- Volume 133, Issue 3, 1987

Flavobacterium F24 metabolized both stereoisomers of phenylglycine and enzyme studies revealed that l-phenylglycine was transaminated by a constitutive enzyme while the d-stereoisomer was oxidized by a phenazine-methosulphate-dependent d-amino-acid dehydrogenase. This latter enzyme was not induced during growth on l-phenylglycine. Phenylglyoxylate formed in the reactions was decarboxylated by an inducible enzyme to benzaldehyde, which was oxidized mainly by an inducible phenazine-methosulphate-dependent benzaldehyde dehydrogenase not described earlier. Benzoate was further metabolized via 3-hydroxybenzoate to gentisate, which in turn was further degraded through a glutathione-dependent pathway.

Two 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolases have been purified to homogeneity from Alcaligenes eutrophus ATCC 17707. One, HODHII, is encoded by the TOL-like plasmid, pRA1000, and the other, HODHI, is chromosomally-encoded. HODHI and HODHII have an M r of 104 × 103 and 116 × 103, respectively, and both are dissociated by SDS into three subunits of equal size which are not linked by disulphide bonds. Both hydrolases are active against a similar range of substrates formed by the action of catechol 2,3-oxygenases, generally being more active against substrates formed from 3-alkyl- rather than 4-alkyl-substituted catechols. HODHI and HODHII had similar K m values for HOD, but HODHI had a significantly lower K m value for, and a higher activity against, 2-hydoxymuconic semialdehyde than HODHII. It was shown by immunodiffusion studies that the two hydrolases were immunologically distinct and that HODHI had no common antigenic determinants with HOD hydrolases from the Pseudomonas putida strains NCIB 9865, NCIB 10015 and mt-2; HODHII gave a line of identity with antiserum to the hydrolase from P. putida NCIB 9865. A comparison of the physico-chemical properties of HODHI and HODHII with the same properties of the HOD hydrolases from the three P. putida strains demonstrated several similarities between the five HOD hydrolases which have been purified.

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage ø105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated ø105J23, ø105J24, ø105J27 and ø105J28, show frequencies of plaque formation that are equal to those of wild-type ø105. This represents at least a 10-fold improvement over ø105J9, the vector used in previous cloning experiments. Two of the new vectors ø105J27 and ø105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.

SUMMARY: Bacteriophage cloning vector ɸ105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis. Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+. Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci. Included are nine loci (spo0D, spo0J, spoIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously. Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.

SUMMARY: Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by l-cysteine. A mutant was isolated which excretes l-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and l-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an M r of 34000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase.

The EnzymeIIbg1 of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of β-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3·6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in β-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66·48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0·62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2·3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the λpL promoter and then transformed into a host strain which produces thermolabile c1857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.

A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the ‘common antigen’ (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

A Pseudomonas aeruginosa transposon insertion mutant with defective release of several exoenzymes has been characterized. The Tn5-751 insertion mutation was located in the previously described xcp-1 locus at 0 min on the chromosomal map and caused several exoenzymes to remain in cell-bound form. At least one of the exoenzymes, elastase, was accumulated in the periplasmic space. The periplasmic elastase had the same M r as the extracellular enzyme produced by the wild-type strain. The virulence of the mutant was comparable to that of wild-type strains in experimental burn infection in mice. The presence of an easily selectable antibiotic resistance marker in the xcp-1 locus offers the possibility of cloning the gene(s) involved in exoprotein secretion.

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3·5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.

Streptomyces coelicolor A3(2) and Streptomyces lividans 66 strains were shown to be sensitive to the galactose analogue 2-deoxy-d-galactose. Spontaneous resistant mutants were isolated that were Gal- and lacked the enzyme galactokinase. The galK gene (structural gene for galactokinase) from S. coelicolor was cloned into S. lividans using the low copy number vector pIJ922. The resulting plasmid (pMT650), which contained a 14 kb insert, complemented gal mutations in both species. The presence of the galK gene on a 28 kb EcoRI fragment was confirmed by expressing it in Escherichia coli where it complemented a well characterized galK mutation.

