IS2-mediated overexpression of kfoC in E. coli K4 increases chondroitin-like capsular polysaccharide production

Transposons are developing molecular tools commonly used for several applications: one of these is the delivery of genes into microorganisms. These mobile genetic elements are characterised by two repeated insertion sequences that flank a sequence encoding one or more orfs for a specific transposase that moves these sequences to other DNA sites. In the present paper, the IS2 transposon of Escherichia coli K4 was modified in vitro by replacing the sequence coding for the transposase with that of the kfoC gene that codes for chondroitin polymerase. KfoC is responsible for the polymerisation of the bacterial capsular polysaccharide whose structure is analogous to that of chondroitin sulphate, a glycosaminoglycan with established and emerging biomedical applications. The recombinant construct was stably integrated into the genome of E. coli K4 by exploiting the transposase from endogenous copies of IS2 in the E. coli chromosome. A significant improvement of the polysaccharide production was observed, resulting in 80 % higher titres in 2.5-L fed-batch cultivations and up to 3.5 g/L in 22-L fed-batch cultures.     

Nuclear magnetic resonance quantification for monitoring heparosan K5 capsular polysaccharide production

Traditional chromatographic quantification methods for heparosan produced from the Escherichia coli K5 strain rely on extensive purification requiring laborious sample preparation. These methods are time-consuming, often resulting in sample loss during purification, and thus might not accurately reflect the amount of heparosan in the original mixture. A simple, sensitive ¹H nuclear magnetic resonance (NMR) quantification method that directly quantifies heparosan K5 polysaccharide present in E. coli fermentation supernatant is described.     

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.     

Improved capsular polysaccharide production by Streptococcus pneumoniae serotype 14 using continuous cultivation

The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and is currently used as antigen in all pneumococcal vaccines. Despite its physiological and epidemiological importance, meager studies have been devoted to improve PS production and understand its relationship with pneumococcal central metabolism. In this study, kinetics of growth and production of PS by Streptococcus pneumoniae serotype 14 (PS14) in batch and continuous cultivation were investigated. Strong cell lysis was observed in batch cultivation, while accumulation of organic acids and autolysis was avoided in continuous cultivation. In the continuous cultivation was possible to achieve higher concentration of biomass and PS14. Calculation of kinetic parameters demonstrated that PS14 is a cell-associated product. The coefficients for growth-associated stoichiometric true yield and maintenance were determined as 0.25 g_glucose g_biomass⁻¹ and 1.24 g_glucose (g_biomass h)⁻¹, respectively. The maximum productivity of PS14 released in the supernatant (PS14_R) and cell-bound PS14 (PS14_C) were obtained at a dilution rate of 0.8 h⁻¹, respectively, 85 and 122 mg  (g_biomass h)⁻¹. Compared to batch fermentation, both PS14_R and PS14_C productivities were increased by about 300% in the continuous process. These findings demonstrate that continuous cultivation is a promising strategy for PS production to be used in pneumococcal vaccines.     

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.     

In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F

The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO(2) did not increase PS yield, glucose was inhibitory above 30 g l(-1), and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N(2) flow using the optimized medium, 240 mg l(-1) of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.     

Development and scale-up of a fed-batch process for the production of capsular polysaccharide from Haemophilus influenzae

The production of the capsular polysaccharide, polyribosylribitolphosphate, from Haemophilus influenzae type b is important for the production of effective conjugate vaccines. Factors limiting the production of this polysaccharide from H. influenzae type b in liquid culture were investigated. A fed-batch fermentation was developed that increased cell density and PRP titer approximately four fold when compared to the batch fermentation. This fed-batch process was successfully scaled from the 1.5 l development scale to the 500 l manufacturing scale. The maximum cell density in the 500 l fermentation was 6 g dry cell weight per liter and the PRP concentration was 1.3 g l(-1).     

Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D approximately P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D approximately P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis.     

Cadmium adsorption by bacterial capsular polysaccharide coatings

Conclusions Excretion of a polysaccharide capsular coating byP.putida provided enhanced cadmium uptake when compared to the noncapsularP.cruciviae. As this advantage is most significant for cadmium concentrations below 2.5 mg/l, levels which are commonly found in waste discharges, it does suggest a potential role for extracellular polysaccharide producers as biosorbents. These encouraging results have led to further work to determine the desorption characteristics, by pH adjustment, ofP.putida along with long term viability after successive cycles of adsorption and desorption.     

Cryptococcal capsular polysaccharide clearance in nonimmune mice

Clearance of cryptococcal polysaccharide (CP) from tissues and body fluids of nonimmune mice was studied. Mice were injected intravenously one time only with one mg of purified CP, and serum, urine and tissues were obtained from each animal at various intervals for a period of 84 days. Tissue extracts, serum and urine were tested for CP content by enzyme-linked immunosorbent assay (ELISA) and latex agglutination. High concentrations of CP were detected by both assays one-half hour after injection in blood (serum), liver, spleen, kidney and lung (extracts). The duration of ELISA detectable CP was longest (70 days) in liver and spleen and shortest (14 days) in lung extract. By 14 days after injection, concentration of CP in the blood fell below that found in the liver and spleen. CP remained detectable (titers 32–64) after all other extracts became negative. These results indicate that CP is stored in tissues (binding mechanism and site unknown), and that the liver and spleen possess greater storage capacity than other tissues. Antibody (IgM) to CP appeared in low titer on the 14th day and thereafter.     

The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti.

The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G. Petrovics, P. Putnoky, R. Reuhs, J. Kim, T. A. Thorp, K. D. Noel, R. W. Carlson, and A. Kondorosi, Mol. Microbiol. 8:1083-1094, 1993; B. L. Reuhs, R. W. Carlson, and J. S. Kim, J. Bacteriol. 175:3570-3580, 1993). Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J. The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, such as KpsS of E. coli. No significant homology was found for RkpI. Biochemical and immunological analysis of Tn5 derivatives for each gene demonstrated partial or complete loss of capsular polysaccharides from the cell surface; on this basis, we suggest that all genes in the fix-23 region are required for K-antigen synthesis or transport.     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。