Journal of General Microbiology

Online ISSN: 0022-1287
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Article
SUMMARY The nutritional requirements of the Gram-negative Bacterium NCIB 8250 (formerly referred to as 'Vibrio 01 ') have been determined. Growth was tested on almost 450 compounds, of which over 100 served as carbon + energy sources and almost 50 as nitrogen sources. The effects of various factors such as pH value, temperature and aeration on the growth rate and cell yield of organism have also been measured.
 
Article
Two chemically different O-polysaccharides, a low molecular mass form of LPS and core LPS produced by chemostat-grown E. coli O157, were analysed by SDS-PAGE, silver staining and immunoblotting. The reactivities of the different O-polysaccharides with antisera prepared against E. coli O157 grown in batch culture, Salmonella O30 or Brucella abortus were very similar, showing that the O-polysaccharides share at least some antigenic determinants. The reactions of the low molecular mass LPS with the antisera indicated it was semi-rough LPS having one repeat unit of the O-polysaccharide attached to core LPS.
 
Article
The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.
 
Article
A description is given of the biological properties of two substances produced extracellularly by a strain of Escherichia coli 078 K 80, grown in aerated liquid culture. One of the materials was of high molecular weight and possessed the properties of endotoxin extracted from whole organisms by chemical methods. In mice this material was toxic, induced non-specific resistance to infection with Salmonella typhi, necrotized Sacroma 180, and elicited the local and general Shwartzman reactions. In rabbits it was strongly pyrogenic, dermonecrotic, and induced formation of precipitating antibodies. Heat, chemicals and enzymes did not affect its toxicity, but acid hydrolysis rendered it non-toxic. We have called this substance ‘free endotoxin’. The other material was of lower molecular weight and apart from inducing nonspecific resistance to infection with S. typhi in mice, it had none of the properties of the endotoxin.
 
Article
Bacteriophage Ω8 is propagated in coli E56b (08:K27 -:H -), a non capsulated strain. Another non capsulated strain, E. coli 2398 (08:K? -:H -), is killed by bacteriophage Ω8 without phage propagation. This strain was formerly believed to be E. coli 093:K? -:H -, cross reacting with strain E56b. The authors established the chemical and serological identity of the 08-specific lipopolysaccharides of the two strains. The 08-specific lipopolysaccharides of both strains inhibited the infection of E. coli E56b with bacteriophage Ω8 equally well. The adsorption rate constants of Ω8 were identical for the two strains of E.coli 08. Evidence was obtained with 32P-labelled bacteriophage Ω8 for penetration of viral DNA into both bacterial strains. In host strain E56b, phage particle synthesis occurred normally. In strain 2398 the viral DNA was not degraded but its expression was blocked. The killing effect of Ω8 on E. coli strain 2398 is supposed to be due to damage of the cytoplasmic membrane, which could not be reversed under the influence of viral information. This was indicated by a blockage of cellular respiration, β-galactoside transport and RNA as well as protein synthesis.
 
Article
His+ hybrids from a cross between a Salmonella typhimurium donor and an Escherichia coli O8 recipient expressed E. coli O8 specificity and in addition Salmonella O4,12-specificity. This indicated that the recipients had received the his-linked donor rfb cluster determining the synthesis of S. typhimurium O-specific repeat units and that the rfb genes of both mating partners are functional in these hybrids. Chemical analyses showed that the hybrids contained an E. coli O8 lipopolysaccharide (O antigen) and a S. typhimurium specific lipopolysaccharide with only one O-specific repeat unit (SR antigen). O8-negative mutants selected from the O8-positive hybrids retained the Salmonella O-specificity and represent semi-rough (SR) forms, because the rfc gene(s) determining the polymerization of repeat units has not been transferred. Attempts to introduce the S. typhimurium rfc locus into E. coli O8 remained unsuccessful. Crosses between a S. typhi donor and E. coli O8 gave rise to smooth (S) and SR His+ recombinants exhibiting only S. typhi O-specificity. The smooth recombinants are assumed to have obtained the his-linked rfb cluster and in addition the rfc gene(s) of the donor. The exchange of the rfb region of such smooth recombinants by that of a S. typhimurium donor led to smooth hybrids with O4,(5), 12-specificity. The phenotypically smooth recombinants exhibited concomitantly S- and SR-lipopolysaccharides of S. typhi and S. typhimurium O-specificity, respectively.
 
Article
The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.
 
Article
The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.
 
