Simultaneous Transport of Two Bacterial Strains in Intact Cores from Oyster, Virginia: Biological Effects and Numerical Modeling

The transport characteristics of two adhesion-deficient, indigenous groundwater strains, Comamonas sp. strain DA001 and Erwinia herbicola OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va. Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 μm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge. The two strains exhibited markedly different behaviors when they were transported through granular porous sediment. To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core. DA001 cells were metabolically labeled with ³⁵S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with ¹⁴C. The fast decay of ³⁵S allowed deconvolution of the two isotopes (and therefore the two strains). Dramatic differences in the transport behaviors were observed. The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively. The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001. Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter. The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells. CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate. All these factors may contribute to the observed differences in transport.     

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.     

Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10⁵ CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.     

Application of a vital fluorescent staining method for simultaneous, near-real-time concentration monitoring of two bacterial strains in an Atlantic coastal plain aquifer in Oyster, Virginia

Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.     

Development of radiographic and microscopic techniques for the characterization of bacterial transport in intact sediment cores from Oyster, Virginia.

The objective of this study was to ascertain the physical and mineralogical properties responsible for the retention of bacteria in subsurface sediments. The sediment core chosen for this study was a fine-grained, quartz-rich sand with minor amounts of Fe and Al hydroxides. A bacterial transport experiment was performed using an intact core collected from a recent excavation of the Butler's Bluff member of the Nassawadox formation in the borrow pit at Oyster, VA. and a 14C-labeled bacterial strain OYS2-A was selected for its relatively low adhesion. After the bacterial breakthrough was observed in the effluent, the intact core was dissected to determine the internal distribution of the injected bacteria retained in the sediment. The sediment was dried, epoxy fixed, and thin sectioned. The distribution of 14C activity in the thin sections was mapped using a phosphor screen and X-ray film. The remainder of the core was subsampled and the 14C activity of the subsamples was determined by liquid scintillation counting. The phosphor imaging technique was capable of directly imaging the distribution of radiolabeled bacteria in thin sections, because of its high sensitivity and linear response over a large activity range. The phosphor imaging signal intensity was utilized as a measure of bacterial concentration. The distribution of bacteria at the millimeter scale in the thin sections was compared to the grain size, porosity, and mineralogy as measured by scanning electron microscopy (SEM) and energy dispersive spectrum (EDS) analyses. No apparent correlation was observed between the retention or collision efficiency of bacteria in the sediment and the amount of Fe and Al hydroxides. This apparent lack of correlation can be qualitatively explained by combination of several factors including a nearly neutral surface charge of the bacterial strain, and texture of the Fe and Al hydroxides in the sediment. The combination of phosphor imaging with SEM-EDS proved to be a robust method for relating the physical and mineralogical microscopic properties of poorly indurated sediment to the distribution of adsorbed bacteria, allowing bacterial retention mechanisms to be unambiguously unraveled.     

Application of a Vital Fluorescent Staining Method for Simultaneous, Near-Real-Time Concentration Monitoring of Two Bacterial Strains in an Atlantic Coastal Plain Aquifer in Oyster, Virginia

Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.     

Comparison of methods for monitoring bacterial transport in the subsurface

The purpose of this study was to compare in a laboratory experiment, a suite of methods developed to track viable bacteria during field transport experiments. The criteria for development and selection of these methods included: (1) the ability to track bacteria within the environment from which they were isolated; (2) the lack of any effect upon the viability or the transport characteristics of the strain; (3) low detection limits; (4) a quantification range that covered several orders of magnitude; and (5) an analytical cost and turnover time commensurate with the analysis of several thousands of samples in a few months. The approaches developed included: enumeration of bacteria labeled with a vital fluorescent stain (CFDA/SE) using microplate spectrofluorometry, flow cytometry, and ferrographic (immunomagnetic) capture; enumeration of highly (13)C-enriched bacteria using combustion-IRMS; and quantitative PCR. These methods were compared to direct microscopic enumeration and plate counts during a bacterial transport experiment performed in an intact sediment core and designed to simulate the field experiment. Four of the seven methods had equivalent recoveries for the breakthrough of a pulse of bacteria eluting from a 50-cm long sediment core, and all of the methods detected the arrival of cells in the effluent prior to the conservative tracer. Combustion IRMS and ferrographic enumeration had the lowest quantification limits (approximately 2 to 20 cells/ml), whereas microplate spectrofluorometry had the highest quantification limit (approximately 10(5) cells/ml). These methods have the potential for numerous applications beyond tracking bacteria injected into the subsurface.     

