Phenol-water Extraction Research Articles

Comparison of lipooligosaccharides from human challenge strains of Neisseria gonorrhoeae.

The alarming rise of antibiotic resistance and the emergence of new vaccine technologies have increased the focus on vaccination to control gonorrhea. Neisseria gonorrhoeae strains FA1090 and MS11 have been used in challenge studies in human males. We used negative-ion MALDI-TOF MS to profile intact lipooligosaccharide (LOS) from strains MS11mkA, MS11mkC, FA1090 A23a, and FA1090 1-81-S2. The MS11mkC and 1-81-S2 variants were isolated from male volunteers infected with MS11mkA and A23a, respectively. LOS profiles were obtained after purification using the classical phenol water extraction method and by microwave-enhanced enzymatic digestion, which is more amenable for small-scale work. Despite detecting some differences in the LOS profiles, the same major species were observed, indicating that microwave-enhanced enzymatic digestion is appropriate for MS studies. The compositions determined for MS11mkA and mkC LOS were consistent with previous reports. FA1090 is strongly recognized by mAb 2C7, an antibody-binding LOS with both α- and β-chains if the latter is a lactosyl group. The spectra of the A23a and 1-81-S2 FA1090 LOS were similar to each other and consistent with the expression of α-chain lacto-N-neotetraose and β-chain lactosyl moieties that can both be acceptor sites for sialic acid substitution. 1-81-S2 LOS was analyzed after culture with and without media supplemented with cytidine-5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which N. gonorrhoeae needs to sialylate its LOS. LOS sialylation reduces the infectivity of gonococci in men, although it induces serum resistance in serum-sensitive strains and reduces killing by neutrophils and antimicrobial peptides. The infectivity of FA1090 in men is much lower than that of MS11mkC, but the reason for this difference is unclear. Interestingly, some peaks in the spectra of 1-81-S2 LOS after bacterial culture with CMP-Neu5Ac were consistent with disialylation of the LOS, which could be relevant to the reduced infectivity of FA1090 in men and could have implications regarding the phase variation of the LOS and the natural history of infection.

Https://microbiolj.org.ua/en/archiv/2022-tom-84/6-nov-dec-tom-84/2022-84-6-02

Bacterial wetwood, bacterial dropsy, or bacterial slime is a common disease caused by Lelliottia nimipressuralis, which affects the central core of many conifers and deciduous trees. Representatives of this species have been isolated from a variety of trees showing symptoms of the disease, as well as from water and, less commonly, from clinical samples. Important aspects of pathogenesis is the process of pathogen recognition and the protection mechanisms of bacterial cells from plant’s antimicrobial substances. It is known that lipopolysaccharides (LPS) take an active part in these processes. They provide the barrier function of the outer membrane, helping to protect bacteria from plant antimicrobial compounds, and the attachment of bacteria to plant cells. Therefore, the aim of the work was to study the peculiarities of the chemical composition and functional and biological characteristics of Lelliottia nimipressuralis F9a1 LPS obtained by different methods. Methods. LPS was isolated from dry bacterial mass by phenol-water method (LPS I), extraction method with 0.85% NaCl solution (LPS II), and phenol-water extraction of LPS insoluble in NaCl solution (LPS III). The carbohydrates were analyzed by Dubois method, nucleic acids ‒ by Spirin, protein content ‒ by Lowry and 2-keto-3-deoxyoctonic acid (KDO) ‒ by Osborn. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromato-mass spectrometry system. The pyrogenicity of LPS was tested keeping the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. Results. LPS II of L. nimipressuralis F9а1 was characterized by low relative yield (2.12%), low content of carbohydrates (9.16%) and nucleic acids (3.7%), and high protein content (26.44%), while the studied preparations of LPS I and LPS III were characterized by a high yield, a rather high content of carbohydrates (46.68 and 38.4%, respectively), an insignificant amount of protein (up to 6.72%) and nucleic acids (up to 4.06%). All LPSs contained up to 0.27% KDO, which is a specific component of the LPS of gramnegative bacteria. The monosaccharide composition indicates that the LPS of the studied L. nimipressuralis strains turned out to be heterogeneous. At the same time, such monosaccharides as fucose, galactose, and glucose were recorded in the LPS of all tested strains. The fatty acid composition of LPS was represented by the presence of fatty acids containing from 12 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids were found. In LPS I and LPS III, the dominant fatty acid was 14:0 (3-OH), which is a kind of marker for the entire family of Enterobacteriaceae. In addition to the marker acid, a 16:1 acid was also predominant in LPS III, whereas in LPS II, 16:1 (32.7%) and 16:0 (22.6%) fatty acids dominated. The pyrogenic effect of L. nimipressuralis LPS studied showed that LPS solutions are pyrogenic. The serological studies showed that tested LPS in homologous systems exhibits antigenic activity. Antisera to L. nimipressuralis F9a1 react with LPS strains IMV 8791, LGK1, and L14b, which may indicate the presence of common antigenic determinants and belonging of these strains to the same serogroup. Conclusions. The LPSs of L. nimipressuralis F9a1 were heterogeneous in both monosaccharide and fatty acid composition, which is explained by the use of different methods for their isolation. To isolate LPS from L. nimipressuralis cells, the water-phenol method is better than sodium chloride extraction since with using the latter, the LPS yield is very low and very contaminated with proteins. At the same time, the isolation method does not affect the serological activity of the studied LPS. The results received during these biological-functional studies of L. nimipressuralis LPS contribute to the biological characteristics of this species.

