Carboxypeptidase Activity of the Zinc Metalloprotease in the Cement Precursor Secretion of the Barnacle, Chthamalus fragilis Darwin

The liquid cement precursor secretion (CPS) of the adult barnacle, Chthamalus fragilis, exhibits Zn-metalloprotease activity. To assess the bond specificity of the Zn-metalloprotease, samples of liquid CPS were collected by glass micropipette from the exposed bases of adult barnacles and incubated in 50 mM Tris buffer, pH 8.0, containing 10 mM Ca⁺⁺, at 27°C or 20°C for 6 or 24 hr. in the presence of fluorescent dye-labeled synthetic C-1 and A-1 PepTag peptides. These peptides have a net positive charge and migrate toward the anode during agarose gel electrophoresis. When incubated with the C-1 PepTag peptide, CPS samples generated F1 hydrolysis fragments that failed to migrate during electrophoresis in 0.8% agarose gels, indicating the presence of proteolytic activity in the CPS. Proteolysis of the C-1 peptide was inhibited by 2.0 mM orthophenanthroline in the presence of 10 mM Ca⁺⁺ ions. No hydrolysis products were generated when samples of CPS were incubated in the presence of the A-1 PepTag peptide. This suggested that the CPS contained a Zn-metalloprotease that exhibited a preference for the carboxy-terminal lysine of the C-1 PepTag peptide. There were no indications of aminopeptidase or endopeptidase activities in the barnacle CPS. When incubated with hippuryl-lysine and hippuryl-arginine substrates, samples of CPS gave activities ranging from 111 to 319 μmol hippuric acid formed/min/mg of CPS protein. These observations indicate that the CPS of C. fragilis contains a Zn-metallo-exoprotease with a preference for carboxy-terminal basic amino acids.     

Consequences of cps mutation of Klebsiella pneumoniae on 1,3-propanediol fermentation

The filtration in 1,3-propanediol (1,3-PD) downstream process is influenced by the large amounts of capsular polysaccharides (CPS) produced by Klebsiella pneumoniae CGMCC 1.6366. The morphological and fermentation properties were investigated with the CPS-deficient mutant K. pneumoniae CGMCC 1.6366 CPS. Similar biomass was obtained with CGMCC 1.6366, and the mutant strain in batch cultures indicating the cell growth was slightly inhibited by CPS defection. The viscosity of fermentation broth by mutant strain decreased by 27.45%. The flux with ceramic membrane filter was enhanced from 168.12 to 303.6 l h⁻¹ m⁻², exhibiting the great importance for downstream processing of 1,3-PD fermentation. The products spectrum of mutant isolate changed remarkably regarding to the concentration of fermentation products. The synthesis of important 1,3-PD and 2,3-butanediol was enhanced from 9.73 and 4.06 g l⁻¹ to 10.37 and 4.77 g l⁻¹ in batch cultures. The noncapsuled K. pneumoniae provided higher 1,3-PD yield of 0.54 mol mol⁻¹ than that of encapsuled wild parent in batch cultures. The fed-batch fermentation of mutant strain resulted in 1,3-PD concentration, yield, and productivity of 78.13 g l⁻¹, 0.53 mol mol⁻¹, and 1.95 g l⁻¹ h⁻¹, respectively.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Anti-Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti-chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)- and weakly (W)-encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H- < W-strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan-activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan-activated serum was incubated with heat-killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O-acetyl groups in their molecules by ¹H-nuclear magnetic resonance spectroscopy. The de-O-acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de-O-acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti-chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O-acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.     

Gel-forming capsular polysaccharide of fast-growing rhizobia: occurrence and rheological properties

Summary In addition to the excretion of soluble acidic polysaccharides many fast-growing rhizobia deposit insoluble neutral capsular polysaccharide (CPS), which is composed of d-mannose, d-galactose, and d-glucose in the ratios 1:4:1. CPS was found to occur in all strains of Rhizobium leguminosarum and R. trifolii. Synthesis takes place in the stationary phase of growth, but the extent of synthesis differs widely for individual strains. CPS was not found in the species R. phaseoli and R. meliloti. CPS can be extracted from the cell pellet with N NAOH and the so obtained material is notable for its gelling character. It is insoluble in cold water and dissolves in hot water to a clear solution. On cooling to room temperature the solution solidifies to a resilient gel at a setting point of 40–45° C, and remelts on heating at 50–55° C. Gel strength of CPS in 500 g/cm² for a 1% suspension.     

Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of eurospora crassa.

Summary As glutamine-dependent carbamoyl phosphate synthetase (CPS) activity in some organisms is composed of a glutaminase and an ammonium-dependent CPS, CPS^- mutants in Neurospora crassa were examined for glutamine- and ammonium-dependent CPS activities. No evidence was found that the genetic location of these two functions were separable. This is discussed with reference to the close genetic proximity of the CPS_pyr and aspartate carbamoyl-transferase (ACT) structural gene (pyr-3) and the arg-2 gene which appears to specify a subunit responsible for glutamine utilisation in CPS_arg.Supported by Science Research Council Grant B/RG/2981     

Effect of light and temperature on biomass, photosynthesis and capsular polysaccharides in cultured phototrophic biofilms

Phototrophic biofilms seem to be suitable candidates for tertiary wastewater treatment due to their high uptake capacity for nutrients and other pollutants, also taking into account the time and cost savings derived from easy procedures for biomass harvesting. Biomass accrual, structure, and physiology of biofilms affect the efficiency of nutrient removal by its microbial community. Here, we construct a biofilm consisting of a cyanobacterium Synechocystis sp. and the green alga Chlorococcum sp. and determine the effect of combined variations of irradiance and temperature on the biofilm structure and function. The two species were isolated from phototrophic biofilms naturally developing in an Italian wastewater treatment plant and grown in a microcosm designed for biofilm investigations. Phototrophic biomass accumulation, percent species composition, photosynthetic response and the amount and composition of capsular polysaccharides (CPS), including anionic residues, are reported. The results showed that biofilm development required relatively moderate irradiances (60 μmol photons m⁻² s⁻¹) below which development was arrested. Both light and temperature had a strong effect on the composition of each species to the biofilm. The CPS compositions also changed with temperature, light and species composition. The CPS of the green-algal-dominated biofilm had the higher uronic acid content indicating a potential to exploit green algae in the treatment of waste contaminated with heavy metals. Given the knowledge of the response of certain species to light and temperature combinations, it may be possible to construct biofilms of known species and CPS composition to use them for specific applications.     

Plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in Spodoptera frugiperda cell cultures

The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1–412), the second leads to the production of a species devoid of the anchor domain (aa 1–391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18–391). All three recombinant CPS were produced at about 3 g per 10⁶ infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - CSP circumsporozoite protein     

Identification and structural determination of the capsular polysaccharides from two Acinetobacter baumannii clinical isolates, MG1 and SMAL

The structures of the capsular polysaccharides (CPSs) of the two clinical isolates Acinetobacter baumannii SMAL and MG1 were elucidated. Hot phenol/water extractions of the dry biomasses, followed by enzymatic digestions and repeated ultracentrifugations led to the isolation of polysaccharides that were negative in Western blot analysis utilizing an anti-lipid A antibody, thus proving that they were not the LPS O-antigens but CPSs. Their structures were established on the basis of NMR spectroscopy and GC-MS analyses. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide with acyl substitution heterogeneity at the N-4 amino group of QuipN4N:4)-α-d-GlcpNAc-(1→4)-α-l-GalpNAcA-(1→3)-β-d-QuipNAc4NR-(1→R = 3-hydroxybutyrryl or acetyl.The repeating unit of the CPS produced by strain SMAL is a pentasaccharide, already reported for the O-antigen moiety from A. baumannii strain ATCC 17961:     

Endothelial retinoblastoma protein reduces abdominal aortic aneurysm development via promoting DHFR/NO pathway-mediated vasoprotection

