Genes encoding bile salt hydrolases and conjugated bile salt transporters in Lactobacillus johnsonii 100-100 and other Lactobacillus species.

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

Microbial biota of the human intestine: a tribute to some pioneering scientists.

Research on the indigenous intestinal microbiota of man was initiated well before the end of the 19th Century. The work continued at a slow but steady pace throughout the first half of the 20th Century. Findings from the effort had little impact on medicine and other aspects of human biology, however, until the 6th decade of the 20th Century. During that decade, research in the area was begun by eight groups of investigators, each of which was led by one or two senior scientists with great experimental talent, creativity and foresight. Their findings added new dimension to knowledge of the microbiota and initiated an explosion of interest in research in the field that has continued to the present day. The research of the groups during the 1960's is described in this review as a tribute to the senior scientists who had such critical impact on this important field of study.

Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli.

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Identification of genes encoding conjugated bile salt hydrolase and transport in Lactobacillus johnsonii 100-100.

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, alpha and beta, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3' partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5' and 3' deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3' of the cloned segment. The predicted amino acid sequence of the 5' partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5alpha cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [14C]taurocholic acid that were increased as much as threefold over control levels. [14C]Cholic acid was taken up in similar amounts by strain DH5alpha pSportI (control) and DH5alpha p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.

Filter paper cortisol profiles in secondary adrenocortical insufficiency.

The use of filter paper four point 24 hour cortisol profiles in the diagnosis and management of secondary adrenocortical deficiency was examined. Eighteen children with familial short stature and isolated growth hormone deficiency had normal 24 hour and morning plasma cortisol concentrations measured. Eight of 11 children with multiple pituitary hormone deficiencies had evidence of hypocorticalism despite previously normal baseline cortisols or responses to insulin hypoglycaemia or tetracosactrin. Nine of 11 children with hypopituitarism on replacement treatment (4.9-12.5 mg/m2/day) had satisfactory concentrations of cortisol, though morning cortisols were often low. Filter paper profiles are a simple, inexpensive, and relatively non-invasive way of managing children with suspected hypocorticalism and of monitoring their treatment.

Presentation, acute illness, and learning difficulties in salt wasting 21-hydroxylase deficiency.

The presentation, pattern of acute illness, and incidence of learning difficulties are described in 63 (33 boys, 30 girls) children with salt wasting 21-hydroxylase deficiency, drawn from a cohort study of congenital adrenal hyperplasia in the South West Region of England between 1968 and 1988. Thirty boys presented with a salt losing crisis from birth whereas the other three boys presented between 2 and 14 months of age with failure to thrive and hyponatraemia. Diagnostic uncertainty led to 13 (43%) of 30 girls developing a salt losing crisis. Five girls were misassigned as boys at birth. There were four deaths in the group, two due to salt losing crisis, one to complications of prematurity possibly compounded by 21-hydroxylase deficiency, and one from heart failure probably related to an excess of steroids. Acute admissions were common, especially during the first year of life, with convulsions in 7% of admissions. The 9% incidence of hypoglycaemia was considered to be an underestimate as blood glucose was measured in only 56 (22%) of 254 admissions. No convulsions occurred in the 38 (15%) admissions where the parents had given intramuscular hydrocortisone before bringing the child to hospital. A high incidence of learning difficulties was found among the 59 surviving children (9/30 (30%) boys and 6/29 (21%) girls), and in only two children could any factor other than 21-hydroxylase deficiency be invoked. Analysis of the subgroup with learning difficulties indicated that they were more ill at presentation with a significantly higher incidence of hypoglycaemia, and that growth in the first year was significantly worse. It is concluded that congenital adrenal hyperplasia remains a formidable disorder with an appreciable mortality and morbidity. The high incidence of learning difficulties seen in salt wasting 21-hydroxylase deficiency needs further attention. A prospective study is indicated to examine the effect of neonatal screening on morbidity from congenital adrenal hyperplasia, particularly the intellectual impairment seen in this study.

Characterization of an activity from the strict anaerobe Roseburia cecicola that degrades DNA when exposed to air.

Roseburia cecicola is an obligately anaerobic bacterium that is extremely sensitive to oxygen. Genomic DNA isolated from cells exposed to air for even a brief period (< 5 min) is partially degraded, while DNA extracted from cells maintained in an anaerobic environment remains intact. Cells exposed to air for longer and longer periods yield DNA which is progressively degraded into fragments with decreasing sizes. Oxygen toxicity for this anaerobe appears to result, at least in part, from degradation of its genomic DNA. Cell lysates of the organism exhibited a similar ability to degrade exogenous sources of DNA when assayed in vitro under aerobic conditions. A substance that degrades both DNA and RNA when incubated aerobically was partially purified from such lysates. It has an approximate molecular weight of 2,800 and is unlikely to be a protein. It requires a reducing agent for activity and can be inhibited by catalase and peroxidase but not superoxide dismutase. The rate at which it degrades DNA in vitro can be enhanced by temperatures above 37 degrees C or by oxygen at partial pressures above atmospheric pressure. These results suggest that this substance degrades nucleic acids by a mechanism involving oxygen radicals.

Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100.

Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of Lactobacillus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalytically similar, except that the Vmax of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated alpha and beta, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations alpha 3, alpha 2 beta 1, alpha 1 beta 2, and beta 3 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all alpha peptide) react in Western blots (immunoblots) only with proteins containing the alpha peptide; such antibodies raised against BSH D (all beta peptide) react only with proteins containing the beta peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.

Characterization of an extracellular factor that stimulates bile salt hydrolase activity in Lactobacillus sp. strain 100-100.

Bile salt hydrolase activity in Lactobacillus sp. strain 100-100 is strictly intracellular. The strain produces an extracellular factor that stimulates the intracellular hydrolase activity. The factor is inducible by conjugated bile salts, has an apparent molecular mass over 12 kDa but less than 25 kDa, is stable in air, and resistant to pronase and heat. It is partially extractable into organic solvents and inactivated by a sulphydryl group inhibitor. We postulate that the factor functions by a novel mechanism to facilitate entry of conjugated bile salts into the bacterial cells.

Growth phase, cellular hydrophobicity, and adhesion in vitro of lactobacilli colonizing the keratinizing gastric epithelium in the mouse.

Lactobacillus strains of numerous species isolated from several animal sources exhibited cellular hydrophobicities that differed from those expected on the basis of their abilities to colonize the keratinizing stratified squamous epithelium in the mouse stomach. Cells of Lactobacillus fermentum 100-33, grown to either exponential or stationary phase, were strongly hydrophilic. By contrast, cells of L. fermentum RI and six transformant derivatives of strain RI and 100-33, strains DM101 through DM106, were hydrophobic to various degrees in either growth phase. Most of them were less hydrophobic, however, when in the stationary phase than in the exponential phase. Cells of strains RI and 100-33 in the exponential phase adhered in the same number in vitro to disks of keratinized mouse gastric mucosa. By contrast, when in stationary phase, strain RI and two transformants, DM103 and DM104, adhered to the surface in higher numbers than 100-33. In contrast to their cellular progenitor, 100-33, the transformant strains share with their DNA donor, RI, the capacity to colonize the keratinizing gastric epithelium in mice. These findings indicate that lactobacilli able to colonize the surface of the keratinocytes in the murine stomach can adhere to that surface by either hydrophilic or hydrophobic molecules.

Adhesion of Candida albicans to epithelial cells effect of polyoxin D.

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.

Persistent hyperthyrotropinaemia since the neonatal period in clinically euthyroid children.

We describe three children, now aged between 5 and 6 years, with a persistent mild hyperthyrotropinaemia since the neonatal period and normal levels of thyroid hormones. The increased thyroid stimulating hormone concentration is not artefactual and is not caused by antibody interferences. Their growth and development is normal and none has received thyroid hormone replacement. We believe that they have compensated hypothyroidism, and that before the advent of screening for congenital hypothyroidism these children would have presented in mid-childhood with juvenile hypothyroidism.

Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100.

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.

The glycaemic effect of simple sugars in mid-morning and afternoon snacks in childhood diabetes.

Eighteen diabetic children ate three different snacks (free sucrose, sucrose plus fibre, fructose plus fibre) or had no snack on each of 4 mornings. Subsequently 10 children from this group took a standard snack (free sucrose) or no snack on two afternoons. In other respects the day of testing was standardised, the children going to school as normal and collecting their blood spots on filter paper for glucose analysis. There was no difference in the blood glucose profiles between different snacks or when no snack was taken either in the mornings or afternoon. This suggests both that moderate amounts of simple sugar do not have a detrimental effect on glycaemic control and that snacks can generally be omitted without serious hypoglycaemic problems.

Influence of Rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses.

Opsonized Rhodococcus equi activated the respiratory burst of resident alveolar macrophages (AM) from adult horses in a logarithmic-linear, mass-related manner. The effect of R equi was not significantly different from that of equal masses of opsonized zymosan A. Therefore, R equi does not appear to attenuate the respiratory burst of equine AM. The stimulatory effect of R equi was not reflected by increased production of superoxide anion (O2-), but increased activity of the hexose monophosphate shunt was observed. These results suggest a similarity between the respiratory burst of AM from horses and that of AM from rabbits. We concluded that resident AM from adult horses do not produce O2- concurrently with an increase in activity of the hexose monophosphate shunt when stimulated with either opsonized zymosan A or opsonized R equi. This suggests that O2- is not an important component of the antibacterial defenses of equine AM. Whether equine AM are incapable of producing O2- or require different stimuli to produce it was not determined.

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33.

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Obesity and diabetic control.

Children with diabetes mellitus are not significantly heavier than their peers, and those who are obese do not have poorer diabetic control.

Thyroid function in survivors of cancer.

Thyroid function was assessed in three selected groups of children who had survived cancer. Children in group 1 had received radiotherapy to the thyroid area, group 2 had radiotherapy to the thyroid area and adjuvant chemotherapy, and group 3 had chemotherapy with or without radiotherapy away from the thyroid area. There were 75 survivors and 63 (40 boys, 23 girls) were available for study. Eighteen (29%) were found to have thyroid dysfunction, and these included all those who had had lymphangiograms or received a radiation dose greater than 40 Gy to the thyroid area. Only nine of the 18 children were already known to have thyroid dysfunction, and only 15 of 44 children who had had irradiation to the thyroid area had had their thyroid function examined. This study shows that children who have received radiotherapy to the thyroid area should have their thyroid function assessed regularly. Chemotherapy does not appear to be a risk factor but longer follow up of these children is necessary.