Article
The cervical microbial flora of 25 females and stock cultures of various micro-organisms which may be present in the human female cervix were examined using a fluorimetric assay for 1,2-propanediol oxidoreductase. Results indicated that only members of the genera Neisseria and Acinetobacter possess appreciable activities of the enzyme, whose physiological function is not yet known. The activity of this enzyme in N. gonorrhoeae appeared to be significantly higher than the activities observed in mot of the other Neisseria species and in the Acinetobacter species. These results indicated that it may be possible to utilize this enzyme as a presumptive diagnostic marker for N. gonorrhoeae in cervical secretions. 1,2-Propanediol oxidoreductase may also be of taxonomic significance for the classification of various bacterial species.
 
Article
An enzyme which oxidizes 1,2-propanediol in the presence of NAD+ has been purified from lysates of Neisseria gonorrhoeae. The enzyme was activated by monovalent cations, had a pH optimum between 9 and 10, and showed a substrate specificity unlike any known alcohol or glycerol dehydrogenase. The enzyme had an apparent Km of 17 mM for 1,2-propanediol and 0 . 37 mM for NAD+. When chromatographed on a Sephadex G-150 column, the enzyme eluted as a single peak in the molecular weight region of a bovine serum albumin marker. An antibody to the purified enzyme was prepared in goats. When antiserum was reacted with the enzyme in immunodiffusion experiments, a single precipitin band was detected. When the enzyme was mixed with an excess of antibody and then reacted with substrate, enzyme activity was completely inhibited.
 
Article
Escherichia coli K12 growing anaerobically on L-fucose excretes L-1,2-propanediol as a fermentation product whose formation is catalysed by an inducible NAD-linked oxidoreductase. The activity of this enzyme is highly induced only during anaerobic growth. Three bacterial strains bearing a hybrid operon with the structural genes for lactose utilization (lacZYA) fused to the promoter of the propanediol oxidoreductase gene (fucO) were constructed to test whether or not transcriptional control was involved. In contrast to propanediol oxidoreductase of wild-type cells, beta-galactosidase in the phi(fuc-lac) strains was induced by fucose to high levels both aerobically and anaerobically. Data from this work are in accord with the previous report that the enzyme protein (assayed by specific antibodies) was induced both aerobically and anaerobically, but that only in anaerobically grown cells was the oxidoreductase catalytically active. In the present study, we found that the oxidoreductase induced anaerobically in wild-type cells remained enzymically active during aerobic growth in the absence of fucose. On the other hand, wild-type cells grown aerobically in the presence of fucose and then allowed limited anaerobic growth on glucose did not gain any oxidoreductase activity. The mechanism of this post-transcriptional control remains to be discovered.
 
Article
The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu::lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his::MudA and pdu::MudA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map.
 
Article
Escherichia coli K12 cannot grow on D-arabitol, L-arabitol, ribitol or xylitol (Reiner, 1975). Using a mutant of E. coli K12 (strain 3; Sridhara et al., 1969) that can grow on L-1,2-propanediol, a second-stage mutant was isolated which can utilize D-arabitol as sole source of carbon and energy for growth. D-Arabitol is probably transported into the bacteria by the same system as that used for the transport of L-1,2-propanediol. The second-stage mutant constitutively synthesizes a new dehydrogenase, which is not present in the parent strain 3. This enzyme, whose native substrate may be D-galactose, apparently dehydrogenates D-arabitol to D-xylulose, and its structural gene is located at 68.5 +/- 1 min on the E. coli genetic map. D-Xylulose is subsequently catabolized by the enzymes of the D-xylose metabolic pathway.
 
Article
Yeast cells of Candida albicans 1001 produced glucan-hydrolysing activity, most of which was due to an exo-1,3-beta-glucanase. The enzyme was periplasmically located; it could be found in culture medium samples, and was secreted by protoplasts when cultured under regeneration conditions. In contrast to most yeast exoglucanases, this enzyme was practically inactive against p-nitrophenyl-beta-D-glucoside, hydrolysis of this substrate being carried out by a beta-glucosidase located inside the cytoplasmic membrane and not secreted to the external medium. Supernatant fluids from cell-free extracts reached their maximum glucanase level after several days at 0 degrees C, suggesting that the active enzyme was formed from an inactive precursor. Glucanase activity substantially decreased and sometimes disappeared from the cells when the yeast-to-mycelium transition was induced, but a significant (though lesser) reduction was also observed in yeast cells incubated in the same medium under conditions (temperature, cell concentration) that did not lead to formation of hyphae. It is suggested that C. albicans exo-1,3-beta-glucanase may not be necessary for mycelial growth.
 
