Biodiversity of microorganisms that degrade bacterial and synthetic polyesters

The biodiversity and occurrence in nature of bioplastic-degrading microorganisms are exemplified by the identification of 695 strains, isolated from different environments, such as soils, composts, natural waters, and sludge, that are able to degrade the bacterial polyester poly(3-hydroxybutyrate)in vitro. These microorganisms belong to at least 57 different taxa, including Gram-negative and Gram-positive bacteria, streptomycetes, and moulds. The literature on the biodiversity of poly(3-hydroxybutyrate)-degrading microorganisms is reviewed. The degrading abilities of 171 streptomycete strains were investigated on four different bacterial poly(3-hydroxyalkanoates), and the synthetic polyesters poly(-caprolactone) and BIONOLLE, and most of these strains degraded at least three different polymers.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Pleiotropic mutations in appR reduce pH 2.5 acid phosphatase expression and restore succinate utilisation in CRP-deficient strains of Escherichia coli

Summary Several strains of Escherichia coli K12 were compared for activity of the periplasmic pH 2.5 acid phosphatase, an enzyme whose expression is regulated negatively by cyclic AMP. Two distinct enzyme levels differing by about four-fold were observed. This strain-dependent difference does not involve modifications in the structure of the enzyme, but results from a difference in its expression. We show that (1) strains with a high- or a low level of enzyme differ in the gene locus appR located in the 59 min region of the chromosome, a site remote from the structural gene appA; (2) the appR ⁺ versus appR enzyme ratio is 3–4 in wild-type strains, adenylate cyclase-deficient strains (cya) or cyclic AMP receptor protein-deficient strains (crp) grown in rich medium or in glucose minimal medium, but is close to 1 in cya strains in the presence of 0.1 mM cyclic AMP and in wild-type strains grown with succinate as carbon source; (3) in a crp genetic background, appR strains, contrary to appR ⁺ strains, are able to grow on minimal medium with succinate as the sole carbon source. The selection, from an appR ⁺ crp strain, of clones growing on succinateminimal medium. yielded mutations in the same region of the chromosome and showing the same phenotype as naturally-occurring appR strains.All appR strains analysed so far showed other similar deficiencies. The possibility that mutated appR gene products might function as weak substitutes for a functional cAMP-CRP complex is discussed.     

Colonization of hydrophilic contact lenses by yeast

The growth of six strains of yeast was analyzed in vitro in order to assess their capacity for colonizing (adhesion and invasion) hydrophilic contact lenses. Lenses with different water content were cultured in two culture media for 3, 7, 14, and 21 days. Only strain 93150 of Candida albicans could adhere to and invade the polymers. Specifically, fungal growth was observed in cultures with Sabourauds broth. The degree of yeast colonization of contact lenses was significantly related to the species, the strain, and the culture medium in which the yeast and lenses were cultured. The results here obtained were compared with those reported for the filamentous fungus Aspergillus niger 2700. For both microorganisms, the strain and the medium in which the lenses and microorganism were cultured influenced the colonization, but the percentage of colonized lenses, the degree of colonization, and the density and size of the internalized colonies were always noticeably lower for C. albicans 93150. Colonization by A. niger 2700 was also related to the type of material of the lenses and the incubation period. For both microorganisms, when the strain is right and the growth and development are correct, colonization of hydrophilic contact lenses occurs.     

DNA relatedness among serovars ofListeria monocytogenes sensu lato

Sixty-six strains ofListeria monocytogenes (as defined in the eighth edition ofBergey's Manual of Determinative Bacteriology) were characterized by DNA relatedness. These strains formed five distinct DNA relatedness groups: (i)L. monocytogenes sensu stricto (30 strains) including the type spain ATCC 15313; (ii) serovar 5 strains (9 strains) corresponding to L. bulgarica; (iii) L. inocua (11 strains of serovars 6a, 6b, 4ab, and undesignated ones) including the reference strains; (iv) six strains of serovars 6a and 6b; (v) ten strains of various serovars. These five groups were clearly distinct fromL. grayi (4 strains) andL. murrayi (3 strains).     

Estimating position-time relationships in steady-state,one-dimensional growth zones

Two methods are described for estimating position-time relationships (pathlines) in steady, one-dimensional growth zones. Pathlines can be used to provide a time base for spatial data in developmental studies. The methods apply within extension-only zones (zones of growth without cell division) and require data for cell-number densities, or cumulative cell numbers, or mean cell lengths, and for the overall elongation rate of an organ. The first method (continuous-pathline method) can be used to estimate spatial velocity fields within extension-only zones and pathlines can then be obtained by integration of the velocity data. This method is based on the continuity equation for cell-number densities. Pathlines can also be estimated using a simple graphical version of this method. The second method (pathline-coordinate method) is based on the approximation that a cell of mean length remains of mean length as it moves through the extension-only zone, and can be used to estimate the coordinates of wall pathlines at discrete intervals. The methods are illustrated using published data from studies of apical growth in Zea mays L. roots and of intercalary growth in Triticum aestivum L. leaves.     