Ruminal Lipid A Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli.

Extraction and Characterization of Lipopolysaccharide from Salmonella typhi

There is no standard process available for high purity LPS isolation, and it may be appropriate to mix two purification steps. For Salmonella typhi LPS that were subjected to normal microbiological & biochemical screening protocols, updated phenol-water extraction protocol & non-phenolic extraction methods were also used in present research. Pellet of crude LPS obtained with wet weight was 2.0 gm. yield of LPS was more by hot phenol method 1.94 mg/ml as compared to non-phenolic method 0.40 mg/ml. relative purity of LPS obtained by hot phenol method was more as compared to non-phenolic method, as protein content was 23.60 mg/ml in LPS extracted from hot phenol method & 26.62 mg/ ml in LPS extracted from non-phenolic method. However, Nucleic acid contain was comparable in LPS extracted from both methods. Qualitative analysis showed ladder like bands of LPS extracted by hot phenol method as compared to single band obtained for LPS extracted by non-phenolic method. Findings of silver staining clearly revealed ladder pattern of multi-rung bands that are characteristics of smooth form of gram negative bacteria due to difference in length of carbohydrate chain of O-antigen component. In order to clarify diseased conditions & better work on LPS profiling, this research may be crucial to lead to more successful diagnosis and care.

Lipopolysaccharide of Pantoea agglomerans 7460: O-specific polysaccharide and lipid A structures and biological activity

Lipopolysaccharide (LPS) was isolated from Pantoea agglomerans 7460 cells by phenol-water extraction. Mild acid degradation allowed to separate OPS and lipid A. Lipid A was analyzed by negative-ion mode ESI MS and found to consist mainly of hexaacylated derivative containing biphosphorylated GlcN disaccharide, four 14:0 (3-OH), 18:0 and 12:0 fatty acids. The structure of the O-specific polysaccharide was established by chemical, NMR and computational methods:▪The LPS of Р. agglomerans 7460 showed low level of toxicity and pyrogenicity to compare with LPS of E. coli O55:B5 and pyrogenal, respectively. The ability of the modified (succinylated) LPS, which have lost its toxicity, to block the toxic effects of native LPS has been shown.

Effect of nifedipine, a calcium channel blocker, on the generation of nitric oxide and interleukin-1β by murine macrophages activated by lipopolysaccharide from Prevotella intermedia.

Nifedipine, a calcium channel blocker, has been reported to possess anti-inflammatory and immunosuppressive effects. The current study was undertaken to explore the influence of nifedipine on the generation of proinflammatory mediators by murine macrophages activated by lipopolysaccharide (LPS) prepared from Prevotella intermedia, a putative periodontal pathogen, and associated mechanisms of action as well. LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1β. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent secreted embryonic alkaline phosphatase (SEAP) levels were estimated by reporter assay. Nifedipine markedly suppressed the generation of iNOS-derived NO and IL-1β together with their mRNA expressions in murine macrophages activated by P. intermedia LPS. LPS-stimulated cells exposed to nifedipine notably increased the mRNA levels of Arg-1, Ym-1, FIZZ1, and TGF-β, which are typical markers for M2 macrophage polarization. Nifedipine induced HO-1 at both gene and protein levels in cells challenged with P. intermedia LPS, and the nifedipine-mediated inhibition of NO generation was significantly abrogated by adding SnPP, an HO-1 inhibitor. Nifedipine inhibited LPS-evoked generation of NO and IL-1β in a PPAR-γ-independent manner. In addition, NF-κB activation as well as phosphorylation of STAT1/3 induced by P. intermedia LPS was suppressed by nifedipine. Nifedipine is an inhibitor of P. intermedia LPS-evoked production of NO and IL-1β in murine macrophages and encourages macrophage polarization toward the M2 phenotype. Nifedipine possibly has potential to be used for host modulation of periodontal disease and is worth being further researched.