Article
Conditions for obtaining stable protoplasts from Sclerotium glucanicum and their reversion to hyphal growth were determined. 1,3-beta-Glucan synthase activity was detected in particulate enzyme fractions from mycelium and protoplasts of Scl. glucanicum. UDP-[U-14C]glucose was linearly incorporated into a beta-glucan for about 20 min at 25 degrees C. Optimum pH and temperature values, as well as thermal stabilities of the 1,3-beta-glucan synthase activity, were determined. High concentrations of EDTA were inhibitory. Enzyme activity was stimulated by ATP and GTP. The apparent Km value for UDP-glucose was 0.54 mM. The reaction product was characterized as 1,3-beta-glucan by 13C NMR spectroscopy and hydrolysis products of an exo-1,3-beta-glucanase.
 
Amino acid composition of the purijied exo-1,3-fi-D-glucanase from S . cerevisiae 
Two-dimensional immunoelectrophoresis of the exo-l,3-fl-D-glUCanaSe purified from S. cuecisiue. Enzyme activity on 4-methyl-umbellipheryl-fl-D-glucoside (a, c) and protein staining (b, d ) ; (u, b) and (c, d ) correspond to different electrophoretic runs with 1.5 and 1.1 pg of antigen, respectively. 
Article
Addition of tunicamycin to the culture medium of growing Saccharomyces cerevisiae protoplasts or cells resulted in the formation of a modified exo-1,3-beta-D-glucanase which was detectable in both extracellular and intracellular fractions. This modified enzyme had a lower molecular weight than the native form and did not bind to concanavalin A. The activation energy and Km values of both enzyme forms were identical. Antibodies raised against the native protein readily precipitated the exo-1,3-beta-D-glucanase produced after tunicamycin treatment. The latter enzyme was comparable, in terms of molecular size and lack of affinity for concanavalin A, to the beta-D-glucanase obtained by treatment of the native form with endoglycosidase H; both lacked the carbohydrate moiety present in the native enzyme. The exo-1,3-beta-D-glucanase obtained in the presence of the antibiotic was more sensitive to variations in temperature and pH than both endoglycosidase H-treated and non-treated enzymes. Our results suggest that the carbohydrate moiety, if not necessary for exo-1,3-beta-D-glucanase secretion, may play a role in the conformation of the protein and in stabilizing the enzymic activity.
 
Article
Nine mutants of Aspergillus nidulans differing in cleistothecia and/or conidia production were investigated. The strains were grown on four media with different levels of glucose (0.8, 3, and 4%) and nitrate (0.6 and 0.15%). The mycelia were analyzed on the basis of the dry weight of the total mycelium and the alkali soluble fraction (containing the α 1,3 glucan) as well as enzyme activities lytic to α 1,3 glucan, laminarin, and starch. Low quantities of α 1,3 glucan present on the third day are correlated with low α 1,3 glucanase activity and absence of cleistothecia formation on the sixth day. α 1,3 Glucan and cleistothecium formation seemed to be inversely related to conidiation.
 
SDS-PAGE of exoglucanase. A Coomassie-Blue-stained gel of 1, purified exoglucanase (5 pg) ; 2, bovine serum albumin, ovalbumin and carbonic anhydrase and 3, purified secreted aspartate proteinase of C. albicans (5 pg). 
Exoglucanase secretion by C. albicans strains and other yeasts. YPD cultures were incubated for 48 h at 30 "C (entering stationary phase, OD,, = 30, after 20 h) and 0.8 ml samples of the culture media were analysed in a Western blot with the exoglucanase antiserum (see Methods). Exoglucanase activity in the culture media was measured with laminaran and is shown with each lane in milliunits m1-l. 
(a) Restriction map of the insert of pXG29. H, HindIII; B, BumHI, Bg, BgBI; X, XbaI; P, PstI; and A, AvaI. The following restriction endonucleases did not cut; SrnaI, SacI, EcoRI, EcoRV and YpnI. (b) Sequencing strategy for the exoglucanase gene. Arrows indicate the directions and the extent of the DNA sequenced in various fragments of the random library generated from the 2.3 kb BglII-XbaI fragment of pXG29. The sequences were compiled into a contiguous sequence using the program VTUTM. Restriction sites of endonucleases used in mapping and the position of the ORF (box) are indicated. 
Expression of the exoglucanase gene. (a) A YPD culture was grown at 30 "C and 0.8 ml samples of the culture media were taken at 0, 1,2,4, 10,24 and 48 h. Growth at these times (OD,,) is given above. The samples were analysed on a Western blot using the exoglucanase antiserum (see Methods). (b) Total RNA was prepared from cells of a YPD culture harvested at various stages of growth. The RNA samples were analysed on a Northern blot using the 2.5 kb BglII-PstI fragment of pXG29 as a probe (see Methods). Each lane contains approximately the same amount of total RNA and the stringency conditions were 65 "C with 0.1 x SSC, 0.5% SDS. 
Article
A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.
 
