General Microbiology

Transfer of bacteriocin factors from bacteriocinogenic to other staphylococci was observed at high frequency on the basis of paradoxical inhibition (pi.) on two media (BTM and MPI). Colonies of non-bacteriocinogenic sensitive bacteria from the central growth of p.i. showed acquisition of bacteriocinogeny and bacteriocin resistance at high frequencies which was not lost in the course of 18 h. Acquired bacteriocinogeny was a stable character and had an overall frequency of I o -~ to I O -~ among different recipient populations. Conjugation, transduction and transfection processes could be excluded, and the gene transfer process was considered to be transformation with plasmid DNA. / I N T R O D U C T I O N A central region of growth has been observed within the zone of inhibition produced by bacteriocinogenic bacteria among Escherichia coli (Fredericq, I 948), Shigella sonnei (Abbott & Shannon, 1958), Pseudomonas aeruginosa (Wahba, I 963), Vibrio cholerae (Chakrabarty, Adhya, Basu & Dastidar, 1970) and among different members of Shigella, Salmonella, Escherichia, Proteus, Vibrio and Alcaligenes spp. (Chakrabarty & Dastidar, 1974). Chakrabarty, Adhya, Basu & Dastidar (1970) called the phenomenon paradoxical inhibition (pi.) and Chakrabarty & Dastidar (I 974) demonstrated that transfer of bacteriocinogeny from the primary to the secondary bacteria which constituted the central growth took place at high frequency through a process of plasmid-mediated genetic transformation. Bacteriocinogeny is also known among staphylococci (Parker, Tomlinson

Protozoa ciliates are the important components of the "microbial loop". Protozoa are unicellular, phagotrophic organisms. They are found in lake, river and pond. Ciliates and other protoctists are believed to play a remarkable metabolic role in freshwater systems. Ciliates are believed not only to route energy through their consumption of primary producers and subsequent ingestion by predaceous and detritivotrous metazoans but also as photosynthesizers via phytoflagellate symbionts. High rates of reproduction and growth make ciliates major organic matter transformations. Ciliates cover the major portion of living organisms in an aquatic ecosystem. Sunlight has found one of the vital factors which control the ciliates temporal distribution. Food has been one of the important factors which influence the distribution and abundance of a species. There are 106 species of ciliates belonging to 58 genera and 36 families are described from the freshwater ecosystems of India so far.

Intravenous inoculation of an attenuated agerminative strain of Candida albicans (PCA-2) of low virulence, but not of two other species of Candida of low virulence (C. parapsilosis and C. viswanathii) into CD2F 1 mice conferred protection against the highly virulent microbes C. albicans CA-6, Staphylococcus aureus and Aspergillus fumigatus. To provide protection, a definite inoculum size ( lo6 cells per mouse) resulting in organ colonization and establishment of a long-lasting chronic infection with PCA-2 was needed. An inoculum of lo5 cells gave rise to transient kidney colonization whereas inocula greater than lo6 cells led to acute septicaemia and eventual death. Chronic infection of mice following inoculation of lo6 PCA-2 cells was accompanied by detectable mannoprotein antigen levels in the serum (30-70 ng ml-I) while specific antibodies did not appear until 14 d after inoculation, at which time low antimannan antibody was present (ELISA titre 1 : 40-1 : 80). Chronic infection was characterized by the presence in the kidneys of 2-3 x lo6 c.f.u. of PCA-2 for at least 40 d after inoculation. Pharmacological modulation of the host through administration of either an anti-Candida drug, amphotericin B, or an immunosuppressive agent, cyclophosphamide, strongly supported the premise that the anti-infectious state conferred by PCA-2 'immunization' correlated with the maintenance of a sufficient number of PCA-2 in vim. Protection was 'switched on' when 2-3 x lo5 cells were present in the kidneys. It was maximal at a kidney count of 2-3 x lo6 c.f.u. of PCA-2, and promptly declined when the number of PCA-2 cells in the kidneys fell below 2 x lo5. Mice chronically infected with PCA-2 had splenic macrophages with pronounced candidacidal activity in vitro. Modulation of the growth of PCA-2 in vivo, which determined activation or deactivation of the protective state, was paralleled by a similar modulation in macrophage activation, showing that in all cases resistance to virulent organisms persisted as long as macrophage activation was present. The results demonstrate that a critical in vivo antigenic load is crucial for the occurrence of resistance to infection and suggests that macrophages could be involved in this protection.

In the presence of S-adenosyl-L-methionine, cell-free extracts of Streptomyces sp. C5, Streptomyces peucetius ATCC 29050, Streptomyces insignis ATCC 31913 and Streptomyces coeruleorubidus ATCC 31276 0-methylated carminomycin and 13-dihydrocarminomycin to daunomycin and 13-dihydrodaunomycin, respectively. With the corresponding aglycones, carminomycinone and 13-dihydrwarminomycinone, as substrates, no methylated products were detected. Other 4-hydroxyanthracyclines such as aklavin and aclacinomycin A, and 4-hydroxyanthracyclinones such as erhodomycinone and aklavinone, were not substrates for the 4-0-methyltransferase. These reaction specificities indicate that glycosylation of the anthracyclinone molecule must occur before 4-0-methylation, which means that 4-0-methylation of carminomycin is probably the terminal step in the biosynthesis of daunomycin, and that daunomycinone is not an intermediate in the pathway.

