Lysis of cells of diverse bacteria by l,d-peptidases of Escherichia coli bacteriophages RB43, RB49 and T5.

AIMS: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. METHODS AND RESULTS: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4ß) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. CONCLUSIONS: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

Two Glycosyl 1-Phosphate Polymers and Teichulosonic Acid from Glutamicibacter protophormiae VKM Ac-2104T Cell Wall.

Two glycosyl 1-phosphate polymers containing monoglycosyl 1-phosphate, -6)-α-D-Glcp-(1-P-, and diglycosyl 1-phosphate, -6)-α-D-GalpNAc-(1→6)-α-D-GlcpNAc-(1-P-, in the repeating unit were identified in the cell wall of Glutamicibacter protophormiae VKM Ac-2104T (formerly, Arthrobacter protophormiae). The structures of these polymers were described for the first time in prokaryotes. Teichulosonic acid, the third identified polymer, with 3-deoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the main chain, →6)-ß-D-Glcp-(1→8)-α-Kdn-(2→, has been previously detected in a number of actinobacteria. The structures of these glycopolymers were established based on the results of chemical analysis and one-dimensional 1H, 13C, and 31P NMR spectroscopy using two-dimensional homonuclear (1H,1H COZY, TOCSY, ROESY) and heteronuclear (1H,13C HSQC, HSQC-TOCSY, HMBC, and 1H,31P HMBC) techniques.

Rhamnomannans and Teichuronic Acid from the Cell Wall of Rathayibacter tritici VKM Ac-1603T.

The structures of three cell wall glycopolymers of the phytopathogen Rathayibacter tritici VKM Ac-1603T (family Microbacteriaceae, order Micrococcales, class Actinobacteria) were established by chemical methods and NMR spectroscopy. Polymer 1 is a branched rhamnomannan with the repeating unit →3)-α-[ß-D-Xylp-(1→2)]-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 2 is a linear rhamnomannan with the repeating unit →2)-α-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→; polymer 3 is a branched teichuronic acid containing monosaccharide residues GlcA, Gal, Man, and Glc at a 1 : 1 : 1 : 5 ratio (see the text for the structures). It has been demonstrated that representatives of four Rathayibacter species studied to date (R. tritici VKM Ac-1603T, R. iranicus VKM Ac-1602 T, R. toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596) contain differing patterns of phosphate-free glycopolymers. At the same time, the above Rathayibacter strains have a common property - the presence of rhamnomannans with D-rhamnose. These rhamnomannans may be linear or branched and differing in the positions of glycosidic bonds and side substituents. The presence in the cell wall of rhamnomannans with D-rhamnose may serve as useful chemotaxonomic marker of the genus Rathayibacter.

Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596.

Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers - a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2 : 4 : 1, respectively (the structure is presented in the text). The "Rathayibacter tanaceti" VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.

Erratum to: "Rhamnose-Containing Cell Wall Glycopolymers from Rathayibacter toxicus VKM Ac-1600 and "Rathayibacter tanaceti" VKM Ac-2596" [Biochemistry (Moscow), 83, 717 (2018)].

This corrects the article DOI: 10.1134/S0006297918060093.

[Molecular biological characterization of biphenyl-degrading bacteria and identification of the biphenyl 2,3-dioxygenase α-subunit genes].

Bacterial isolates from soils contaminated with (chlorinated) aromatic compounds, which degraded biphenyl/chlorinated biphenyls (CB) and belonged to the genera Rhodococcus and Pseudomonas were studied. Analysis of the 16S rRNA gene sequences was used to determine the phylogenetic position of the isolates. The Rhodococcus cells were found to contain plasmids of high molecular mass (220-680 kbp). PCR screening for the presence of the bphA1 gene, a marker indicating the possibility for induction of 2,3-dioxygenase (biphenyl/toluene dioxygenase subfamily) revealed the presence of the bphAl genes with 99-100% similarity to the homologous genes of bacteria of the relevant species in all pseudomonad and most Rhodococcus isolates. A unique bphA1 gene, which had not been previously reported for the genus, was identified in Rhodococcus sp. G10. The absence of specific amplification of the bphA1 genes in some biphenyl-degrading bacteria (Rhodococcus sp. B7b, B106a, G12a, P2kr, P2(51), and P2m), as well as in an active biphenyl degrader Rhodococcus ruber P25 indicated the absence of the genes encoding the proteins of the biphenyl/toluene dioxygenase subfamily and participation of the enzymes other than this protein family in biphenyl/CB degradation.

Structure of hexasaccharide 1-phosphate polymer from Arthrobacter uratoxydans VKM Ac-1979(T) cell wall.

A hexasaccharide 1-phosphate polymer of original structure and two teichoic acids (TA) belonging to different structural types were found in Arthrobacter uratoxydans VKM Ac-1979(T) cell wall. The poly(hexasaccharide 1-phosphate) combines features of teichuronic acids and glycosyl 1-phosphate polymers, and its structure has never been reported earlier. Its composition includes residues of α- and ß-D-glucuronic acid as well as α-D-galacto-, ß-D-gluco-, α-D-mannopyranose, and 6-O-acetylated 2-acetamido-2-deoxy-α-D-glucopyranose. The phosphodiester bond in the polymer joins the glycoside hydroxyl of α-D-glucuronic acid and O6 of α-D-galactopyranose. TA 1 is ß-D-glucosylated 1,3-poly(glycerol phosphate), and TA 2 is 3,6-linked poly[α-D-glucosyl-(1→2)-glycerol phosphate]. The phosphate-containing polymers were studied by chemical methods and on the basis of one-dimensional 1H-, 13C-, and (31)P-NMR spectra, homonuclear two-dimensional (1)H/(1)H COSY, TOCSY, ROESY, and heteronuclear (1)H/(13)C HSQC, HSQC-TOCSY, HMBC, and (1)H/(31)P HMBC experiments. The set and structure of the polymers revealed as well as the cell wall sugars (galactose, glucose, mannose, glucosamine) and glycerol can be used in microbiological practice for taxonomic purposes.

[Isoprenoid pigments in representatives of the family Microbacteriaceae].

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.

[Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups].

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.

[Terminal oxidases in different genera of the family Microbacteriaceae]. / Terminal'nye oksidazy predstavitelei razlichnykh rodov semeistva Microbacteriaceae.

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa3-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb3- and aa3-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.

[A new aerobic gram-positive bacterium with a unique ability to degrade ortho- and para-chlorinated biphenyls]. / Novyi aérobnyi grampolozhitel'nyi mikroorganizm s unikal'nymi svoistvami destruktsii orto- i para-khlorirovannykh bifenilov.

Strain B51 capable of degrading polychlorinated biphenyls (PCB) was isolated from soil contaminated with wastes from the chemical industry. Based on its morphological and chemotaxonomic characteristics, the strain was identified as a Microbacterium sp. Experiments with washed cells showed that strain B51 is able to degrade ortho- and para-substituted mono-, di-, and trichlorinated biphenyls (MCB, DCB, and TCB, respectively). Unlike the known PCB degraders, Microbacterium sp. B51 is able to oxidize the ortho-chlorinated ring of 2,2'-DCB and 2,4'-DCB and the para-chlorinated ring of 4.4'-DCB. The degradation of 2,4'-DCB and 4,4'-DCB was associated with the accumulation of 4-chlorobenzoic acid (4-CBA) in the medium in amounts comprising 80-90% of the theoretical yield. The strain was able to utilize 2-MCB, 2,2'-DCB, and their intermediate 2-CBA and to oxidize the mono(ortho)-chlorinated ring of 2,4,2'-TCB and the di(ortho-para)-chlorinated ring of 2,4,4'-TCB. A mixed culture of Microbacterium sp. B51 and the 4-CBA-degrading bacterium Arthrobacter sp. H15 was found to grow well on 1 g/l 2,4'-DCB as the sole source of carbon and energy.

Agreia bicolorata gen. nov., sp. nov., to accommodate actinobacteria isolated from narrow reed grass infected by the nematode Heteroanguina graminophila.

Agreia bicolorata gen. nov., sp. nov. (type strain VKM Ac-1804T=UCM Ac-620T) is proposed to accommodate aerobic, oxidase- and catalase-positive, weakly motile, coryneform actinobacteria isolated from leaf galls induced by the plant-parasitic nematode Heteroanguina graminophila in narrow reed grass, Calamagrostis neglecta. Bacteria assigned to Agreia bicolorata gen. nov., sp. nov. form a distinct lineage within the phylogenetic branch of the family Microbacteriaceae and possess the following chemotaxonomic characteristics: B-type peptidoglycan containing 2,4-diaminobutyric acid, ornithine, alanine, glycine, glutamate and hydroxyglutamate; cell wall sugars rhamnose, fucose and mannose; MK-10 as major menaquinone; phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids; and 12-methyltetradecanoic acid (anteiso-15:0), 14-methyl-pentadecanoic acid (iso-16:0) and 14-methyl-hexadecanoic acid (anteiso-17:0) as predominant fatty acids. The DNA G+C content of Agreia bicolorata is about 67.0 mol %.

Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa annua, and reclassification of 'Corynebacterium aquaticum' Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov.