Structural and genetic characterization of the colitose-containing O-specific polysaccharide from the lipopolysaccharide of Herbaspirillum frisingense GSF30T

The lipopolysaccharide (LPS) of Herbaspirillum frisingense GSF30T (HfGSF30), a non-pathogenic diazotrophic endobiont, was isolated by phenol-water extraction from bacterial cells and was characterized by chemical analyses and SDS PAGE. The O-specific polysaccharide (OPS, O-antigen), obtained by mild acid hydrolysis of the LPS, was examined by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The OPS was found to consist of branched tetrasaccharide repeating units of the following structure:→3)−α−L−Rhap−1→3−β−D−Galp−1→3−β−D−GlcpNAc−(1→2↑1α−ColpThis structure is unique among the known bacterial polysaccharide structures. Analysis of the HfGSF30 genome showed that it contained a set of sequentially arranged operons (presumably a cluster of genes) associated with the O-antigen. Amino acid sequence analysis using the BLAST program demonstrated the specificity of this putative cluster for Herbaspirillum spp. The genes responsible for the biosynthesis of the OPS of HfGSF30 were dispersed in the genome, constituting small operons. A putative O-antigen gene cluster of HfGSF30 was identified and found to be consistent with the OPS structure.

The Marine Dinoflagellate Alexandrium minutum Activates a Mitophagic Pathway in Human Lung Cancer Cells.

Marine dinoflagellates are a valuable source of bioactive molecules. Many species produce cytotoxic compounds and some of these compounds have also been investigated for their anticancer potential. Here, we report the first investigation of the toxic dinoflagellate Alexandrium minutum as source of water-soluble compounds with antiproliferative activity against human lung cancer cells. A multi-step enrichment of the phenol–water extract yielded a bioactive fraction with specific antiproliferative effect (IC50 = 0.4 µg·mL−1) against the human lung adenocarcinoma cells (A549 cell line). Preliminary characterization of this material suggested the presence of glycoprotein with molecular weight above 20 kDa. Interestingly, this fraction did not exhibit any cytotoxicity against human normal lung fibroblasts (WI38). Differential gene expression analysis in A549 cancer cells suggested that the active fraction induces specific cell death, triggered by mitochondrial autophagy (mitophagy). In agreement with the cell viability results, gene expression data also showed that no mitophagic event was activated in normal cells WI38.

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Structure of the O-polysaccharide of Yersinia frederiksenii H56-36/81 (serotype O:60) containing 4-deoxy-d-arabino-hexose

Aiming at creation of the chemical basis for serotyping of strains of Yersinia spp., we studied the O-specific polysaccharide (OPS) of Yersinia frederiksenii H56-36/81 belonging to serotype O:60. A branched polysaccharide was obtained by mild alkaline degradation of the lipopolysaccharide (LPS) isolated from bacterial cells by the phenol—water extraction and found to contain d-ribose, l-rhamnose, and 4-deoxy d-arabino-hexose (d-ara4dHex). The last monosaccharide occurs rarely in nature and earlier has been found only in OPS of Citrobacter spp. Mild acid hydrolysis of the LPS resulted in cleavage of the d-ara4dHex side-chains to give a linear polysaccharide. Structure of the hexasaccharide repeating unit of the OPS was established by studies of both isolated polysaccharides by 1D and 2D 1H and 13C NMR spectroscopy.

Elucidation of a masked repeating structure of the O-specific polysaccharide of the halotolerant soil bacteria Azospirillum halopraeferens Au4.

An O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide isolated by the phenol–water extraction from the halotolerant soil bacteria Azospirillum halopraeferens type strain Au4. The polysaccharide was studied by sugar and methylation analyses, selective cleavages by Smith degradation and solvolysis with trifluoroacetic acid, one- and two-dimensional 1H and 13C NMR spectroscopy. The following masked repeating structure of the O-specific polysaccharide was established: →3)-α-L-Rhap2Me-(1→3)-[β-D-Glcp-(1→4)]-α-D-Fucp-(1→2)-β-D-Xylp-(1→, where non-stoichiometric substituents, an O-methyl group (~45%) and a side-chain glucose residue (~65%), are shown in italics.

Structure of fructans from three strains of Cronobacter dublinensis

Partially O-acetylated fructan was isolated by phenol—water extraction of cells of Cronobacter dublinensis G2732 followed by anion-exchange chromatography on DEAE-Toyopearl 650M. Structure of the isolated fructan was established by 2D 1H and 13C NMR spectroscopy. The same fructan but lacking O-acetyl groups was isolated from two other C. dublinensis strains, G3947 and G4061. Genes for the fructan biosynthesis were not found at the typical Cronobacter O-antigen gene cluster located between the conserved genes galF and gnd and are evidently located elsewhere on the chromosome.

Structure elucidation of the capsular polysaccharide of Acinetobacter baumannii AB5075 having the KL25 capsule biosynthesis locus

Capsular polysaccharide was isolated by the phenol–water extraction of Acinetobacter baumannii AB5075 and studied by 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established:→3)-β-d-ManpNAcA-(1→4)-β-d-ManpNAcA-(1→3)-α-d-QuipNAc4NR-(1→where R indicates (S)-3-hydroxybutanoyl or acetyl in the ratio∼2.5:1. The genes in the polysaccharide biosynthesis locus designated KL25 are appropriate to the established CPS structure.

Structure of the capsular polysaccharide of Acinetobacter baumannii 1053 having the KL91 capsule biosynthesis gene locus

Acinetobacter baumannii 1053 is the type strain for the maintenance of specific bacteriophage AP22, which infects a fairly broad range of A. baumannii strains circulating in Russian clinics and hospitals. A capsular polysaccharide (CPS) was isolated from cells of strain 1053 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit was established: [Display omitted] where ManNAcA and FucNAc indicate 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2,6-dideoxygalactose, respectively. A polysaccharide having the same repeating unit but a shorter chain was isolated by the phenol–water extraction of bacterial cells. Sequencing of the CPS biosynthesis gene locus showed that A. baumannii 1053 belongs to a new group designated KL91. The gene functions assigned putatively by a comparison with available databases were in agreement with the CPS structure established.

Structure and gene cluster of the O antigen of Escherichia coli L-19, a candidate for a new O-serogroup.

Escherichia coli L-19 isolated from a healthy individual did not agglutinate with any of 21 polyvalent antisera that cover 174 E. coli O-serogroups. The strain was studied in respect to the O-antigen (O-specific polysaccharide, OPS) structure and genetics. The LPS was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the OPS. The OPS was studied by sugar and methylation analyses, along with 1D and 2D (1)H and (13)C NMR spectroscopy. The established structure of the linear tetrasaccharide repeating unit was found to be unique among known bacterial polysaccharide structures. A peculiar component of the L-19 OPS was an amide of glucuronic acid with 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol) (GroN). The O-antigen gene cluster of L-19 between the conserved genes galF and gnd was sequenced, and gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the OPS structure. Except for putative genes for synthesis and transfer of GroN, the sequences in the L-19 O-antigen gene cluster were little related to those of reference strains of the 174 known E. coli O-serogroups. The data obtained suggest that L-19 can be considered as a candidate for a new E. coli O-serogroup.

Lipopolysaccharide and outer membrane proteins of Leptospira interrogans induce macrophage apop-tosis via Fas/FasL pathway

