光强和氮源对念珠藻胞外多糖分泌的影响

胞外多糖（EPS）是结皮蓝藻形成生物结皮的胶结剂,为了理解常球状存在的丝状蓝藻Nostoc胶结沙粒的机理,探讨了光强40、80 E/（m2s）和氮源（气态氮,硝态氮）对结皮优势种Nostoc sp.分泌EPS（包括荚膜多糖CPS和释放多糖RPS）的影响规律及其内在机理。结果发现:Nostoc sp.在气态氮和硝态氮下都有相似的快速生长,但其分泌的RPS、CPS及EPS量,在硝态氮下均随光强的增加而增加,在气态氮下却与光强没有关系。相关代谢研究发现,在硝态氮下细胞内有更高含量的可溶性糖和蔗糖。进一步的相关分析发现,在两种氮源下,蔗糖量与RPS量或CPS量间的显著正相关都只发生在80 E/（m2s）下,在气态氮中,两光强下的胞内总糖量都与CPS量显著负相关。以上结果说明,Nostoc sp.在氮源利用和光强适应方面都有明显优势,它即使在快速生长的对数期,也可同时分泌相当量的EPS,这使其在球状藻殖段形成之前胶结沙粒成为可能。由此可推知,Nostoc sp.在贫瘠沙土表面的最初生长过程中,其胞外的EPS均来自胞内的固碳产物,在高光强下,蔗糖很可能是其EPS合成的原料。       

Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides

Abstract The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-β-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolli (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.     

Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids.

The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

Staphylococcus aureus growth and type 5 capsular polysaccharide production in synthetic media.

The production of type 5 capsular polysaccharide by Staphylococcus aureus in synthetic media was investigated. The influence of medium components on capsular polysaccharide synthesis appeared to relate to the presence or absence of the component rather than to concentration gradient. The production of type 5 capsular polysaccharide was linked to energy availability and energy source, but not to carbohydrate concentration or carbon/nitrogen ratio. Regulation of capsular polysaccharide production by S. aureus in response to medium changes would appear to differ from that typically displayed in other organisms that produce polysaccharides.     

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.     

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.     

NOSTOC (CYANOPHYCEAE) GOES NUDE: EXTRACELLULAR POLYSACCHARIDES SERVE AS A SINK FOR REDUCING POWER UNDER UNBALANCED C/N METABOLISM1

Many species of the filamentous N₂‐fixing heterocyst‐forming Cyanobacteria of the genus Nostoc produce large amounts of extracellular polymeric substances (EPS), but hitherto no general model has been proposed of the factors that control their synthesis. Previously, we demonstrated a strong correlation between the presence of a glycocalyx (or EPS capsule) and diazotrophic growth in the genus Nostoc. When grown with nitrate, nude morphotypes lacking a glycocalyx were obtained for all the capsulated strains tested. CO₂ availability was pro‐posed as a key factor that controls the synthesis of the capsule. To test this hypothesis, Nostoc PCC 7936 was cultured diazotrophically (N₂) or with nitrate with different CO₂ supplies. By tuning the pH and the supply of CO₂, capsulated or nude mor‐photypes were obtained irrespective of the source of nitrogen. Exocellular polysaccharides were synthesized only when the fixed carbon exceeded the amount of nitrogen available. The glycocalyx is not needed for the optimal functioning of nitrogenase because diazotrophic cultures grew equally well, irrespective of whether they were capsulated or nude. Capsulated cultures possessed protein to carbohydrate ratios that ranged between 1 and 1.5, whereas in nude cultures the ratio ranged between 2 and 2.5. Low protein to carbohydrate ratios were indicative for either nitrogen‐limited or carbon‐oversaturated cultures. The results demonstrate that in Nostoc EPS serve as a sink for the excess fixed carbon under unbalanced C/N metabolism.     

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.     

Characterization of exopolysaccharides produced by seven biofilm-forming cyanobacterial strains for biotechnological applications

The molecular identification of seven biofilm-forming cyanobacteria and the characterization of their exopolysaccharides were made and considered in terms of potential biotechnological applications. The studied strains were isolated from phototrophic biofilms taken from various Italian sites including a wastewater treatment plant, an eroded soil, and a brackish lagoon. The polysaccharides were characterized by use of ion exchange chromatography, circular dichroism, and cytochemical stains. All strains produced exopolysaccharides with differing ratios of hydrophobic and hydrophilic moieties depending on the species, the polysaccharide fraction (i.e., whether capsular or released), and the ambient conditions. It was shown that the anionic nature of the exopolysaccharides was due to the presence of carboxylic and sulfated groups and is likely the main characteristic with industrial applicability. Potential biotechnological applications are discussed.     

Biofilm formation by exopolysaccharide mutants of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NRRL B-1355

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.     

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.     

Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti.

The pathways of polysaccharide biosynthesis were investigated in cells of Sinorhizobium meliloti (strain Su47) using a stable isotope approach. The isotopic labeling of the periplasmic beta-1,2-glucans synthesized from glucose labeled at various positions evidenced the involvement of catabolic pathways, namely the pentose-phosphate and Entner-Doudoroff pathways, into the early steps of polysaccharide synthesis. The exopolysaccharides produced at the same time had a labeling pattern similar to that of the beta-glucans, indicating similar early steps for both polysaccharides. The results emphasized a cyclic organization of the carbohydrate metabolism in S. meliloti, in which the carbons of the initial hexose were allowed to re-enter the catabolic pathways many times. The metabolic incidences of such metabolic topology are discussed.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Characterization of Neisseria meningitidis capsular polysaccharides containing sialic acid by pyrolysis mass spectrometry

The group B, C, W-135, and Y capsular polysaccharides of Neisseria meningitidis which contain sialic acid were differentiated by Curie-point pyrolysis low-voltage mass spectrometry. A large series of partially purified group B polysaccharide preparations obtained from pathogenic as well as nonpathogenic strains were analyzed by the same technique. It was shown that the carbohydrate structure of these group B polysaccharides appears to be the same throughout the whole series. Slight immunogenicity of some of the group B polysaccharide preparations tested is probably due to protein impurities. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turns out to be a rapid method of characterizing microgram samples of large series of polysaccharide preparations.     

Effects of nitrogen source on the synthesis of the UV-screening compound, scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102

The effects of nitrogen source (N(2), NO(3)(-) and NH(4)(+)) on scytonemin synthesis were investigated in the heterocystous cyanobacterium Nostoc punctiforme PCC 73102. With the required UVA radiation included, Nostoc synthesized three to seven times more scytonemin while fixing nitrogen than when utilizing nitrate or ammonium. A similar increase in scytonemin synthesis occurred when nitrate or ammonium became depleted by growth and Nostoc switched to diazotrophic metabolism with the differentiation of heterocysts. In addition, UVA-exposed cultures grown in medium with both NO(3)(-) and NH(4)(+) synthesized some scytonemin but synthesis increased when NH(4)(+) was depleted and growth had become dependent on NO(3)(-) reduction. Although the mechanism is unclear, these results suggest that the greater the restriction in nitrogen accessibility, the greater the production of scytonemin. Perhaps the entire response may be an interaction between this restriction and a resultant sensitivity to UV radiation that acts as a cue for determining the level of scytonemin synthesis. Scytonemin is a stable UVR screening compound and appears to be synthesized by cyanobacteria as a long-term solution for reducing UVR exposure and damage, but mainly or solely, when metabolic activity is absent. It is likely that during metabolic resurgence, the presence of a dense scytonemin sheath would facilitate the recovery process without the need for active defenses against UV radiation.