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.     

Determination of in situ bacterial growth rates in aquifers and aquifer sediments

Laboratory and field-scale studies with stained cells were performed to monitor cell growth in groundwater systems. During cell division, the fluorescence intensity of the protein stain 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) for each cell is halved, and the intensity can be tracked with a flow cytometer. Two strains of bacteria, Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107, both isolated from a shallow aquifer, were utilized in this study. The change in the average generation or the average fluorescence intensity of the CFDA/SE-stained cells could be used to obtain estimates of doubling times. In microcosm experiments, the CFDA/SE-based doubling times were similar to the values calculated by total cell counting and were independent of cell concentration. Intact and repacked sediment core experiments with the same bacteria indicated that changes in groundwater chemistry were just as important as growth rates in determining planktonic cell concentrations. The growth rates within the sediment cores were similar to those calculated in microcosm experiments, and preferential transport of the daughter cells was not observed. The experiments indicated that the growth rates could be determined in systems with cell losses due to other phenomena, such as attachment to sediment or predation. Application of this growth rate estimation method to data from a field-scale bacterial transport experiment indicated that the doubling time was approximately 15 days, which is the first known direct determination of an in situ growth rate for bacteria in an aquifer.     

Determination of In Situ Bacterial Growth Rates in Aquifers and Aquifer Sediments

Laboratory and field-scale studies with stained cells were performed to monitor cell growth in groundwater systems. During cell division, the fluorescence intensity of the protein stain 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) for each cell is halved, and the intensity can be tracked with a flow cytometer. Two strains of bacteria, Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107, both isolated from a shallow aquifer, were utilized in this study. The change in the average generation or the average fluorescence intensity of the CFDA/SE-stained cells could be used to obtain estimates of doubling times. In microcosm experiments, the CFDA/SE-based doubling times were similar to the values calculated by total cell counting and were independent of cell concentration. Intact and repacked sediment core experiments with the same bacteria indicated that changes in groundwater chemistry were just as important as growth rates in determining planktonic cell concentrations. The growth rates within the sediment cores were similar to those calculated in microcosm experiments, and preferential transport of the daughter cells was not observed. The experiments indicated that the growth rates could be determined in systems with cell losses due to other phenomena, such as attachment to sediment or predation. Application of this growth rate estimation method to data from a field-scale bacterial transport experiment indicated that the doubling time was approximately 15 days, which is the first known direct determination of an in situ growth rate for bacteria in an aquifer.     

Physical and chemical factors influencing transport of microorganisms through porous media.

Resting-cell suspensions of bacteria isolated from groundwater were added as a pulse to the tops of columns of clean quartz sand. An artificial groundwater solution (AGW) was pumped through the columns, and bacterial breakthrough curves were established and compared to test the effects of ionic strength of the AGW, cell size (by using strains of similar cell surface hydrophobicity but different size), mineral grain size, and presence of heterogeneities within the porous media on transport of the bacteria. The proportion of cells recovered in the effluent ranged from nearly 90% for AGW of a higher ionic strength (I = 0.0089 versus 0.00089 m), small cells (0.75-micron-diameter spheres versus 0.75 by 1.8-micron rods), and coarse-grained sand (1.0 versus 0.33 mm) to less than 1% for AGW of lower ionic strength, large cells, and fine-grained sand. Differences in the widths of peaks (an indicator of dispersion) were significant only for the cell size treatment. For treatments containing heterogeneities (a vein of coarse sand in the center of a bed of fine sand), doubly peaked breakthrough curves were obtained. The first peak represents movement of bacteria through the transmissive coarse-grained vein. The second peak is thought to be dominated by cells which have moved (due to dispersion) from the fine-grained matrix to the coarse-grained vein near the top of the column and thus had been retarded, but not retained, by the column. Strength of effects tests indicated that grain size was the most important factor controlling transport of bacteria over the range of values tested for all of the factors examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical and chemical factors influencing transport of microorganisms through porous media.