The classification of plant-pathogenic coryneform bacteria has been revised many times. All plant-pathogenic coryneform bacteria were once classified in the genus Corynebacterium. Currently, all are classified in the genera Arthrobacter, Cluuibacter, Curtobacterium or Rhodococcus. Difficulties still remain regarding the characterization of species and subspecies within these groups. Analysis of cellular fatty acids by high-resolution capillary gas chromatography has recently become a standard method of bacterial identification in many laboratories. In the studies reported here, cellular fatty acid analyses were performed to examine the plant-pathogenic coryneform bacteria as an autonomous group. A total of 189 bacterial strains were profiled. Results from these studies support the division of plant-pathogenic coryneforms into four major groups (genera). Further subdivision was not required for Rhodococcus and Arthrobacter, as each genus contains only one species of plant-pathogenic bacteria. Some, but not all, Curtobacterium pathovars could be differentiated from one another based on fatty acid analysis alone. Infrasubspecific classification of plant-pathogenic Curtobacterium organisms appears to be appropriate. Most of the species or subspecies belonging to the genus Cluuibacter could be differentiated using fatty acid profiles. Other chemotaxonomic methods also differentiate organisms within Cluuibacter. Reclassification is warranted within Cluuibacter michigunense. Cluuibacter michigunense subspecies should be returned to full species status. CsCA A

Fingerprinting by random amplification of polymorphic DNA (RAPD) was compared with existing molecular typing systems for Candida albicans. Fifteen isolates were chosen, including three from the same patient; these gave 14 distinct karyotypes by pulsed-field gel electrophoresis (PFGE) and 7 different DNA types by EcoRI-generated restriction fragment length polymorphisms (RFLPs). RAPD with primer I (5'GCTGGTGG3') gave 5 types, whereas primer II (5'GCGCACGG3') yielded 11 types. Combining the results from both primers, all isolates were unique by RAPD with the exception of the three from the same patient. RAPD provided a fast, economical and reproducible means of typing C. albicans with a level of discrimination approaching that of PFGE.

Lipoarabinomannan (LAM), a major lipoglycan of the mycobacterial cell envelope, was previously recognized as existing in two major forms: LAM with arabinofuranosyl (Arafl-containing termini (AraLAM) and a mannosecapped version (ManLAM) in which the majority of these termini are modified by additional mannose residues. Since ManLAM was first recognized in the virulent (Erdman) strain of Mycobacteriurn tuberculosis and the noncapped version in a rapidly growing, attenuated, H37Ra strain, it was thought that mannose capping may be a key factor in virulence. In the present study, LAM from M. bovis BCG was isolated and the non-reducing termini sequenced through differential 0-akylation, partial depolymerization and gas chromatography-mass spectrometric analyses of fragments. LAM from M. bovis BCG contains a short mannan backbone, highly branched arabinofuranosyl-containing side chains and several mannosyl residues capping the non-reducing termini of these side chains. Thus, LAM from M. bovis BCG is of the ManLAM type, showing no major structural differences at the non-reducing ends from the M. tuberculosis Erdman product. This observation led us to examine the earlier strain and to conclude that it showed little resemblance to conventional strains of M. tuberculosis. Thus, the absence of mannose caps may be more a feature of rapid growth than of avirulence. These results demonstrate that the relationship between mannose capping and disease induction is not a simple one. However, use of a panel of LAM-specific monoclonal antibodies showed antigenic differences between the BCG and the Erdman products, suggesting the presence of features specific to the different strains and pointing to LAM as a molecule within which further species and strain variations reside.

The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, a- difluoromethylomithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMhl, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMhl. The pSAMhl cDNA is missing 72 nucleotides from the 5' end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMhl in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.

Polyacrylamide gel electrophoresis of extracellular pectinase and amylase isozymes of 170 mainly terverticillate Penicillium strains was undertaken. The data were coded and subjected to numerical analysis. Variation in intensity of isozymes was observed in repeat analyses of some strains, although most were consistent. Variation was also observed between some representative strains of species. P . viridicatum was more variable than P . brevicompactum and P . hordei for intensity of pectinase activity. There was a correlation between the grouping of the strains on the basis of the isozymes and the species concepts only in some cases. The method proved useful for the identification of strains producing intense activity which provided clear patterns, for example, P. brevicompactum and P . chrysogenum and to a lesser extent P . solitum var. crustosum and P . hordei. The method was also exclusionary in that some species were restricted to a particular cluster or subcluster. Amylase patterns confirmed that strains referred to as single species are not all homogeneous genetically, and that some strains are not simply haploid homokaryons. The genetic heterogeneity of the strains explains some of the problems in the systematics of the terverticillate penicillia. The material and methods used for the growth of the fungi, and electrophoresis, were adapted from Cruickshank & Pitt (1987). The main modifications were (a) different electrophoresis tank and cooling system, (b) thinner gels, (c) using filter paper for sample application, and ( d ) different amylase production medium.

Ammonia assimilation in Rhizobium leguminosarum biovar viceae strain RCRlOO 1 (hereafter called R . leguminosarum) appears to take place only through the glutamine synthetase/glutamate synthase pathway since (a) no glutamate dehydrogenase was detected in crude extracts of bacteria grown in different nitrogen sources, and (b) the growth rate on glutamine as a nitrogen source was faster than that observed on NH4Cl. In contrast to reports for other Rhizobium species, R . leguminosarum can definitely utilize NH4C1 for growth. R. Ieguminosarum contains two glutamine synthetase isoenzymes, GSI and GSII, which can be detected in the presence of each other by differential heat stability, or separated by affinity chromatography or immunoabsorption with an antiserum raised against pure GSI. GSII does not cross-react with an anti-GSI antiserum. GSI was shown to be reversibly adenylylated and it was also shown that adenylylation inhibits the biosynthetic activity of this enzyme, in a similar way to that reported for Escherichia coli glutamine synthetase and in contrast to that observed for glutamine synthetase of Rhizobium sp. strain ANU289. The apparent adenylylation level in different growth conditions changes from 21% to 99%, indicating a physiological role of this posttranslational modification in the in vivo regulation of GSI activity. The intracellular concentration of GSI varies very little when R . leguminosarum is grown on different nitrogen sources (twofold when measured by the transferase assay, or fourfold when measured by ELISA). In addition, the concentration of mRNA specific for GSI in different nitrogen sources does not show appreciable differences. The intracellular concentration of GSII varies from a specific activity value higher than 1000 when R . leguminosarum is grown on glutamate or nitrate, to an undetectable level when grown on NH4C1. When NH4C1 is added to a culture growing in glutamate, GSII activity is rapidly diluted out, suggesting a post-translational mechanism of enzyme inhibition or inactivation. Chloramphenicol prevents the disappearance of GSII activity, thus suggesting that protein synthesis is required for this process.

Resistant strains of Aspergillus nidulans were obtained by one-step selection on high concentrations of various inhibitors; each strain was mutant in a different gene conferring resistance to actidione (Act), p-fluorophenylalanine ( p f ) , teoquil (te), iodoacetate (Iod) or malachite green (mg). Some mutant alleles have been firmly, others tentatively, located. For comparative purposes attempts were made to find instances of multi-step or non-genic increases in resistance to malachite green and to teoquil, by prolonged exposure to low concentrations of inhibitor. No such increases were found. Iodl is fully dominant, Act1 is semidominant, pf21, t e l and rngl are recessive. mgl confers resistance to acriflavine to about the same degree as the non-allelic am 2 but these two mutant alleles do not show additivity. pf21 confers resistance to iodoacetate and also suppresses requirement for nicotinic acid (nic8). Iod I strains, which are not resistant to fluoroacetate, are able to use acetate as sole carbon source. Nutritionally balanced heterokaryons, between Act I and sensitive strains, show a gradual (and reversible) increase of the Act component on increasing actidione concentrations. Ultimately a plateau is reached ; this presumably represents the nutritional limits of each particular combination of nutritional markers.

Mucoid variants of Pseudomonas aeruginosa isolated in vitro or in vivo could be classified into two phenotypic groups based on whether alginate was produced on a chemically defined medium. Mucoid strains yielded lower recombination frequencies than the noninucoid parent when used as donors in FP2-mediated plate matings. The mucoid characteristic (muc) was co-inherited by a proportion of recombinants selected for the inheritance of chromosomal markers his-5075+ or cys-5605f. The results of further experiments using either a mucoid recipient or a mucoid donor carrying plasmid R68.45 suggested that the control of alginate production in P. aeruginosa involves at least one chromosomal locus.

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas Jluorescens, P. putida and P. mendocina at a frequency of 1 in lo8 by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopoly saccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia. Only two species of bacteria, Azotobacter vinelandii (Gorin & Spencer, 1966) and Pseudomonas aeruginosa , are known to produce alginic acid, a ( 1 4)-linked linear copolymer of p-D-mannuronic and a-L-guluronic acids. Alginate production in P. aeruginosa is restricted to mucoid variants which are rarely encountered except in association with chronic pulmonary infection in patients with cystic fibrosis (Doggett, 1969). We have previously reported that mucoid mutants of P. aeruginosa can be isolated in vitro from wild-type, non-mucoid strains by selecting for carbenicillin resistance or resistance to a virulent phage . Mutations responsible for this change in phenotype can be mapped on the chromosome (Fyfe & Govan, 1980). This evidence suggested that wild-type, non-mucoid strains of P. aeruginosa carry the genetic information necessary for alginate production but it is normally repressed. The aim of the present study was to determine whether alginate-producing mutants occur in other species of Pseudomonas. We report for the first time the isolation of mutant strains of P. putida, P. Jluorescens and P. mendocina producing an alginate-like exopolysaccharide similar to that obtained from A. vinelandii and mucoid P. aeruginosa.