Carbamoyl phosphate synthetase I (CPS1) represents an important regulatory enzyme of the urea cycle that mediates the ATP-driven reaction ligating ammonium, carbonate, and phosphate to form carbamoyl phosphate. The freeze-tolerant wood frog (Rana sylvatica or Lithobates sylvaticus) accumulates high concentrations of urea during bouts of freezing to detoxify any ammonia generated and to contribute as a cryoprotectant thereby helping to avoid freeze damage to cells. Purification of CPS1 to homogeneity from wood frog liver was performed in control and frozen wood frogs by a three-step chromatographic process. The affinity of CPS1 for its three substrates was tested in the purified control and freeze-exposed enzyme under a variety of conditions including the presence and absence of the natural cryoprotectants urea and glucose. The results demonstrated that affinity for ammonium was higher in the freeze-exposed CPS1 (1.26-fold) and that with the addition of 400 mM glucose it displayed higher affinity for ATP (1.30-fold) and the obligate activator N-acetylglutamate (1.24-fold). Denaturation studies demonstrated the freeze-exposed enzyme was less thermally stable than the control with an unfolding temperature approximately 1.5 °C lower (52.9 °C for frozen and 54.4 °C for control). The control form of CPS1 had a significantly higher degree of glutarylated lysine residues (1.42-fold increase) relative to the frozen. The results suggest that CPS1 activation and maintenance of urea cycle activity despite the hypometabolic conditions associated with freezing are important aspects in the metabolic survival strategies of the wood frog.

     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

Background  

The bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L^-1 K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.     

Clinical application of the SurePath liquid-based Pap test in cytological screening of bronchial brushing for the diagnosis of lung cancer

The SurePath liquid-based Pap test (LPT) is successfully and widely used to assess sputum cytology. This study aimed to compare the cytological findings and diagnostic sensitivity of LPT with those of the conventional Pap smear (CPS) method for diagnosing lung cancer. Bronchial brushing specimens from 204 patients diagnosed with lung cancer were studied. LPT slides showed decreased areas of cell monolayers, a clearer background and distinct, stereoscopic cytological features. The LPT had a significantly higher diagnostic sensitivity for lung cancer (71.6%) than the CPS method (57.8%, p < 0.05), particularly for small cell lung carcinoma and >2 cm lesions (p < 0.05). Combination of the LPT with the CPS method showed obviously higher diagnostic sensitivity for the detection of adenocarcinoma (63.6%), central lesions (85.0%) and >2 cm lesions (81.4%) compared with the CPS method alone (p < 0.05, p < 0.01). Thus, LPT is a useful and easily performed technique that can be widely applied, and is suitable for the early diagnosis of lung cancer.     

Structure of the K82 Capsular Polysaccharide from <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> LUH5534 Containing a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose 4,6-Pyruvic Acid Acetal

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of D-galactose 4,6-(R)-pyruvic acid acetal.     

STRUCTURE OF THE CAPSULAR AND EXTRACELLULAR POLYSACCHARIDES PRODUCED BY THE DESMID SPONDYLOSIUM PANDURIFORME (CHLOROPHYTA)1

The filamentous desmid Spondylosium panduriforme (Heimerl) Teiling var. panduriforme f. limneticum (West & West) Teiling (Desmidiaceae), strain 072CH-UFCAR, is surrounded by a well-defined, mucilaginous capsule consisting of a capsular polysaccharide (CPS). This microalga also produces an extracellular polysaccharide (EPS), which can be isolated from the culture medium. Analysis of the carbohydrate composition of the two polymers by gas chromatography showed that they were different. Both were composed, of galactose, fucose, xylose, arabinose, rhamnose, and glucose but in different amounts. For example, glucuronic acid accounts for 24% of the EPS material but only traces were found in the CPS. Significant differences were also found during methylation analysis. Fucose appeared to have a higher degree of branching in the EPS than in the CPS. These branches were located on C-3 and could be the position for the attachment of the glucuronic acid units in the EPS. The glucuronic acid was present as 1→4-linked and terminal units. A possible explanation for the formation of the EPS is suggested.     

Identification and expression pattern analysis of PtCarA and PtCarB genes in Populus trichocarpa under different nitrogen treatments