Light and dark metabolism ofl- andd-amino acids in 5 strains ofChlorella vulgaris and 4 strains of the genusAnkistrodesmus

In the light, 2 out of the 4 newly testedChlorella vulgaris strains were found to use about as many amino acids as a source of nitrogen as the previously investigated strain Delft; the other 2C. vulgaris strains and 3 of theAnkistrodesmus strains used only a few. The 4thAnkistrodesmus strain used none.On the average,l-amino acids supported better growth thand-amino acids, butd-serine was preferred tol-serine by 3Ankistrodesmus strains.In the dark, growth was only obtained withC. vulgaris strain Delft, and only on a few of thel-amino acids,l-leucine in particular.The author is indebted to the Direction of the Academic Hospital Dijkzigt, Rotterdam and to Prof. Dr. H. Esseveld, Head of the Central Bacteriological Laboratory, Rotterdam, for providing facilities for the performance of this study.He thanks Mrs. Dr. H. J. Leijnse-Ybema for her help in making the chromatograms, and Mr. J. B. Lenstra, pharmacist, for advice in matters of organic chemistry.     

Structure and biosynthesis of teichoic acids in the cell walls of Staphylococcus xylosus DSM 20266

The simultaneous occurrence of a N-acetylglucosaminyl poly(ribitolphosphate) (-GlcNAc) and a N-acetylglucosaminyl poly(glycerolphosphate) (-GlcNAc) in the cell walls of Staphylococcus xylosus DSM 20266 was demonstrated by different experimental lines:(1) Fractionation of extracted cell wall teichoic acid on DEAE-cellulose, (2) investigation of the composition of cell walls in the growth cycle, (3) in vitro biosynthesis using crude membranes as the source of enzyme.The polymerization of these polymers starts from CDP-ribitol and CDP-glycerol, respectively. In the presence of UDP-N-acetylglucosamine both polymers are substituted with N-acetylglucosamine at a level and with the identical anomeric configuration found in the native cell wall teichoic acids. The in vitro biosynthesis of poly(glycerolphosphate) was unique in that it was highly stimulated by UDP-N-acetylglucosamine and to a lower extent by other UDP-activated sugars. Kinetic studies have provided evidence that this stimulation is due to an increase of V _max while K _m is unchanged. Competition experiments have indicated that poly(ribitolphosphate) and poly(glycerolphosphate) were synthesized in the in vitro system in a close spatial relationship.Abbreviations ADP adenosine 5-diphospho - CDP cytidine 5-diphospho - GDP guanosine 5-diphospho - GalNAc N-acetyl-galactosamine - Glc glucose, glucosyl - GlcNAc N-acetyl-glucosamine - N acetylglucosaminyl - GlcUA glucuronic acid - Gro glycerol - Man mannose, mannosyl - Rit ribitol - SDS sodium dodecyl sulfate - UDP uridine 5-diphospho     

Examining serological diversity of “cowpea” rhizobia by the ELISA technique

To appraise the usefulness of the enzyme-linked immunosorbent assay (ELISA) technique for examining the serological diversity of slow-growing rhizobia, twelve diverse strains from three countries were examined with four antisera. Eleven of the strains were from the cowpea miscellany, and the twelfth was a Rhizobium japonicum strain. Some cowpea strains showed no antigenic relatedness with each other while others were closely related, and some showed a greater affinity with the R. japonicum strain than with other cowpea strains. All of the strains showed antigenic homology to an isolate from a wild Arachis sp., while two strains isolated from adjacent plants of the same cultivar had little homology. These patterns ofrelatedness and diversity clearly demonstrated the utility of the ELISA method, and so it was used to examine 53 strains isolated from cowpeas grown at three West African locations, Maradi (Niger), Ibadan and Onne (Nigeria). Broad ranges of serological diversity were found in the rhizobia at each location, moreover each population had its own general characteristics. Maradi strains were highly reactive with the five antisera used, Onne strains less so, and Ibadan strains even less so. ELISA reactivity correlated with colony morphology but not with nodulation potential.     

Rhodoferax fermentans gen. nov., sp. nov., a phototrophic purple nonsulfur bacterium previously referred to as the “Rhodocyclus gelatinosus-like” group

Strains of the phototrophic bacteria previously referred to as the rhodocyclus gelatinosus-like (RGL) group were taxonomically studied in comparison with Rhodocyclus species. Cells of the RGL strains were curved rods and motile by means of polar flagella. They contained bacteriochlorophyll a and carotenoids of the spheroidene series. The intracytoplasmic membrane system was absent. Photoorganotropho with various organic compounds as carbon sources was the preferred mode of growth. Aerobic growth at full atmospheric oxygen tension and fermentative growth under anaerobic-dark conditions were also possible. The major cellular fatty acids were palmitoleic acid and palmitic acid, and 3-hydroxylated fatty acids with octanoic acid predominating were also found. Both ubiquinone-8 and rhodoquinone-8 occurred as major quinones. The mol% guanine plus cytosine of the DNAs varied between 59.8 and 60.3. DNA-DNA hybridization studies showed that the RGL strains were highly related to each other but exhibited low levels of the homology to Rhodocyclus species. These data allow the establishment of the RGL group as a new taxon of the purple nonsulfur bacteria, for which the name Rhodoferax fermentans gen. nov., sp. nov. is proposed.     

Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Hydrophobicity of ruminant mastitisStaphylococcus aureus in relation to bacterial aging and slime production

Hydrophobicity of 72 bovine and 53 ovine mastitisStaphylococcus aureus strains was studied throughout the logarithmic growth phase with a water-xylene two-phase system. Hydrophobicity increased during this growth phase. Freshly isolated strains were more hydrophobic than old strains (p<0.01). Old (bovine) strains became more hydrophobic (i.e., refreshed) after passage through the mouse mammary gland (p<0.01). Bovine strains were more hydrophobic than ovine strains (p<0.01). For the majority of strains, bacteria became more hydrophilic (p<0.001) after growth in exopolysaccharide-inducing media (Columbia and modifiedStaphylococcus 110 broth). This could be expected, since exopolysaccharides are hydrophilic. However, in these media, the strains that were able to produce slime in Congo red agar or in tryptic soy broth supplemented with 2% glucose (w/v) did not become more hydrophilic.It is proposed that different mechanisms may be involved in triggering exopolysaccharide production when different exopolysaccharide-inducing media are used.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol     

Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases

The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate-co-3-mercaptopropionate) [poly(3HB-co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75°C to 80°C and was stable at 70°C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and ¹H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB-co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB-co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.This publication is dedicated to Prof. Dr. Hans G. Schlegel in honor of his 80th birthday     

Secondary alcohol dehydrogenase from a vinyl alcohol oligomer-degrading Geotrichum fermentans; stabilization with Triton X-100 and activity toward polymers with polymerization degrees less than 20

An NAD+-dependent secondary alcohol dehydrogenase has been found in the cells of Geotrichum fermentans WF9101 grown on 2,4-pentanediol (the constitutional unit of vinyl alcohol polymers). Although the enzyme was unstable and lost its activity very rapidly, we found that it could be purified after stabilization in a buffer containing 0.01% Triton X-100. The molecular weight of the purified enzyme was 88,000 with subunit size of molecular weight 42,000. The enzyme, which was specific for the (S)-(+)-enantiomers, was active on various secondary mono-ols and diols, notably, 2-hexanol. The K_m values at 30 °C, pH 8.0, were 240 m for 2-hexanol and 48.7 m for NAD⁺. This enzyme showed activity toward vinyl alcohol polymers with average degrees of polymerization of less than 20, and was assumed to contribute on the degradation of poly(vinyl alcohol) in synergism with other microorganisms.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

Partial characterization of anrpoD-like gene oflactoccocus lactis subsp.lactis ML3 with a polymerase chain reaction-based approach

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of anrpoD-like gene ofLactoccocus lactis subsp.lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical RpoD box of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs inPediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, andLactococcus lactis subsp.cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.     

Microbial populations in trifluralin-treated soil

Summary This study examined the effects of trifluralin (,,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), a soil incorporated herbicide, on soil microflora both in the general soil environment and in the rhizosphere of trifluralin damaged wheat roots. Two Dark Brown Chernozemic soils were treated with various trifluralin rates in the growth chamber and wheat [Triticum aestivum L. Neepawa] was seeded. Trifluralin generally had no effect on fungi, bacteria, or actinomycete populations in either the general soil or in the rhizosphere. CO₂ evolution was unchanged when trifluralin was added to the soil. In wheat plots, at two field locations, there were no significant effects of trifluralin (1.0 kg ha^–1) on soil fungi, bacteria, actinomycete, denitrifying bacteria, and nitrifying Nitrobacter propulations. A pure culture study with 42 soil microorganisms showed that many isolates were inhibited at 400 to 100,000 g g^–1 but not at concentrations <16 g g^–1. Similar data were obtained from tests on four different soils. These studies indicate that trifluralin is unlikely to cause changes in the numbers of soil microorganisms when used at recommended levels.