Members of the species Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due, in part, to the secretion of pectolytic enzymes. We studied the secretion of pectate lyases by using Mu-lac insertion mutagenesis in E. chrysanthemi strain 3937. Analysis of β-galactosidase expression of the out—lac fusions, in different growth conditions, showed that the expression of the out gene is constitutive. Compartmentation of pectate lyases during growth suggests that these enzymes are first exported to the periplasm with formation of an intracellular pool of active pectate lyases and then released into the extracellular medium. In the out mutants, pectate lyases are retained in the periplasm. Analysis by electrofocusing showed that the pectate lyases are not modified during the transfer via the outer membrane. A 65 kDa protein is absent from the periplasm of the out mutants; this protein could have a role in the secretory system of E. chrysanthemi.

The membrane-bound ribosome protein (MBRP)-complex of Staphylococcus aureus was studied using antibodies to its individual components. The four polypeptides of the complex were firmly held together, and none were present in large excess. The membrane-bound fraction of the MBRP-complex was accessible to trypsin only after removal of the membrane-bound ribosomes; it also remained associated with the membrane-bound ribosomes even after solubilization of the membranes with Triton X-100. Furthermore, the amount of MBRP-complex in the membrane was proportional to the rate of exoprotein synthesis. These results strongly suggest a role for the MBRP-complex in protein secretion.

A series of genomic DNA fragments that encode gonococcal pilins from four well-characterized pilus variants of Neisseria gonorrhoeae strain P9 have been cloned in Escherichia coli K12. At least nine classes of cloned P9 pilin genes have been identified on the basis of restriction mapping of cloned pilin-encoding DNA and physical and immunological analysis of expressed pilin proteins. Each antigenic variant of strain P9 possesses many genomic segments of pilin gene information, although our results suggest that strain P9 contains only a single pilin-expressing (pilE) locus.

Candida albicans produces extracellular polymeric material (EP) which contains a mannoprotein adhesin. EP isolated from culture supernatants of C. albicans GDH 2346 consisted of a mixture of glycoprotein components and inhibited yeast adhesion to buccal epithelial cells by up to 60 %. Partial purification of the adhesin was achieved by a two-step procedure involving chromatography of EP on concanavalin A-Sepharose and DEAE-cellulose. The purified adhesin inhibited adhesion to buccal cells 30 times more efficiently (on a weight basis) than unfractionated EP. Pretreatment of EP with heat, dithiothreitol or proteolytic enzymes either partially or completely destroyed its ability to inhibit adhesion, whereas pretreatment with sodium periodate or α-mannosidase had little or no effect. These results suggest that the protein portion of the mannoprotein adhesin is more important than the carbohydrate moiety in mediating yeast attachment to buccal epithelial cells.

The effect of various lectins and sugars on adhesion of five strains of Candida albicans to buccal and vaginal epithelial cells in vitro was investigated. Adhesion of C. albicans GDH 2346 was inhibited primarily by l-fucose and winged-pea lectin, whereas adhesion of strain GDH 2023 was inhibited by N-acetyl-d-glucosamine, or d-glucosamine, and wheat-germ agglutinin. Three other strains of C. albicans (MRL 3153, GRI 681 and GRI 682) gave results similar to those obtained with strain GDH 2346. Extracellular polymeric material (EP) isolated from strain GDH 2346 inhibited adhesion of strains MRL 3153, GRI 681 and GRI 682 by more than 50%, but that of strain GDH 2023 by only 30%. EP from strain GDH 2023 had little or no effect on the adhesion of any other yeast strain. Lectin-like proteins with affinities for l-fucose, N-acetyl-d-glucosamine and d-mannose were detected in EP from all five strains in different amounts. These results indicate that there are at least two types of adhesion mechanism and that glycosides containing l-fucose or N-acetyl-d-glucosamine can function as epithelial cell receptors for C. albicans.

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 107 phase I C. burnetii.