• Carbamoyl phosphate synthetase (CPS) catalyses the synthesis of ammonia carbamoyl phosphate (CP), which plays a key role in the biosynthesis of arginine and pyrimidine nucleotides. There are two subunits of the CPS enzyme in Populus trichocarpa, CarA (small subunit) and CarB (large subunit). Only when they coexist can CPS catalyse synthesis of CP. However, it is not clear how CPS responds to nitrogen (N) to affect arginine and pyrimidine nucleotide biosynthesis.
• In this study, bioinformatics methods were used to analyse the expression patterns of genes encoding CarA and CarB, and qRT-PCR and RNA-seq were used to investigate their molecular responses under different N concentrations.
• Phylogenetic analysis revealed that the phylogenetic trees of CarA and CarB had similar topologies. qRT-PCR showed that the PtCarA and PtCarB genes were regulated by N, while their N-regulated patterns differed in different tissues. The expression patterns of PtCarA and PtCarB show a significant positive correlation according to qRT-PCR and RNA-seq. The analysis of promoter cis-acting elements showed that the promoter regions of PtCarA1, PtCarA2 and PtCarB contained some identical cis-acting elements. According to analysis of the phylogenetic tree, expression patterns and promoter elements, we speculate that there might be coevolution among PtCarA1, PtCarA2 and PtCarB.
• This study provides valuable information for further understanding the function of CPS in poplar, especially for N response, and provides new ideas for studying the evolution of gene families related to heteromultimers.

     

Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2m3

Summary The conversion of glycerol to 1,3-propanediol (PD) by Clostridium butyricum DSM 5431 was studied in anaerobic culture. Growth and product formation were optimal at pH = 7.0 and T = 35° C, while aeration rate and stirrer speed were found to have no significant influence. As increasing amounts of initial glycerol led to inhibition of growth, cultivations were done in fed-batch operation. Comparative cultivations were carried out in an air-lift (ALR) and a stirred-tank reactor (STR) having equal working volumes (V _L = 30 l) and no difference in product formation was found. The process was scaled up to reactor sizes of 1.2 m³ (ALR) and 2.0 m³ (STR). The same results were obtained irrespective of reactor volume as well as reactor type (STR/ALR). PD concentrations of approximately 50–58 g·l^–1 and overall productivities of 2.3–2.9 g·l^–1 ·h^–1 could be reached. Offprint requests to: W.-D. Deckwer     

Adhesion of Acinetobacter venetianus to Diesel Fuel Droplets Studied with In Situ Electrochemical and Molecular Probes

The adhesion of a recently described species, Acinetobacter venetianus VE-C3 (F. Di Cello, M. Pepi, F. Baldi, and R. Fani, Res. Microbiol. 148:237–249, 1997), to diesel fuel (a mixture of C₁₂ to C₂₈ n-alkanes) and n-hexadecane was studied and compared to that of Acinetobacter sp. strain RAG-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. Oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains. The dropping-mercury electrode (DME) was used as an in situ adhesion sensor. In seawater, RAG-1 was hydrophobic, with an electrophoretic mobility (μ) of −0.38 × 10⁻⁸ m² V⁻¹ s⁻¹ and zeta potential (ζ) of −4.9 mV, while VE-C3 was hydrophilic, with μ of −0.81 × 10⁻⁸ m² V⁻¹ s⁻¹ and ζ of −10.5 mV. The microbial adhesion to hydrocarbon (MATH) test showed that RAG-1 was always hydrophobic whereas the hydrophilic VE-C3 strain became hydrophobic only after exposure to n-alkanes. Adhesion of VE-C3 cells to diesel fuel was partly due to the production of capsular polysaccharides (CPS), which were stained with the lectin concanavalin A (ConA) conjugated to fluorescein isothiocyanate and observed in situ by confocal microscopy. The emulsan from RAG-1, which was negative to ConA, was stained with Nile Red fluorochrome instead. Confocal microscope observations at different times showed that VE-C3 underwent two types of adhesion: (i) cell-to-cell interactions, preceding the cell adhesion to the n-alkane, and (ii) incorporation of nanodroplets of n-alkane into the hydrophilic CPS to form a more hydrophobic polysaccharide–n-alkane matrix surrounding the cell wall. The incorporation of n-alkanes as nanodroplets into the CPS of VE-C3 cells might ensure the partitioning of the bulk apolar phase between the aqueous medium and the outer cell membrane and thus sustain a continuous growth rate over a prolonged period.     

Chemical Structures of Streptococcus pneumoniae Capsular Polysaccharide Type 39 (CPS39), CPS47F,and CPS34 Characterized by Nuclear Magnetic Resonance Spectroscopy and Their Relation to CPS10A

Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: Open in a separate window Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the α1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with α1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with α1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from α-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity.