabbits and was immunologically distinct from the toxin.

The reaction components and conditions affecting CAMP factor (Streptococcus agalactiae) induced lysis of target cells have been investigated. Both the amount of sphingomyelinase used and the time of preincubation with sphingomyelinase directly affected the rate of haemolysis by CAMP factor. The CAMP factor induced lysis was temperature dependent between 15 and 30 °C and was proportional to the amount of CAMP factor added.

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production.

Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively K m = 14 mm and 46 mm, V max = 208 nkat g–1 and 25 nkat g–1. The optimum temperature and pH for production were 30 °C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.