Objective To investigate the role of pathogenic Leptospira interrogans lipopolysaccha-ride （L-LPS） and outer membrane proteins （L-OMP） in the apoptosis of mouse macrophages （J774A.1） and their association with Fas/FasL pathway .Methods Phenol-water extraction and Triton X-114 phase separation were used to extract L-LPS and L-OMP from L.interrogans serogroup icterohaemorrhagiae serovar Lai strain Lai, respectively.Polymyxin B （ PMB） and protease K （ PK） were used to treat L-LPS and L-OMP, respectively.J774A.1 cells were stimulated by L.interrogans strain Lai with or without ultraviolet inactivation.In parallel, the cells were stimulated by extracted L-LPS and L-OMP with or without PMB and PK treatments .The apoptosis and necrosis of J 774 A.1 cells before and after treatment were detected by flow cytometry.The siRNAs were used to silence the expression of Fas or FasL gene in J 774A.1 cells and their inhibitory effects were further validated by using real-time fluorescent quantitative RT-PCR.Flow cytometry was used to detect the effects of L-LPS or L-OMP on the apoptosis of J774A.1 cells with Fas or FasL gene-knockdown .Results L.interrogans strain Lai with or without ultraviolet inactivation could cause similar early apoptosis rates （47.1%and 55.6%） and late apoptosis/necrosis rates （7.6%and 7.9%）.The ear-ly apoptosis rates of 1×10^5 J774A.1 cells were 40.4%and 34.0%after the treatment with 100 ng of L-LPS and 100 μg of L-OMP for 4 h.The late apoptosis/necrosis rates of the cells were 7.5%and 6.9%upon the treatments with L-LPS and L-OMP, respectively.However, the apoptosis or necrosis of the cells was not ob-served when using L-LPS and L-OMP pre-treated by PMB and PK, respectively.Silenced expression of Fas or FasL gene reduced the L-LPS-induced J774A.1 cells apoptosis （P〈0.05）, while decreased early apopto-sis rate of J774A.1 cells mediated by L-OMP was only observed in Fas gene-knockdown cells （P〈0.05）. Conclusion Both L-LPS and L-OMP can cause the Fas/FasL-associated apoptosis of macrophages , which is beneficial for L.interrogans to establish the productive infection in hosts . Key words: Fas/FasL; Leptospira interrogans; Lipopolysaccharide/outer membrane proteins; Macrophage/apoptosis; Fas/FasL

Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14

Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol–water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2: [Display omitted] Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko et al., 2011]6 whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Structure of the capsular polysaccharide of Acinetobacter baumannii ACICU containing di-N-acetylpseudaminic acid

Capsular polysaccharide was isolated by the phenol–water extraction of Acinetobacter baumannii ACICU cells and studied by sugar analysis, partial acid hydrolysis, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide was found to contain 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic or di-N-acetylpseudaminic acid (Pse5Ac7Ac), and the following structure of the branched tetrasaccharide repeating unit was established: [Display omitted] The genes present in the polysaccharide gene cluster of A. baumannii ACICU are appropriate to the structure established.

Antioxidant capacity in urban soils

The quality of soils in 31 urban green areas in Pisa was assessed in terms their soil antioxidant systems by measuring antioxidant capacity (TEAC) and phenol substances, soil basal respiration, community level physiological profile (CLPP), expressed as average well color development (AWCD), community metabolic diversity (CMD), the Shannon–Weaver index (H), and soil enzyme activities. The urban results were compared to an extra-urban control area (near the S. Rossore-Migliarino-Massaciuccoli Natural Park).The soils of the greenspaces of Pisa city were mostly sandy, subalkaline, lightly calcareous, with a rather high (mean of 3.27±1.24gC100g−1drysoil) and variable (1.32–7.57gC100g−1drysoil) organic matter content.There were little differences in the functional diversity (AWCD) of soil microbial communities. Dehydrogenase, catalase, alkaline phosphatase, β-glucosidase and lipase showed little variability among soils while arylsulphatase, protease and urease activities varied within a fairly wide range of values. Values of the alkali-and water-soluble TEAC of urban soils varied within quite large ranges (2.53–11.45mMg−1soil and 0.11–2.91mMg−1drysoil, respectively) and were generally higher than those of control. TEAC and phenol substances, both in alkaline and water extracts, were closely correlated (r>0.850, P≤0.01) and were also positively correlated with soil organic C, AWCD, CMD and, H. With the exception of dehydrogenase, the soil antioxidant system showed positive correlations with the enzymatic activities and soil basal respiration. The antioxidant system and soil basal respiration can be considered useful indicators of the soil biological status and soil quality in the examined urban soils.

Structural studies of the O-specific polysaccharide(s) from the lipopolysaccharide of Azospirillum brasilense type strain Sp7

Lipopolysaccharide was obtained by phenol–water extraction from dried bacterial cells of Azospirillum brasilense type strain Sp7. Mild acid hydrolysis of the lipopolysaccharide followed by GPC on Sephadex G-50 resulted in a polysaccharide mixture, which was studied by composition and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy. The following polysaccharide structures were established, where italics indicate a non-stoichiometric (∼40%) 2-O-methylation of l-rhamnose.▪