Resting-cell suspensions of bacteria isolated from groundwater were added as a pulse to the tops of columns of clean quartz sand. An artificial groundwater solution (AGW) was pumped through the columns, and bacterial breakthrough curves were established and compared to test the effects of ionic strength of the AGW, cell size (by using strains of similar cell surface hydrophobicity but different size), mineral grain size, and presence of heterogeneities within the porous media on transport of the bacteria. The proportion of cells recovered in the effluent ranged from nearly 90% for AGW of a higher ionic strength (I = 0.0089 versus 0.00089 m), small cells (0.75-micron-diameter spheres versus 0.75 by 1.8-micron rods), and coarse-grained sand (1.0 versus 0.33 mm) to less than 1% for AGW of lower ionic strength, large cells, and fine-grained sand. Differences in the widths of peaks (an indicator of dispersion) were significant only for the cell size treatment. For treatments containing heterogeneities (a vein of coarse sand in the center of a bed of fine sand), doubly peaked breakthrough curves were obtained. The first peak represents movement of bacteria through the transmissive coarse-grained vein. The second peak is thought to be dominated by cells which have moved (due to dispersion) from the fine-grained matrix to the coarse-grained vein near the top of the column and thus had been retarded, but not retained, by the column. Strength of effects tests indicated that grain size was the most important factor controlling transport of bacteria over the range of values tested for all of the factors examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium falciparum glycolipid synthesis: constant and variant molecules of isolates and of strains with differing knob and cytoadherence phenotype

Plasmodium falciparum-infected erythrocytes were metabolically labeled with tritiated glucosamine. Lipid extracts were analyzed by high-performance thin-layer chromatography to compare labeled molecules of eleven isolates from patients, six cytoadherent in vitro strains, and two knobbed and two knobless strains from Aotus monkeys. Up to nineteen labeled bands were identified. Glycolipid GL1, previously identified in Malayan Camp, was present in all isolates and strains. Other molecules, between CG and GM1 and between GM1 and GD1a, varied in mobility or presence. There was no apparent association between labeled molecules and the presence of knobs or the property of cytoadherence.     

Site specific glycosylation patterns of H-2K: effects of allelic polymorphism and mitogenic stimulation

The site-specific glycosylation patterns of two H-2K alleles, k and b, were determined on splenic T cells metabolically labeled with [3H]mannose. Cells from B10, B10.A, (B10 X B10.A)F1, and C3H mice were examined, along with the effect of short- (8 hr) and long-term (36 hr) mitogenic stimulation. For both glycosylation sites (Asn86 and Asn176) of both antigens, 80% of the structures consisted of mono- and bisialylated biantennary N-linked complex oligosaccharides, with the remaining consisting of smaller (probably high mannose) structures. Asn176 of both H-2Kk and H-2Kb contained the same ratio (2.8 to 1) of bi- to monosialylated chains. However, Asn86 of H-2Kb contained a higher ratio (5 to 1), while Asn86 of H-2Kk a lower ratio (1.5 to 1). This difference was seen on antigens isolated from cells of the parental strains as well as from the F1 cross. The glycosylation of H-2Kk did not vary between B10.A and C3H mice. Mitogenic stimulation increased markedly both total [3H]mannose incorporation and the spectrum of N-linked oligosaccharides labeled. For H-2Kk, it had no effect on sialylation, but resulted in a slight under galactosylation of the monosialylated structures at both sites. A comparison of the patterns seen here, determined on nontransformed T cells, with those previously determined on H-2Kk from a B lymphoma line, revealed marked differences in sialylation and branching patterns at both sites. These data indicate that glycosylation differences may be found between highly homologous (91%) alleles of an H-2 gene, even when co-dominantly expressed by F1 cells; however, the patterns do change with mitogenic stimulation, and between normal and transformed cells.     