Mucoid variants of Pseudomonas aeruginosa isolated in vitro or in vivo could be classified into two phenotypic groups based on whether alginate was produced on a chemically defined medium. Mucoid strains yielded lower recombination frequencies than the noninucoid parent when used as donors in FP2-mediated plate matings. The mucoid characteristic (muc) was co-inherited by a proportion of recombinants selected for the inheritance of chromosomal markers his-5075+ or cys-5605f. The results of further experiments using either a mucoid recipient or a mucoid donor carrying plasmid R68.45 suggested that the control of alginate production in P. aeruginosa involves at least one chromosomal locus.

Electrophoretic patterns of esterases, phosphatases, of malate, glucose-6phosphate and a-glycerophosphate dehydrogenases, and of other soluble proteins of 69 isolates representing 2 I species of Xanthomonas were determined. Electrophoretic protein patterns were generally identical for all isolates of the same species but dissimilar for different species. Most species showed multiple forms of enzymes. The pattern of a-glycerophosphate dehydrogenase was identical in all species and may be genus-specific. Esterases and phosphatases varied widely among species. In most cases, however, taxon-specific patterns, with slight quantitative variations, were evident. No lactate dehydrogenase activity was detected. This technique appears useful in differentiating among Xanthomonas species which until now have been identifiable only by their specific pathogenicity.

The genetic diversity of symbiosis (Sym) plasmids was investigated in samples from two field populations of Rhizobiurn legurninosarurn biovar viceae that had previously been characterized for chromosomally-encoded enzyme electrophoretic polymorphism. Five overlapping cloned DNA fragments from the Sym plasmid pRLlJI were used as hybridization probes to identify restriction fragment variation in the homologous genes of the isolates. In addition, a clone of the P-galactosidase gene region was used as a probe, extending the data on chromosomal relatedness. The plasmid-encoded Sym region was very polymorphic (1 1.4% average DNA sequence divergence). Some isolates had the same Sym fragment pattern as pRLl JI, but most were very different. One was closely similar to pRL6J1, another widely-studied viceae plasmid. The distribution of plasmids across chromosomal backgrounds was far from random, as though the species were subdivided into compartments with largely separate plasmid pools. Nevertheless, indistinguishable plasmids were found in quite different genetic backgrounds, implying that plasmid transfer must occur in the field. This has implications for the population genetics and evolution of bacteria and for the release of genetically engineered strains.

Growth assays reveal that Rhodococcus rhodochrous IGTSS uses a wide range of organosulphur compounds as the sole source of sulphur, yet none of the compounds serve as carbon sources. Compounds that are utilized include thiophenes, sulphides, disulphides, mercaptans, sulphoxides, and sulphones. A convenient spectrophotometric assay (Gibbs assay), based on the chromogenic reaction of 2,6-dichloroquinone-4-chloroimide with aromatic hydroxyl groups, was developed and used in conjunction with GC/MS analyses to examine the kinetics of dibenzothiophene metabolism by axenic and mixed cell cultures of Rhodococcus rhodochrous IGTSS. The desulphurization trait is expressed at increasing levels during the exponential phase of growth and then declines in stationary-phase cells. Mixtures of streptomycin-resistant Rhodococcus rhodochrous IGTSS and Enterobacter cloacae (an organism incapable of cleaving carbon-sulphur bonds in relevant test compounds) were prepared in ratios that varied over six orders of magnitude. Growth studies revealed that E. cloacae was able to gain access to sulphur liberated from organosulphur compounds by IGTSS ; however, cell-to-cell contact appears to be required. These experiments also indicate that the desulphurization activity, on a per cell basis, is higher in mixed cultures than in axenic cultures.

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae 0 1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classicallike CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM, ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined. I N T R O D U C T I O N YAMAMOTO, T., GOJOBORI, T. & YOKOTA, T. (1987). Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae 0 1 .

The basis of tetracycline resistance mediated by TetA determinants and joint resistance to tetracycline and minocycline coded by TetB determinants was investigated. The TetA class of determinants was represented by those carried on plasmids pSC101, RPl and pIP7 and TetB by TnlO. The relationships between expression of tetracycline and minocycline resistance and accumulation of these antibiotics suggest that there are three mechanisms of plasmid-determined resistance conferring (1) about a 10-to 20-fold increase in resistance to tetracycline that is not associated with decreased antibiotic accumulation, (2) a 4-to ?' -fold increase in resistance to tetracycline that is associated with decreased drug accumulation, and (3) about a 2-to 3-fold increase in resistance to both tetracycline and minocycline that is not associated with decreased accumulation of either antibiotic. Mechanism 1 was coded by the tetracycline resistance determinant of pSClOl (TetA), mechanisms 1 and 2 by the determinants in RP1 and pIP7 (TetA) and all three mechanisms by TnlO (TetB).

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.

A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2p.m. Hyphal colonies resistant to the neomycin analogue G-4 18 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 transformant, 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.

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkaliinsoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferri tin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps : firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall. Abbreciations: Au-WGA, wheat-germ agglutinin complexed with colloidal gold ; Con A-fe, concanavalin A-ferritin; I-glucan, alkali-insoluble glucan; S-glucan, alkali-soluble glucan.

Sfreptomyces lividans was investigated for extracellular proteinases. The major extracellular proteinase activity detected under all conditions tested was L-leucine aminopeptidase activity. Slight extracellular L-proline aminopeptidase activity was also detected. No clear evidence for the presence of serine proteinases in S. lividans culture broths was found using several different methods. The major extracellular proteinase of S. lividans, i.e. the L-leucine aminopeptidase, was purified 33-fold to homogeneity. The purified enzyme was found by SDS-PAGE to have an M, of 34000. The purified enzyme had a final specific activity of 3-6 units mg-', a K, for L-leucine-pnitroanilide of 300 p~ and a kax of 4.2 pmol min-' mg-'. The pure enzyme did not exhibit proteolytic activity on azocasein or L-proline-p-nitroanilide, substrates for other proteinase activities observed in crude extracts of S. lividans.

Streptomyces sp. strain C5, an organism that normally produces baumycins, daunomycin and E-rhodomycinone, was treated with N-methyl-N'-nitro-N-nitrosoganidine (NTG). Mutants blocked at various points in daunomycin and baumycin production were isolated by screening for altered pigmentation and absence of bioactivity against Staphyfococcus aureus. Examination of the mutants by thin-layer chromatography of their accumulated anthracycline metabolites, by cosynthesis assays, and by extract feeding experiments allowed a classification into six groups. These were: dauA, strains that accumulated no anthracyclines but with other blocked mutants cosynthesized anthracyclines (polyketide-synthase-minus mutants); dauG, regulatory mutants that, either alone or mixed with other blocked mutants, accumulated no anthracyclines; d a d , mutants that accumulated aklanonic acid; dauE, mutants that accumulated maggiemycin; dauF, mutants that accumulated aklavinone; and dauH, mutants that accumulated only E-rhodomycinone. Mutant SC5-24 (dauE), which accumulated the shunt product maggiemycin, was re-mutagenized with NTG to obtain blocked mutants in preceding biosynthetic steps; the three groups of double mutants obtained accumulated aklanonic acid (dauC,E), aklanonic acid methyl ester (dauD,E) and aklaviketone (dauE,F).

Rapidly reannealing DNA, containing repeated base sequences, was shown to be present in the genomes of four Rhizobium bacteriophages by studies of renaturation kinetics. Estimations of the mol. wt. of the DNA by measurement of kinetic complexity gave values of I "3 × IoS for phage c, 1.6 × lO 8 for phage I, 9"4 × lO7 for phage e and 8.2 × io 7 for phage a. Comparison of the phage DNAs by annealing studies confirmed the classification of the phages into two groups.

A laboratory yeast isolate identified as Exophiala jeanselmei synthesized melanin and showed polyphenol oxidase activity with dihydroxyphenylalanine (DOPA) and pyrogallol. Electrophoresis on native and denaturing gels, and activity staining with DOPA revealed two immunologically distinguishable bands of M , about 100000 and 120000, which could be separated by (NH,)2S04 fractionation. The enzyme isolated from this yeast gave optimal activity at higher pH (8.5-10) and temperature (3740°C) than other known melaninsynthesizing activities.

A genomic library of Yersiniapestis EV76c created in a cosmid vector was screened for clones capable of binding type IV collagen. An unexpectedly high number of such clones was observed. One recombinant plasmid was selected for further study, and the locus controlling collagen binding was mapped by subcloning, transposon mutagenesis and exonuclease digestion. The outer-membrane protein profiles of transposon insertion mutants were correlated with phenotype to implicate a 36 kDa polypeptide in type IV collagen binding. Fine substructure restriction mapping and limited DNA sequence analysis showed the cloned locus to be identical to the locus (pla) for the plasminogen activator, previously characterized genetically and biochemically. The pla locus is resident on a 9-5 kb plasmid in wild-type Y. pestis strains. Curing of this plasmid resulted in negligible reduction in collagenbinding capacity, implying the existence of a chromosomally located determinant for collagen binding. The affinity of the plasminogen activator for collagen was relatively weak. When the cloned pla locus was introduced into E. cufi, it conferred upon the cell the ability to bind to cells from a number of cell lines. Binding to glycolipids separated by thin-layer chromatography demonstrated that the receptor was a member of the globo-series of glycolipids. Since it has been reported that mutation of pla dramatically reduces virulence, we propose that this hitherto undescribed function of the gene product could contribute to the biological activities necessary for full virulence.