Article
Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.
 
Article
An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.
 
Fig. 2 Fig. 3 Fig. 1. PAGE of purified exo-l,3-&glucanase (after the gel filtration step: see Table 1) (a) stained with 
PAGE analysis and MUG-hydrolysing activity of exo-l,3-glucanase in different cellular fractions. (a) Purified preparation (after gel filtration : see Table 1); (b) protoplast lysates; (c) cell-free extracts; (d) culture fluids; (e) partially purified C. albicam /3-glucosidase. 
Article
An exo-1,3-beta-glucanase was purified from blastoconidia of Candida albicans 1001. The purified enzyme appeared as a single protein band by PAGE, and split into two subunits (Mr approximately 63,000 and 44,000) when analysed by SDS-PAGE. The pI of the enzyme was 4 and a Km of 1.7 mg ml-1 was estimated for laminarin as substrate. Despite its very reduced activity on the synthetic substrate p-nitrophenyl beta-D-glucoside, C. albicans exo-1,3-beta-glucanase hydrolysed 1,3-beta-glucan by an exo-splitting mechanism and was inhibited by glucono-delta-lactone and by Hg2+ and Ag+ cations. The active exo-glucanase was mainly located in the periplasm, but it was also present inside the cytoplasmic membrane in small amounts and was secreted into the culture medium. The electrophoretic mobility of the enzyme from all three locations was the same.
 
Article
EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location. beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.
 
Article
Two genes encoding distinct 1,3-beta-glucanases have been cloned from Bacillus circulans and expressed in Escherichia coli. A cosmid library of B. circulans WL-12 DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in E. coli for clones which exhibited 1,3-beta-glucanase activity. Two 1,3-beta-glucanase-positive clones were identified which contained genes encoding two independent 1,3-beta-glucanases as shown by biochemical, physical and molecular analyses. The cosmids, designated pMON5401 (27.1 kb insert) and pMON5402 (24.7 kb insert), encoded 68 kDa and 40 kDa 1,3-beta-glucanases, respectively. Both 1,3-beta-glucanases were purified from their respective E. coli strains, characterized biochemically, and were shown to exhibit lytic activity against purified yeast cell wall preparations.
 
Effect of various chemicals on the activity of A. furnigatus glucan synthase. Activity is expressed as percentage incorporation of ['4C]glucose into 1,3-P-glucans. Control in RM displaying 100 % activity [27 nmol min-' (mg protein)-']. 
Coomassie blue-stained 10 Yo gel (lane 1) and its corresponding autoradiographs (lanes 2 4 ) of the P30 fraction (40pg protein per lane). Lanes 2 and 3, photolabelling using 5-N,-[32P]UDP-Glc. Lane 4: photolabelling using 5-'251ASA-UDP-Glc. The reaction mixture not illuminated with UV light is shown in lane 2. 
Inhibition of photolabelling with 5-N,-[32P]UDP-Glc (a) and 5-[1251]ASA-UDP-Glc (b). P30 fraction ( 1 7-h-old mycelium) was photolabelled in the presence of increasing UDP-Glc concentrations (0 mM, lanes a l , bl ; 6 mM; lane a2; 12 mM, lanes a3,62), or in the presence (lanes a4, b l ) or absence of GTPyS (lanes a5 and b3). Lane a6, P30 fraction from a 5-d-old mycelium. 
Article
1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors--GTP, NaF, sucrose and EDTA--added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.
 