The new genus Leifsonia gen. nov. with two new species, Leifsonia poae sp. nov. (type strain VKM Ac-1401T) and Leifsonia aquatica (ex Leifson 1962) nom. rev., comb. nov. (the type species, with VKM Ac-1400T = DSM 20146T = JCM 1368T as type strain), is proposed to accommodate bacteria found in Poa annua root gall, induced by the nematode Subanguina radicicola, and 'Corynebacterium aquaticum' Leifson 1962. Further, it is proposed to reclassify Clavibacter xyli Davis et al. 1984 with two subspecies in the new genus as Leifsonia xyli (Davis et al. 1984) comb. nov., Leifsonia xyli subsp. xyli (Davis et al. 1984) comb. nov. and Leifsonia xyli subsp. cynodontis (Davis et al. 1984) comb. nov. Members of the proposed genus are characterized by coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, the major menaquinone MK-11, phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids, the high content of anteiso- and iso-branched saturated fatty acids, and a DNA G+C base composition of 66-73 mol%. They form a distinct phylogenetic branch attached to the line of descent of Agromyces spp. The new and reclassified species of the new genus clearly differ from each other phylogenetically and phenetically and can be recognized by their morphologies, the cell wall sugar composition, the requirement of complex media for growth, and numerous physiological characteristics, including the oxidase reaction.

Further-attenuated measles vaccine: virus passages affect viral surface protein expression, immunogenicity and histopathology pattern in vivo.

Measles vaccine viruses Leningrad-16 (L-16) and Moscow-5 (M-5, an L-16-derived clonal variant), at passage levels used for vaccination and after ten further low-multiplicity passages on quail embryo (QE) cells, were compared for (1) immunogenicity, (2) histopathological lesions induced in vivo and (3) surface protein expression within infected cells and on the virion surface. At the 10th passage, viruses evoked a poorer neutralizing antibody response in guinea pigs, induced an earlier appearance of more pronounced pathological lesions and replicated faster in Vero cells than the original viruses. H protein expression increased 1.8-2.3-fold after 10 passages of the L-16 variant, but remained virtually unaltered for the M-5 variant. F protein expression of both 10th-passage variants was 0.5-0.8 that of the original virus variants. A similar two-fold decrease in F protein expression was noted after a single virus passage in guinea pigs. The data implicate the loss of F protein as a cause of reduced immunogenicity of further attenuated measles vaccines.

Experimental-scale measles and mumps vaccine production on microcarrier-grown cells.

Small-scale measles and mumps virus propagation in microcarrier-grown cells was studied to assess putative advantages over conventional roller-type virus propagation. Significantly higher virus yields could not be attained with microcarrier cultures in cell stirrers, therefore making the advantages purely technological. The pattern of measles virus production was slightly different for the three types of microcarriers used. Experimental measles and mumps vaccine lots obtained met vaccine quality control requirements.

[Effect of magnesium sulfate on the reproduction of the measles virus in cell culture]. / Vliianie sul'fata magniia na reproduktsiiu virusa kori v kul'ture kletok.

Magnesium sulphate in concentrations of 25-50 mM induced reproducible increase in titers of extracellular measles virus (by 0.5-2.0 1g TCD50/0.5 ml) in Japanese quail embryo cells. MgSO4 effect was observed with all methods of cell cultivation: stationary, roller, or on microcarriers. Its effect was associated not with its stabilizing influence on the extracellular virus but rather with the stimulation of the synthesis of intracellular viral proteins.

Salt-induced enhancement of measles virus yields in cultured cells.

Supplementation of culture medium with MgSO4 or Na2SO4 in millimolar concentrations caused an enhanced measles virus (MV) yield from cultured quail embryo cells. MgSO4 at 25-50 mM concentrations exhibited the most pronounced and consistent stimulatory effect. MV infectivity increases ranged from 2- to 200-fold; the effect was highly reproducible for stationary monolayer, roller or microcarrier-grown cell culture types. MgSO4 also improved MV plaque development and caused MV plaque size enlargement on Vero cell monolayers. At mM concentrations MgSO4 was not operative as a MV thermostabilizing agent; rather, salt-induced enhancement of MV yields appeared to be due to intracellular events, e.g., augmented viral protein synthesis.

[Study of measles virus (strain L-16) in J-96 cell culture by electron microscopy and immunofluorescence]. / Issledovanie virusa kori (shtamm L-16) v kul'ture kletok J-9 6 metodami élektronnoi mikroskopii i immunfliuorestsentsii

The vaccine L-16 strain of measles virus was studied in a continuous line of J-96 cells (clone L-41) by the electron microscope and fluorescent antibody techniques. Cytological studies revealed a direct correlation between the intensity of symplast formation and the infective virus dose. The fluorescent antibody technique established cytoplasmic localization of the specific virus antigen. The results of electron microscope examinations of ultrathin sections of J-97 culture revealed in the cell cytoplasm and intercellular space some structures morphologically similar to virus-like particles previously described.