Plasmodium falciparum Glycolipid Synthesis: Constant and Variant Molecules of Isolates and of Strains with Differing Knob and Cytoadherence Phenotype

ABSTRACT. Plasmodium falciparum -infecled erythrocytes were metabolically labeled with tritiated glucosamine. Lipid extracts were analyzed by high-performance thin-layer chromatography to compare labeled molecules of eleven isolates from patients, six cytoadherent in vitro strains, and two knobbed and two knobless strains from Aotus monkeys. Up to nineteen labeled bands were identified. Glycolipid GL1, previously identified in Malayan Camp, was present in all isolates and strains. Other molecules, between CG and GM1 and between GM1 and GD1a, varied in mobility or presence. There was no apparent association between labeled molecules and the presence of knobs or the property of cytoadherence.     

Proteomic analysis of secreted muscle components: search for factors involved in neuromuscular synapse formation

Denervated but not innervated skeletal muscles secrete polypeptides that are involved in neuromuscular synapse formation. With the aim of identifying such components, metabolically labeled polypeptides in extracts from denervated and innervated muscles were submitted to two-dimensional gel electrophoresis, and the abundance of individual molecular species was compared. Consistent differences between the proteomic maps from the two sources of muscles were seen. Likewise, proteomic maps of polypeptides from organ culture media conditioned by chronically denervated muscles and by control muscles revealed consistent differences, but the abundance of material within individual spots from conditioned media was not sufficient for analysis by mass spectrometry. Since it was not possible to match the patterns from muscle extracts and from conditioned media, it has been established that extract of Sol8 muscle cells was a satisfactory source of material for analysis. From 1,200 spots identified on the proteomic map from Sol8 cells by image analysis, some 140 have been defined by mass spectrometric analysis. In order to identify the components that are shared by secreted molecules from denervated muscles and Sol8 cells, a mixture of extracts from the two sources was co-electrophoresed and a shared proteomic pattern was established by visualization of metabolically labeled spots from the conditioned medium and of silver stained spots from the Sol8 cells. More than 100 spots sharing x/y coordinate localization could be seen on the pattern. Of these, fourteen were among those identified by mass spectrometry. It is concluded that co-electrophoresis of radioactively labeled polypeptides from conditioned media with extracts from Sol8 cells can be used to mark in the proteome of Sol8 cells those polypeptides that are secreted at low abundance by adult muscles. Their higher abundance in Sol8 cells opens the possibility for further scrutiny of spots by mass spectrometry or by microsequencing.     

Differences in avidity and epitope recognition of CD8(+) T cells infiltrating the central nervous systems of SJL/J mice infected with BeAn and DA strains of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection induces immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infectious model for human multiple sclerosis. To investigate the pathogenic mechanisms, two strains of TMEV (DA and BeAn), capable of inducing chronic demyelination in the central nervous system (CNS), have primarily been used. Here, we have compared the T-cell responses induced after infection with DA and BeAn strains in highly susceptible SJL/J mice. CD4(+) T-cell responses to known epitopes induced by these two strains were virtually identical. However, the CD8(+) T-cell response induced following DA infection in susceptible SJL/J mice was unable to recognize two of three H-2K(s)-restricted epitope regions of BeAn, due to single-amino-acid substitutions. Interestingly, T cells specific for the H-2K(s)-restricted epitope (VP1(11-20)) recognized by both strains showed a drastic increase in frequency as well as avidity after infection with DA virus. These results strongly suggest that the level and avidity of virus-specific CD8(+) T cells infiltrating the CNS could be drastically different after infection with these two strains of TMEV and may differentially influence the pathogenic and/or protective outcome.     

Occurrence of secretory glycoprotein-specific GalNAc beta 1-->4GlcNAc sequence in N-glycans in MDCK cells

Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and (3)H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, +/-Sia alpha 2-->6GalNAc beta 1-->4GlcNAc beta 1-->R, which linked only to a non-bisected trimannosyl core. beta1-->4N-acetylgalactosaminyltransferase (beta 4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding beta 4GalNAc-T are involved in transport of secretory glycoproteins.