Article
SUMMARY Protoplast release from young mycelium of Schizophyllum commune by a lytic enzyme preparation from Trichoderma viride was accompanied by degradation of the three wall polymers, S-glucan, R-glucan and chitin. Part of the S-glucan was resistant. The resistance of S-glucan to enzymatic degradation increased with culture age and concomitantly the yield of protoplasts was reduced. Isolated S-glucan was also partly resistant to degradation, probably due to its crystallinity. In living cells S-glucan protected chitin and possibly R-glucan against degradation by external enzymes. S-glucanase, R-glucanase, chitinase and exo-laminarinase were purified from the Trichoderma enzyme mixture. Addition of only S-glucanase and chitinase was essential for protoplast release ; R-glucan was degraded endogenously.
 
Article
Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in Escherichia coli HB101 and a Lac- mutant thereof. Twenty-eight clones with carboxymethylcellulase (CMCase) activity were selected from two libraries by means of the Congo Red plate assay. Restriction enzyme analysis indicated that the CMCase+ clones contained a total of 13 unique DNA inserts. Hybridization of recombinant plasmids with chromosomal DNA confirmed the physical maps in all but one case and was further used to demonstrate the absence of homology between the HindIII restriction fragments of similar size which occurred in many of the clones. Without exception, CMCase+ E. coli clones expressed endoglucanase activity, but differed with respect to the amount and nature of the enzyme activity produced; additionally, some clones had exoglucanase activity which, in at least one case, was not attributable to the production of a second enzyme. For a few selected clones, the partially purified CMCase was analysed by electrophoresis. A temperature profile characteristic of a thermostable enzyme was demonstrated for the endoglucanase of one of the most active clones. Based on the evidence presented here, it is probable that the 13 unique DNA fragments described do not contain any of the C. thermocellum endoglucanase genes previously cloned.
 
Article
1,4-beta-D-Xylanase (1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) has been detected in both cell-free extracts and culture fluids of the yeast Cryptococcus albidus var. aerius grown on glucose as the only carbon source. Mild acid treatment of whole cells proved that the enzyme was extracellularly located. The activity remained almost completely linked to the wall after cell breakage, only being liberated in the presence of salt at high concentration. After release, the enzyme became very unstable and so has been characterized in situ in 'permeabilized' cells. The maximum production took place at the beginning of the exponential growth phase. The optimum pH and temperature for activity were 5.0 and 40 degrees C, respectively. The enzyme degraded xylan and xylo-oligosides by an endo-splitting mechanism giving xylobiose, xylotriose and xylose as the main end-products. Activation energy and kinetic constants for xylan degradation were determined. Several metal ions such as Ag+ and Hg2+ inhibited the enzyme. The possible function of this endo-xylanase in Cr. albidus var. aerius is discussed.
 
Article
A novel acid cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) was found in a culture of Bacillus sp. KSM-330 isolated from soil. One-step chromatography on a column of CM-Bio-Gel A yielded a homogeneous enzyme, as determined by silver staining of both sodium dodecyl sulphate (SDS) and nondenaturing gels. The enzyme had a molecular mass of 42 kDa, as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was higher than pH 10. The N-terminal amino acid sequence of the enzyme was Val-Ala-Lys-Glu-Met-Lys-Pro-Phe-Pro-Gln-Gln-Val-Asn-Tyr-Ser-Gly-Ile-Leu- Lys-Pro . This enzyme had an optimum pH for activity of 5.2, being active over an extremely narrow range of pH values, from 4.2 to 6.9; below and above these pH values no activity was detectable. The optimum temperature at pH 5.2 was around 45 degrees C. The enzyme efficiently hydrolysed carboxymethylcellulose (CMC) and lichenan, but more crystalline forms of cellulose, curdlan, laminarin, 4-nitrophenyl-beta-D-glucopyranoside and 4-nitrophenyl-beta-D-cellobioside were barely hydrolysed. The enzymic activity was inhibited by Hg2+ but was not affected by other inhibitors of thiol enzymes, such as 4-chloromercuribenzoate. N-ethylmaleimide and monoiodoacetate. N-Bromosuccinimide abolished the enzymic activity, and CMC protected the enzyme from inactivation by this tryptophan-specific oxidant. It is suggested that a tryptophan residue(s) is involved in the mechanism of action of the Bacillus cellulase and that the inhibition of enzymic activity by Hg2+ is ascribable to interactions with the tryptophan residue(s) rather than with thiol group(s).
 
