The effect of slaframine on salivary output and subacute and acute acidosis in growing beef steers.

Experiments were conducted to determine 1) the effect of injecting slaframine (SF) on salivary output in growing beef steers and 2) whether increased salivary output after SF injection would inhibit the decrease in ruminal pH that occurs after experimentally induced subacute and acute ruminal acidosis. In Exp. 1 and 2, we measured ruminal pH and salivary output in ruminally and esophageally cannulated beef steers fed an 88% concentrate diet. Injections of 66 or 100 micrograms of SF/kg BW increased salivary flow approximately 50% compared with controls. Those doses were tested in subacute and acute acidosis models using ruminally cannulated beef steers in Exp. 3 and 4, respectively. In these experiments, salivation was assessed indirectly using a visual scoring system. In the subacute acidosis model, SF reduced (P < .10) the decrease in ruminal pH (1.1, .7, and .6 pH units for control, 66, and 100 micrograms of SF/kg BW doses, respectively), and excessive salivation was observed in all SF-injected steers. In the acute acidosis model, there were no differences (P > .10) in ruminal pH at 12 h after injection between control and SF-treated steers. Mean ruminal lactate concentrations for all treatment groups were between 87 and 112 mM. Although treatment with 66 micrograms of SF/kg BW reduced (P < .10) ruminal lactate concentrations, all ruminal lactate concentrations were indicative of acute acidosis. These results indicate that SF will reduce the decrease in ruminal pH associated with subacute acidosis in growing beef steers, but SF does not attenuate acute ruminal acidosis.

Ion chromatographic methods for the detection of starch hydrolysis products in ruminal digesta.

Dionex high-performance ion chromatographic methods were evaluated for separation and quantitation of plant sugars and starch digestion products in the ruminal digesta of cattle. Mono- and disaccharides were eluted from a Dionex CarboPac PA1 column with sodium hydroxide used isocratically or as a pH gradient. Maltooligosaccharides which had a degree of polymerization (DP) less than 30 glucose residues were eluted in 60 min by a sodium hydroxide eluent containing a sodium acetate gradient. Carbohydrates were detected amperometrically. Responses were linear (r2 greater than 0.99) for glucose, disaccharides and maltooligosaccharides (DP less than 8). Precipitation and solid-phase extraction methods were evaluated for clean-up of samples of feedstuffs, ruminal contents, and bacterial culture fluids. Perchloric acid precipitation hydrolyzed sucrose but did not affect recoveries of cellobiose, isomaltose or maltose. Ethanol in concentrations of 79 and 86% precipitated maltooligosaccharides having chain lengths larger than 14 and 9 glucose residues, respectively. Maltooligosaccharide recoveries from solid-phase extraction columns varied with maltooligosaccharide size and column packing. Recoveries were greater than 94% for short chains (DP less than 6) eluted from phenyl-substituted columns and variable for all oligosaccharides eluted from C18 columns. Applications of these methods are presented and include: (1) detection of sugars in ruminant feed, (2) monitoring changes in ruminal sugars after feeding and (3) monitoring changes in extracellular sugars and oligosaccharides in the culture fluids of the ruminal bacterium, Bacteroides ruminicola.

Effect of long generation times on growth of Bacteroides thetaiotaomicron in carbohydrate-induced continuous culture.

We investigated the ability of Bacteroides thetaiotaomicron, an obligate anaerobe from human colonic microflora, to grow in a carbohydrate-limited continuous culture at generation times ranging from 3.5 to 28 h per division. Four carbohydrates were tested: glucose, N-acetylglucosamine, glucuronic acid, and glucosamine. At a generation time of 3.5 h per division, the growth yields for bacteria growing on glucose, N-acetylglucosamine, and glucuronic acid were 76, 68, and 50 g of cells (dry weight) per mol of substrate, respectively. Growth yields at 28 h per division were 61, 52, and 37 g/mol of substrate, respectively. When glucosamine was the carbohydrate source, a stable population of bacteria was attainable only at generation times longer than 12 h per division. Growth yields at 15 and 32 h per division were 11 and 33 g/mol of substrate, respectively. There was no significant variation with increasing generation times in the specific activities of selected glycolytic enzymes, of disaccharidases such as alpha- and beta-glucosidases and alpha- and beta-galactosidases, or of the polysaccharidase chondroitin sulfate lyase. By contrast, the pattern of fermentation products varied with both the generation time and the carbon source. At a generation time of 3.5 h per division, the main products from the fermentation of glucose were acetate and succinate, with a trace of propionate. At 28 h per division, propionate concentrations were higher and succinate concentrations were lower than at 3.5 h per division. The products from the fermentation of glucosamine were the same as those from glucose fermentation. However, when N-acetylglucosamine was fermented, the concentration of acetate was much higher at all generation times than when glucose was the carbon source. When glucuronic acid was the carbon source, acetate was the main fermentation product, and only traces of propionate and succinate were detected. Another characteristic that varied with the growth rate was the ability of B. thetaiotaomicron to produce the inducible enzyme alpha-glucosidase when exposed to maltose. The ability of the organism to produce this enzyme declined with increasing generation times.

Induction of chondroitin sulfate lyase activity in Bacteroides thetaiotaomicron.

Chondroitin sulfate lyase (EC 4.2.2.4) was present constitutively at low levels (0.06 to 0.08 U/mg of protein) in cells of Bacteroides thetaiotaomicron which were growing on glucose or other monosaccharides. When these uninduced bacteria were incubated with chondroitin sulfate A (5 mg/ml), chondroitin sulfate lyase specific activity increased more than 10-fold within 90 min. Synthesis of ribonucleic acid and of protein was required for induction, and induction was sensitive to oxygen. The disaccharides which resulted from chondroitinase action did not act as inducers, nor did tetrasaccharides or hexasaccharides obtained by digestion of chondroitin sulfate with bovine testicular hyaluronidase. None of these substances was taken up by uninduced cells; they may not have been able to penetrate the outer membrane. The smallest oligomer capable of acting as an inducer was the outer membrane. The smallest oligomer capable of acting as an inducer was the octassacharide. Oligomers larger than the octassacharide induced chondroitin lyase activity nearly as well as intact chondroitin sulfate.

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.

Models for study of the specificity by which indigenous lactobacilli adhere to murine gastric epithelia.

A Lactobacillus strain isolated from a mouse (indigenous) and another strain isolated from swine feces (nonindigenous) were compared in two model systems for their ability to adhere in vitro and in vivo to keratinizing squamous and columnar epithelia of mouse stomachs. In one model, stomachs dissected from specific-pathogen-free or germfree mice were injected with suspensions of lactobacilli labeled with [(3)H]thymidine and incubated at 37 degrees C. Thereafter, the non-secreting and secreting tissues were separated and washed vigorously. The radioactivity remaining with each tissue was counted by liquid scintillation spectrometry. When the radioactivity remaining with these tissues ranged between 500 and 100,000 cpm, the calculated radioactivity (disintegrations per minute) was related linearly to the number of lactobacilli adhering to the tissue. The estimate of the number of bacteria adherent to the tissue was not influenced significantly by artifacts in the techniques used. In this model, both Lactobacillus strains adhered in equally high numbers to both types of epithelial surfaces from stomachs from germfree mice. In contrast, in the second model, in which germfree mice were monoassociated with one or the other of the Lactobacillus strains, only the strain indigenous to the mouse formed dense layers on the epithelia of the nonsecreting portions of the stomachs, although both strains maintained high population levels throughout the gastrointestinal tracts of the animals. The capacity to adhere to the mucosal surface is undoubtedly necessary for lactobacilli to colonize gastric epithelia in mice. Our findings suggest, however, that nutritional or environmental conditions dictate whether particular Lactobacillus strains can colonize particular surfaces in the stomachs of living animals.

Starch hydrolysis by the ruminal microflora.

The effects of grain type and processing on ruminal starch digestion are well documented but poorly understood at the biochemical and molecular levels. Waxy grains have starches high in amylopectin and are more readily digested than nonwaxy grains. However, the composition of the endosperm cell matrix and the extent to which the starch granules are embedded within it also affect starch digestion rates. Continued work is needed to determine the influence of specific cell matrix proteins, protein-starch interactions and cell wall carbohydrates on starch availability. The microbial populations that metabolize starch are diverse, differing in their capacities to hydrolyze starch granules and soluble forms of starch. Surveys show that the amylases are under regulatory control in most of these organisms, but few studies have addressed the types of amylolytic enzymes produced, their regulation and the impact of other plant polymers on their synthesis. Research in these areas, coupled with the development and use of isogeneic or near-isogeneic grain cultivars with biochemically defined endosperm characteristics, will enhance our ability to identify mechanisms to manipulate ruminal starch digestion.

Utilization of chondroitin sulfate by Bacteroides thetaiotaomicron growing in carbohydrate-limited continuous culture.

When Bacteroides thetaiotaomicron, an obligate anaerobe from the human colonic flora, was grown in continuous culture with the mucopolysaccharide chondroitin sulfate as the limiting source of carbohydrate, growth yields ranged from 48 g of cell dry weight per mol of equivalent monosaccharide at a growth rate of 3.5 h per generation to 32 g per mol at a growth rate of 24 h per generation. The theoretical maximum growth yield (61 g of cell dry weight per mol of equivalent monosaccharide) was comparable to that of 54 g per mol, which was obtained previously when glucuronic acid, a component of chondroitin sulfate, was the limiting carbohydrate (S. F. Kotarski and A. A. Salyers, J. Bacteriol. 146:853-860, 1981). However, the maintenance coefficient was three times higher when chondroitin sulfate was the substrate than when glucuronic acid was the substrate. The specific activity of chondroitin lyase (EC 4.2.2.4), an enzyme which cleaves chondroitin sulfate into disaccharides, declined by nearly 50% as growth rates decreased from 3.5 to 24 h per generation. By contrast, the specific activities of several glycolytic enzymes and disaccharidases remained constant over this range of growth rates. Although chondroitin sulfate was growth limiting, some carbohydrate was detectable in the extracellular fluid at all growth rates. At rapid growth rates (1 to 2 h per generation), this residual carbohydrate included fragments of chondroitin sulfate having a wide range of molecular weights. At slower growth rates (2 to 24 h per generation), the residual carbohydrate consisted mainly of a small fragment which migrated on paper chromatograms more slowly than the disaccharides produced by chondroitin lyase but faster than a tetrasaccharide. This small fragment may represent the reducing end of the chondroitin sulfate molecule.

Galactosamine inhibition of protein synthesis in Bacteroides thetaiotaomicron.

Galactosamine does not support growth of Bacteroides thetaiotaomicron. Despite this, galactosamine was more effective than utilizable carbohydrates such as glucose in preventing synthesis of the inducible enzymes alpha-glucosidase and chondroitin lyase. Galactosamine also stopped overall protein synthesis. By contrast glucose and other utilizable carbohydrates increased the rate of protein synthesis. Addition of glucose to bacteria which had been treated with galactosamine restored the ability of the bacteria to synthesize protein and to produce inducible enzymes. Moreover, when B. thetaiotaomicron was incubated with [1-14C]galactosamine for 30 min at 37 degrees C, about one-third of the label which was taken up by the cells comigrated with glucosamine-6-phosphate on a thin-layer chromatogram. Thus galactosamine appears to be phosphorylated by the bacteria. After 2 h incubation of the bacteria with [1-14C]galactosamine, there was a significant increase in the amount of label which could be extracted from acidified extracellular fluid with diethyl ether. This indicates that galactosamine can be metabolized to the level of volatile fatty acids. The rate of uptake of galactosamine and the amount of labeled fatty acids produced from galactosamine were both much lower than the values obtained when glucosamine was the substrate. Thus, although some metabolism of galactosamine occurs, the rate is apparently too slow to enable galactosamine to support growth of B. thetaiotaomicron.

Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron.

When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated.

Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate.

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.

Genetic studies of kanamycin resistance in Campylobacter jejuni.

Campylobacter jejuni 3H40 and 4B20 harbored 59-kilobase (kb) self-transmissible plasmids encoding resistance to kanamycin and tetracycline. Although the two antibiotic resistances were more frequently inherited together, some transconjugants and ethidium bromide segregants which were resistant to only one of these antibiotics were recovered. The kanamycin-susceptible, tetracycline-resistant segregants carried plasmids 4 kb smaller than the 59-kb plasmids of their parents, whereas the kanamycin-resistant, tetracycline-susceptible segregants contained no detectable plasmid DNA. Restriction endonuclease maps of deleted forms of the 59-kb plasmids revealed that deletions and rearrangements of 4-kb lengths of DNA were associated with loss of kanamycin resistance. Translocation of the kanamycin resistance determinant between plasmid and chromosomal DNA was demonstrated. Such phenomena have not been previously described in C. jejuni spp. and are consistent with the interpretation that the kanamycin resistance determinant is encoded by a translocatable element.

Absence of persistence and transfer of genetic material by recombinant Escherichia coli in conventional, antibiotic-treated mice.

Strain BST-1 is a derivative of Escherichia coli K-12 that carries a plasmid designated pURA-4 and is the expression system used by The Upjohn Company in the production of recombinant bovine somatotropin (rbSt). This plasmid also encodes an ampicillin resistance gene. The plasmidless carrier strain, BST-1C, contains a gene for tetracycline resistance which is provided by the chromosomal insertion of the transposon Tn10. Therefore, BST-1 is resistant to ampicillin and tetracycline, while BST-1C is resistant only to tetracycline. The Food and Drug Administration requested that we conduct an environmental assessment study to monitor the 'persistence of the recombinant live K-12 E. coli organism compared to the host E. coli organism'. In addition, we were requested to monitor 'the potential transfer of genetic material from (our) recombinant organism to the indigenous microflora' of the mouse gastrointestinal (GI) tract. The differences in persistence were determined by monitoring shedding of BST-1 and BST-1C in the feces of conventionally reared, outbred mice inoculated with either of the two strains. Even with antibiotic selective pressure applied (tetracycline in the water), BST-1 did not persist as well as the non-plasmid carrying parental stain, BST-1C. In the gene transfer experiments, transfer of pURA-4 was monitored by the appearance of the ampicillin resistance marker and/or by hybridization assays for the rbSt gene in indigenous, mouse-colonizing E. coli strains which had been made streptomycin resistant. At the limit of detection, no transfer of pURA-4 was detected either in vitro or in vivo. These data support an interpretation that BST-1 does not present an environmental hazard as measured by colonization/persistence in the gut of conventionally reared mammals.