Twenty-two monoclonal antibodies (mAbs) specific for smooth lipopolysaccharide (S-LPS) were generated by fusion of spleen cells from mice infected with the rough Brucella melitensis strain B115 with the NSO myeloma. According to reactivity in enzyme-linked immunosorbent assay (ELISA) with 0-polysaccharide (0-PS) and absence of reactivity with rough lipopolysaccharide (R-LPS), it was postulated that these mAbs recognized epitopes present on the 0-PS. Most of the mAbs reacted equally well in ELISA and immunoblotting with S-LPS types of Brucella A and M dominant strains and were designated as specific for common (C) epitopes. Three mAbs were highly specific for M dominant S-LPS. All these mAbs, in contrast to a mAb specific for the A epitope, showed little or no cross-reactivity with Yersinia entevocolitica 0 : 9 S-LPS. S-LPS of B. melitensis B115 was extracted and analysed by immunoblotting and ELISA with mAbs specific for A, M and C epitopes. Reactivity of the mAbs with this S-LPS was compared to reactivity with S-LPS of A and M dominant smooth Brucella strains. The results suggest that S-LPS of B. melitensis B115 bears mainly C epitopes and a few M epitopes. The very weak reactivity of this S-LPS with the mAb specific for the A epitope and the fact that the mAbs specific for C and M epitopes showed little or no cross-reactivity with Y. enterocolitica 0 : 9 S-LPS suggest that 0-PS from this rough strain could be used to distinguish Y. enterocolitica 0 : 9 infection from Brucella infection. The potential value of the rough B. melitensis strain B115 as a vaccine strain is also discussed.

Plasmid pEST4011 enables Pseudomonas putida Paw85 to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA). This new 2,4-D degradative plasmid has considerable homology with the regions of pJP4 containing the 2,4-D degradative genes (tfd). Restriction fragment BamHI-B of plasmid pEST4011, which has homology with this region, was cloned into the broad-host-range vector pKT240 and studied in P. putida PaW85. Restriction mapping, hybridization analysis and enzyme assays established the location of the genes for 2,4-D monooxygenase (tfdA), 2,4-dichlorophenol hydroxylase (tfdB), chlorocatechol 1,2-dioxygenase (tfdC) and the tfdR and tfdS regulatory genes on this fragment. Plasmid pEST4012 is a derivative of pEST4011 derived through the spontaneous deletion of a 42 kbp DNA fragment, which results in the loss of the 2,4-D+ and 3-CBA+ phenotype. We present here the physical maps of pEST4011 and pEST4012. In spite of the similarities in functions, the size (70 kbp), order of catabolic genes and restriction pattern of pEST4011 are clearly different from those of pJP4.

The immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibodyinduced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a nonagglutinating emulsan-producer (AB 1 9 , and (2) an agglutinating mutant (1 6TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in nonagglutinating mutants. Component C3 may be identical with or attached to the 300nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism.

Sexual adhesion in the green alga Chlamydomonas eugametos is mediated by hydroxyproline-containing glycoproteins (agglutinins). The agglutinins were metabolically labelled with [35S]sulphate. The sulphur-containing amino acids methionine and cysteine were not detectably labelled; little or no sulphated tyrosine was detected. This indicated that the radioactive label had been largely or exclusively incorporated into sulphated sugar residues. Acid hydrolysis released essentially all radioactivity as free sulphate. Short periods of hydrolysis released sulphated oligosaccharides. /%Elimination released about 20-25 % of the sulphated saccharides, indicating that these are 0-linked to serine or threonine or both. The remaining sulphated sugar chains are presumably 0-linked to hydroxyproline. It is proposed that the sulphated sugars of the agglutinins play a role in sexual adhesion.

The g t f S gene, coding for a glucosyltransferase which synthesizes water-soluble glucan and previously cloned from Streptococcus downei strain MFe28 (mutans serotype A) into a bacteriophage vector, was subcloned into a plasmid vector. The g t f S gene products expressed in Escherichia coli were compared to the primer-independent, oligoisomaltosaccharide synthase in Sfreptococcus sobrinus strain AHT (mutans serotype g) and shown to resemble it closely in molecular mass, isoelectric point, immunological properties, optimum pH and K , values. The glucans produced from sucrose by the g t f S gene products are a-1,6-linked linear oligo-isomaltosaccharides without any branching sites. A similar g t f S gene was also detected on chromosomal DNA from S. sobrinus strain AHT.

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, g t f l , previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage 2 vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtjlr was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d ) and 6715.(serotype g ) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-Cr-Dglycosyl residues (-90%). An internal fragment of the g t f l gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group. The glucosyltransferases (GTFs) of the mutans group of oral streptococci are extracellular enzymes responsible for the synthesis of complex mixtures of water-soluble and insoluble glucans. These glucans are believed to be important in the formation and metabolism of dental plaque . It was first shown by Guggenheim & Newbrun (1969) that a single strain could produce a number of electrophoretically distinct GTFs. Ciardi et al. (1976Ciardi et al. ( , 1977) ) showed that some of these enzymes synthesized soluble, and others insoluble, polymers but progress towards understanding the reason for the multiplicity of enzymes has been considerably hampered by technical problems, including the fact that GFTs exist in highmolecular-mass aggregates and have a strong tendency to bind to separation matrices commonly used in column chromatography (

The contribution of activated macrophages to protection against Escherichia coli was studied in mice. Mice treated intraperitoneally with killed Corynebacterium paruurn organisms 1 d prior to challenge showed an increased resistance to intraperitoneal infection with E. coli; the predominant leucocytes in the peritoneal cavity of these animals were polymorphonuclear cells. However, treatment with C. parvum 4 d prior to challenge induced mainly activated macrophages in the peritoneal cavity and host resistance to the infection was not increased. Activated macrophages from such mice showed both enhanced phagocytic activity in vivo and a high degree of intracellular killing of E. coli in uitro. At the same time these cells became more susceptible to the cytotoxic effect of endotoxin. After challenge with E. coli there was a marked decrease in the number of peritoneal macrophages in mice that were treated with C. parvum 4 d prior to challenge. Increased susceptibility of activated macrophages to the cytotoxic effect of endotoxin could explain the absence of enhanced resistance to E. coli infection in such animals.

Two families of glycoprotein are defined in DictyosteZium discoideum by the presence of different glycoconjugates, both of which are highly immunogenic in mice. The previously described monoclonal antibodies MUD50 and MUD62 recognize the glycoconjugates and identify the respective glycoprotein families. Both types of glycosylation occur on vegetative and developmentally regulated glycoproteins. The immunodominant components of both families are reportedly 0-linked sugars, but Western blots do not identify any glycoprotein that has both 0-glycans, suggesting that there are two independently processed types of 0-linked glycosylation in D. discoideum. The synthesis of the two 0-glycan families is affected by glycosylation-defective mutations. Strains with a mutation at the modB locus lack one of these glycosylation types (that recognized by MUDSO) and this mutation alters the size of two minor glycoproteins in the second family. Two new mutants, HU2470 (mod-352) and HU2471 (mod-353), lack the epitope recognized by MUD62. The two mutations map to different chromosomes. The mod-353 mutation also affects the size of PsA, a cell surface glycoprotein carrying the modB-dependent 0-glycan.

Mutants of Bacillus subtilis 168 were isolated which allow adsorption but not infection by bacteriophage SPPl. The adsorbed phages can be subsequently recovered in active form. From ten other bacteriophages tested, only the SPP1-related phages 41c, 22a, p15 and SF6 failed to plate on the mutant cells. The mutation responsible for this behaviour (pha-2) was mapped by PBSl transduction, showing 95% cotransduction with ald-1.

Removal of the growth medium and resuspension of BfastocfdefZa emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2 % of its initial value within 1Omin. The concentrations of hexose 6-phosphate and of fructose l,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1 -b a s e and fructose-l,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Fourteen yeast strains from six genera were analysed for the presence of long-chain alcohols. Six strains from three genera contained long-chain alcohols, highest levels being found in Candida afbicans. The alcohols were identified and determined by TLC, GLC and GLC-MS. The major long-chain alcohols synthesized by these organisms were saturated, primary alcohols with C1 4, C I 6 or C18 chain length. Unsaturated long-chain alcohols were not detected. In all strains that produced long-chain alcohols, the relative proportions were C I 6 > C18 > C14. Long-chain alcohol contents were higher in organisms from anaerobically, as compared with aerobically, grown cultures reaching about 650 pg (g dry wt organisms)-' in stationary-phase cultures of C. afbicans. In cultures of C . albicans, synthesis of long-chain alcohols occurred only after the end of exponential growth. The alcohols were predominantly present as free alcohols. The fatty-acyl chain-length profile of the triacylglycerol and to a lesser extent the sterol/wax ester fractions from C . albicans reflected that of the long-chain alcohols produced by this yeast.

The genus Bacteroides contains a number of biochemically and physiologically heterogeneous groups of organisms and needs taxonomic revision. In this study cellular fatty acids from a number of Bacteroides spp. were identified and quantified using gas chromatography and gas chromatography-mass spectrometry. The chemical data were then subjected to principal components analysis. In B. fragilis, which is the type species of the genus Bacteroides, C3-OH-iso, was the predominant fatty acid (38.0%) and Cantel was present in higher amounts (32.7 %) than Ciso, (14.6 %). B. fragiZis thus differed from all the other species examined: Prevoteffa (Bacteroides) buccae, P. (B.) oralis, P. (B.) oris, P. (B.) disiens, P. (B.) veroralis, P. (B.) heparinolytica and Porphyromonas (Bacteroides) endodontalis. Principal components analysis also enabled the closely related P. buccae, P. oralis and P. oris to be differentiated.

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physicallyundetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVPl and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmidminus mycelium. Conjugational crosses were used to construct a linkage map of S . venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.

Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.

The Dictyosteliurn discoideum glycogen phosphorylase-1 (gp-1) exhibits a complex pattern of developmental expression in which differential temporal regulation of enzyme activity, protein levels and mRNA levels is observed. This pattern of expression implies that gp-1 regulation occurs at multiple levels, probably involving both transcriptional and post-transcriptional events. Post-translational control of gp-1 activity, in effect, actually regulates the protein from a developmental perspective. In this report we have examined several facets of this regulation. We show that addition of exogenous cAMP to cells in suspension culture caused changes in gp-1 enzyme activity and mRNA levels that are identical to those observed during normal development, suggesting that cAMP is involved in the regulation of gp-1. Exogenous cAMP could regulate gp-1 mRNA expression at concentrations as low as 1.0 PM. cAMP regulation of gp-1 mRNA appeared to occur through a mechanism that required intracellular cAMP signalling. We identified regions of the promoter necessary for gp-1 expression by using gp-1 promoter deletions to drive the expression of a luciferase reporter gene. Results of these experiments suggested that developmental and CAMP-mediated changes in gp-1 mRNA levels were the result of alterations in transcription. The promoter analysis also suggested that a vegetative specific element is located between -785 and -1894 nucleotides from the transcriptional start site. Elements necessary for maximal developmental and CAMPmediated expression appear to be located between -1153 and -1894 nucleotides from the cap site. Sequence elements located between -180 and -1153 appear to be required for a basal level of late developmental expression.

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified.

Cassava (Manihot esculenta Crantz) is a major carbohydrate source in the human diet, yet certain varieties harbor high cyanogenic glycoside concentrations, posing risks of cyanide toxicity. This study sought to determine cyanide concentration levels in leaves, peels, and pulps of cassava varieties selected from earlier morphological characterization studies carried out in Kenya. A total of 32 varieties were selected for cyanide content analysis using picrate paper and spectrophotometric methods. Similarly, an on-field study was conducted in Migori County, Kenya to assess cyanide concentrations in cassava varieties grown by farmers using picrate paper method. Using the spectrophotometric method, the leaves, peels and pulps had overall average cyanide concentrations of 5.89 mg/L CN-, 7.42 mg/L CN-and 8.20 mg/L CN-respectively. The same trend was realized using the picrate paper analysis where the leaves had average cyanide concentration of 3.13 mg/L CN-, the peels had average 5.44 mg/L CN-and the pulp had the highest average cyanide concentration of 7.97 mg/L CN-. Using the spectrophotometer analysis of the leaves, the variety Nyarkadera exhibited the highest cyanide concentration of 26.93 mg/L CN-, while the lowest was observed in the varieties Nyatanga-002, Nyakanyamkago and Kasukali (0.40 mg/L CN-). Peels of Kazanzwara recorded the highest cyanide concentration of 17.82 mg/L CN-, whereas Kasukali showed the least cyanide concentration of 1.19 mg/L CN-. Results obtained from the pulps showed that the variety Nyatanga had the highest cyanide concentration of 26.93 mg/L CN-while the least cyanide concentration was recorded on the variety Mzungu (0.40 mg/L CN-). Based on the two techniques of analyses, the study established moderate positive correlations (r=0.547 and r=0.570) between cyanide levels in leaves and peels of different cassava varieties respectively. A strong positive correlation of r=0.936 was found in the analysis of pulps indicating high consistency of the two methods. In the field study 15 varieties out of 32 had high cyanide concentrations between 10-30 mg/L CN-while 17 varieties had low cyanide concentration. This showed that farmers still planted high and low cyanide varieties in almost equal proportions. The picrate paper analysis was found appropriate for use effectively in assessing cyanide concentrations in the field. The variability in cyanide levels among the cassava accessions appeared to emphasize need to characterize varieties by cyanogenic potential for breeding programs and consumer guidance.

A pleiotropic dinitrogen and nitrate assimilation mutant was obtained by mutagenesis of Nostoc sp. strain ATCC 29133 with N-methyl-N'-nitro-N-nitr~~dine followed by penicillin counterselection in the presence of NO, and N2. Mutant strain UCD 223 was capable of reducing acetylene in the free-living growth state only under anaerobic conditions, or under atmospheric conditions when in symbiotic association with Anthoceros punctatus. Heterocysts of strain UCD 223 were noticeably lacking the cohesive outer polysaccharide layer of wild-type heterocysts. Oxygen microelectrode profiles of symbiotic AnthocerosNostoc tissue revealed an anaerobic environment in the symbiotic cavities containing Nostoc. The acetylene-reducing activities of strain UCD 223, and of its spontaneously-arising Fix+ revertant strain UCD 236, were not repressed by the presence of 10 mM-No; when in the free-living growth state, in contrast to wild-type Nostoc ATCC 29133. However, in situ activities of acetylene reduction by symbiotically associated Nostoc ATCC 29133 and strains UCD 223 and UCD 236 were repressed by the presence of 10 mM-No5. It appears that the symbiotic cavities of Anthoceros punctatus can physiologically replace the function of the heterocyst outer wall and that the repression of nitrogenase activity in symbiotic Nostoc in response to the presence of NO?, and probably NHi, is mediated by Anthoceros.

Schizosaccharomyces pombe cell walls contain two major glycoprotein species, I and 11, with molecular masses of 2 x lo6 and 5 x lo5 Da respectively, as determined by gel filtration chromatography and PAGE. The ratio of sugar to protein is higher in species I than in species 11. Much of the sugar in both glycoproteins (about 85% in wild-type cells) is 0-linked to the peptide moiety. The morphological sphl mutant is resistant to papulacandin B, and its cell wall contains less glycoprotein I1 (but not less glycoprotein I) than the parental wild-type strain, although glycoprotein I1 is still synthesized and released into the growth medium. Papulacandin B largely reverses the morphological alteration of the mutant, and returns the ratio between species I and I1 to about that found in the parental strain, although the absolute amount of species I1 is still lower in the mutant. The results point to the importance of the relative amounts of the Merent wall polymers in determining cell morphology.