Article
Hyphal development in Candida albicans was selectively blocked by the ornithine decarboxylase competitive inhibitor 1,4-diaminobutanone (DAB). Inhibition of hyphal development required DAB during both yeast inoculum growth and subsequent incubation at 37 degrees C to induce mycelial growth. This effect was not due to general growth inhibition since DAB did not inhibit yeast growth, and reduced protein synthesis by 30% at most. Moreover, protein synthesis was unaffected by DAB when cells were pre-grown in drug-containing media. Since DAB inhibited dimorphic transition at 37 degrees C, morphology- and temperature-dependent protein synthesis could be distinguished. DAB stimulated the synthesis of several yeast wall-proteins, irrespective of morphology or growth temperature, and two at 37 degrees C only, but it inhibited the synthesis of a single mycelial-specific glycoprotein species.
 
Article
Mutants deficient in branching enzyme activity (glgB) were isolated and classified into four groups. They accumulated different amounts of a linear polysaccharide similar to amylose. The polysaccharides were extracted and purified. Some of their properties were similar, but gel filtration on Agarose fractionated them into three peaks whose size seemed to be related to α amylase activity. Electron microscopy showed that amylose formed inclusions of varying density and small separated granules; the proportions of these types of aggregates varied from one mutant to another. Amylase deficiency and the glgC mutation (altered ADP glucose pyrophosphorylase) are important factors in amylose accumulation and aggregation.
 
Article
4,9-Dihydroxyphenazine-1,6-dicarboxylic acid dimethylester, the ester form of a proposed 'missing link' in the biosynthesis of phenazines, has been isolated from a strain of Pseudomonas cepacia.
 
Article
Alkaliphilic Bacillus species that grow at pH 10.5 must cope with a low protonmotive force (-50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mM buffered medium, while the actual medium pH remains above 10.2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10.4 and 10.6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7.5 or 10.5. Its molar growth yield (23 mg dry wt mmol-1) on malate (Ymal) was the same at the two pH values and was comparable to Ymal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10.5 also showed Ymal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7.5 (45.2 kJ mol-1) or pH 10.5 (46 kJ mol-1) was in a conventional range for bacteria.
 
SSS calibration : the recovery of a known amount of donor and recipient cells 
Article
A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. Using this 'surface slide system' (SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined. In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (= colony density) and comparable to those obtained in liquid culture. For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer was similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats. In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density. In contrast, liquid matings were virtually unaffected by initial cell density. The transfer advantage of the permanently depressed over the repressed plasmid was much less apparent for lower colony densities. I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.
 
Article
Exoprotease production by Pseudomonas aeruginosa ATCC 10145 was growth-associated when cultures were grown on complex substrates such as proteins but it occurred during the decelerating growth phase when the organism was grown on amino acids, mixtures of amino acids or simple carbon sources. NH4Cl and simple carbon sources caused repression. Exoprotease was produced in chemostat cultures in response to growth under any of the nutrient limitations studied (carbon, nitrogen or phosphate). Furthermore, by growing at rates less than approximately 0.1 h-1, the repression of enzyme production could be overcome to a large degree. At low growth rates there was an inverse relationship between growth rate and exoprotease production. Thus, exoprotease production was depressed by available energy sources and was increased in response to any nutrient limitation.
 
Article
Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.
 
Article
One hundred and fifty-three isolates of coagulase-negative staphylococci obtained from human skin failed to transfer resistance to either cadmium ions (46 isolates), trimethoprim (37 isolates), erythromycin (25 isolates) or tetracycline (45 isolates) to Staphylococcus aureus strain 1030 or to each of 10 of its lysogenic derivatives in mixed cultures. Thirty-three trimethoprim-resistant coagulase-negative staphylococcal isolates obtained from the human intestine also failed to transfer this resistance to the recipients in mixed cultures.
 
Article
The mechanism by which Lactobacillus fermentum strain 104-S adheres to porcine squamous epithelium was investigated by studying the adsorption to epithelial cells, and control surfaces, of radioactively labelled material released from the bacterial cells by water extraction. The released material was fractionated by gel filtration and the adsorption of pronase-sensitive and -resistant material in the various fractions to porcine gastric tissue and the control surfaces of polystyrene and immobilized bovine serum albumin (BSA) was determined. The fraction with affinity for the epithelium was characterized by enzymic degradation, periodate oxidation, lipid extraction, and protein and carbohydrate analyses. The adsorption pattern of radioactively labelled crude released material mimicked the adhesion of whole labelled cells to polystyrene and to gastric squamous tissue pieces. On fractionation, the pattern of adsorption to polystyrene and BSA was different from that obtained for the tissue pieces. Considerably less labelled pronase-stable material bound to surfaces of polystyrene and BSA, as compared with the tissue, suggesting that the pronase-resistant component has a tissue-specific affinity. After pronase treatment of the fraction of M(r) about 20,000 (20 K) containing labelled components with affinity for the epithelium, only saccharides were detected. Radioactivity was lost after hydrolysis with HCl, and therefore this pronase-resistant labelled component must be a saccharide. It is concluded that protein moieties in the extract have an affinity for several surfaces, including polystyrene, and that saccharide moieties have a specific affinity for the gastric squamous epithelium.
 
Article
High yields of staphylocoagulase from Staphylococcus aureus 104 were obtained in a simple salts medium supplement with glycerol, casein hydrolysate and three vitamins. Conditions of oxygen-limitation (dissolved oxygen concentration less than 2%), a pH of 7.4, a temperature of 35 degrees C and a 1 in 10 inoculum of overnight culture were required for optimal yields of staphylocoagulase.
 
Homology between R. meliloti car plasmids and E. coli car genes. Lane A contains 1 ng of a Hind111 + BstEII digest of pMC40 (Nyunoya & Lusty, 1983). The other lanes contain HindIII digests of 1 pg DNA from plasmids pTKllA (B), pTK11M (C), pTKllY (D), pTKl3C (E), pTK12N (F), pTKl2X (G), pTK12K (H), pTK12S (I) and pTK12L (J). The fragments were separated on a 1 % (w/v) agarose gel. (a) The carA probe was a 1.6 kb fragment from a Hind111 + BstEII digest of pMC40 that had been labelled by nick translation with [LX-~*P]~ATP. Hybridization was at 55 "C for 12 h in 3 x SSC. The filters were washed several times in 3 x SSC at room temperature and twice in 0.3 x SSC at 55 "C. The bound radioactivity was determined by autoradiography. The positions of car.4 HindIII fragments H 1H5 (Fig. 2) are indicated to the right of the autoradiogram. The probe fragment is indicated by an asterisk. The degree of contamination of this probe by the carB fragment can be estimated by the degree of hybridization to the band marked +. We attribute the hybridization in lanes E-J to the contaminating carB fragment. (b) The carB probe was a 4.2 kb fragment from a HindIII + BstEII digest of pMC40. The positions of carB HindIII fragments Hl-H8 (Fig. 3) are indicated to the right of the autoradiogram. The position of the probe fragment is indicated by an asterisk. The degree of contamination of this probe by the carA fragment can be estimated by the degree of hybridization to the band marked +. We attribute the hybridization in lanes B-D to the contaminating carA fragment. 
Article
We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.
 
Article
The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.
 
Article
The lys gene of Bacillus subtilis was inserted into prophage phi 105. The recombinant phage (phi 105dlys) contained DNA which was about 2 MDal smaller than the wild-type phage DNA, and the phage particles had no tails. The phage did not plaque but, when provided with tails in vitro, it transduced both lys-1 and lys-3 strains of B. subtilis to Lys$. The lys$ gene was located on a 2.5 MDal EcoRI restriction fragment. Subsequently this phage was phi 105 105dspoIIIB, was also defective, i.e. without tails. The DNA was 1.5 MDal smaller than the wild-type phage DNA and the spoIIIB2$ gene was located on a 3 MDal EcoRI fragment. When provided with tails in vitro, phage phi 105dspoIIIB transduced cells of a spoIIIB2 recipient to Spo$. In these transductants the spoIIIB2 mutation was complemented, and the cells sporulated normally.
 
Article
A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.
 
Article
A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 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.
 
Article
The cells of Actinomyces viscosus ATCC 19246 (Av19246) and Streptococcus sanguis ATCC 10557 (Ss10557) coaggregated immediately after mixing in 40 mM-Tris/HCl buffer. Optimal conditions were pH 7.5 in the presence of Ca2+ at 0.1 mM or higher. Na2 EDTA and its analogues, Na2MgEDTA and Na2MnEDTA at 7.5 mM inhibited the coaggregation. Trypsin and heat treatment impaired the reactive site on Av19246 cells, but not on Ss10557 cells. The coaggregates, once formed, dissociated gradually during extended incubation at 37 degrees C; this was prevented by addition of sufficient Ca2+. The disaggregation appears to be a spontaneous denaturation of the proteinaceous reactive site on Av19246 cell surface. Thus, the coaggregation involves the interaction of a lectin-like substance on the surface of Av19246 with a carbohydrate site on Ss10557. Native Ss10557 cell walls possessed reactivity with Av19246 cells but 5% (w/v) TCA-extracted cell wall residues did not. A carbohydrate moiety extracted from Ss10557 exhibited a high potency in blocking coaggregation, and coaggregates were dissociated upon addition of the carbohydrate. Lactose, galactose and N-acetyl-D-galactosamine (the latter two are major constituents of the antigen extract) also significantly inhibited the coaggregation, but the other antigen components, glucose and rhamnose, did not. Relative inhibitory activity, expressed as molar potency, of carbohydrate antigen, lactose, galactose and N-acetyl-D-galactosamine respectively, was approximately 26 X 10(3):16:4:1. Ss10557 cells and cell walls reacted only with a Ricinus communis (castor bean) agglutinin-120 but not with Glycine max (soybean) agglutinin, Arachis hypogaea (peanut) agglutinin or Phaseolus vulgaris agglutinin (phytohaemagglutinin).(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Bacteriophage cloning vector phi 105J27, 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, spoIIIA, 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.
 
Activities o f enzjwies of the pentose phosphate pathway in Candida 107 
Article
Enzymes of glycolysis, pentose phosphate pathway, gluconeogenesis, tricarboxylate acid cycle, glyoxylate by-pass and fatty-acid biosynthesis were assayed in extracts from Candida 107 grown continuously on glucose under carbon limitation, nitrogen limitation and on n-alkanes. The yeast was therefore either in a lipogenic or lipolytic state. Phosphofructokinase was absent under all conditions whereas enzymes of gluconeogenesis, including fructose 1,6-bisphosphatase and the pentose phosphate cycle, were all present. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were specific for NADP+ and were inhibited in a non-competitive manner by NADPH and NADH. Phosphoenolpyruvate, citrate, ATP and acetyl CoA had no inhibitory effects. Thus glucose metabolism appears to be by the pentose phosphate pathway which will rapidly produce NADPH. This can readily be consumed during fatty-acid biosynthesis and, as there appears to be no inhibition of the flow of carbon from glucose to acetyl CoA, fatty-acid synthesis can continue for as long as there is a supply of glucose. These results help to explain the probable causes of fat build-up to high concentrations (about 40% of the cell dry weight) in this and other organisms. In alkane-grown cells, lipogenesis is repressed and carbon is able to flow from the alkanes via acetyl CoA, oxaloacetate and pyruvate into pentoses and hexoses in a unidirectional manner, because of the strong repression of pyruvate kinase and the increased activities of phosphoenolpyruvate kinase and fructose 1,6-biosphosphatase under these conditions. Although there was little change in the total activity of the TCA cycle enzymes under the various growth conditions, isocitrate lyase was induced under lipolytic conditions.
 
Article
Growth of Candida 107 on n-alkanes (C10, C16 or a mixture) completely repressed formation of acetyl-CoA carboxylase and partially repressed the fatty acid synthetase complex. As all fatty acids must then be derived directly from the substrate no matter what its chain length, the yeast must be able to elongate even-chain acids (C10 to C14) and modify, by unknown reactions, odd-chain acids to give even-chain acids. Short-term control of fatty acid biosynthesis appears to be by long-chain (C16 or C18) fatty acyl-CoA esters feedback-inhibiting the activities of both acetyl-CoA carboxylase and fatty acid synthetase. n-Alkanes, n-alcohol, free fatty acids or C12 and C14 acyl-CoA esters, had little or no effects on these enzymes. Extracts from n-alkane-grown yeast inhibited the carboxylase in extracts from glucose-grown yeast, the pattern of inhibition being similar to that observed with hexadecyl-CoA.
 
Article
Phosphofructokinase was not detected in extracts of Candida 107 prepared in a variety of ways but was highly active in cells treated with toluene. Disruption of these cells destroyed activity of phosphofructokinase indicating that the enzyme is extremely labile. As patterns of labelling from [I-14C]glucose and [6-14C]glucose showed that 60% of glucose was metabolized via the pentose cycle, augmentation of this cycle is necessary to account for the high molar growth yields of this yeast. Phosphoketolases, reacting with xylulose 5-phosphate and fructose 6-phosphate, were found but the extent to which they contribute to glucose metabolism was not assessed.
 
Top-cited authors
Michael Herdman
  • Institut Pasteur International Network
Michael Goodfellow
  • Newcastle University
Rosmarie Rippka
  • Institut Pasteur
David E Minnikin
  • University of Birmingham
David Alan Hopwood
  • John Innes Centre