No / Streptococcus mutans is frequently identified on the basis of phenotypic characteristics such as the ability to ferment carbohydrates. The usefulness of some of these identification tests may be limited in the case of isolates which are atypical with regard to their fermentation properties. We previously identified isolates of S. mutans which were unable to ferment melibiose, a characteristic which is included in some typing schemes. In all of these isolates there was a large chromosomal deletion which included the multiple sugar metabolism (msm) operon which encodes several genes involved in the uptake and metabolism of a number of sugars including melibiose. In the present study, sugar fermentation tests, ribotyping, colony hybridisation with DNA probes and polymerase chain reaction (PCR) were used to investigate the relatedness of these atypical isolates. The PCR and colony hybridisation procedures were based on amplification and detection of two genes: the wapA gene which encodes a surface protein found in all S. mutans strains and the gtfA gene which lies within the msm operon. The colony hybridisation and PCR results confirmed loss of the gtfA gene in the melibiose-negative isolates. Three new melibiose-negative isolates were also identified, but in only 2 of these was the gtfA gene absent, the third did not appear to have lost this region of the chromosome. Biotyping, as well as ribotyping based on an EcoRl digest of chromosomal DNA, revealed that the melibiose-negative isolates fell into a number of distinct groups. The identification of an isolate which is unable to ferment melibiose but does not appear to have lost the msm operon indicates that the melibiose-negative phenotype can arise from more than one type of genetic event.

<p><strong>Apstrakt je obrađen tehnologijama za optičko prepoznavanje teksta (OCR).</strong></p><p>Analizom podloge iz standardizovanog fiziolo&scaron;kog testa fermentacije rafinoze<br />primenom HPTLC metode, utvrđeno je da se u podlozi javlja jedinjenje sa<br />retencijom većom od rafinoze, dok svi konstituenti rafinoze imaju manju retenciju.<br />Za nastajanje takvog jedinjenja tokom testa fermentacije rafinoze i njenih<br />konstituenata kvascima nije bilo literatume potvrde.<br />Ispitivano je kod kojih kvasaca i kod fermentacije kojih ugljenih hidrata se u<br />podlozi javlja ispitivano jedinjenje. Zatim je ispitivana veza određenih parametara<br />procesa fermentacije sa nastajanjem zapaženog jedinjenja. Pored toga, ispitivanom<br />jedinjenju su, nakon izolovanja iz fermentacione podloge i preči&scaron;ćavanja radi<br />dobijanja čiste supstance, određeni neki elementi strukture.<br />Utvrđeno je da se samo kod kvasaca koji imaju fiziolo&scaron;ko svojstvo fermentacije<br />celog molekula rafinoze i svojstvo fermentacije melibioze tokom procesa<br />javlja ispitivano jedinjenje i da je ono intermedijer metabolizma. Činioci značajni<br />za proces fermentacije - inokulum (količina i podloge za njegovu pripremu),<br />sastav podloge (sadržaj rafinoze i melibioze, kao i različiti izvori azota) i uslovi<br />gajenja, varirani u odnosu na standardizovani fiziolo&scaron;ki test fermentacije, ne<br />prouzrokuju promenu kvalitativnog sastava jedinjenja u podlozi tokom fermentacije<br />rafinoze i melibioze. Dakle, ispitani intermedijer je uvek prisutan u podlozi.<br />Na povećanje njegovog sadržaja u podlozi utiče dodatak galaktoze tokom fermentacije<br />rafinoze. Najveća koncentracija ispitivanog intermedijera (0,87 mg/ml) je<br />trećeg dana fermentacije melibioze, odnosno drugog dana fermentacije rafinoze<br />(1,48 mg/ml). Sadržaj ispitivanog intermedijera na kraju procesa fermentacije<br />rafinoze je 6 puta veći nego kod melibioze (1,04 prema 0,17 mg/ml). Ispitivani<br />intermedijer je redukujući ugljeni hidrat molekulske mase 504 &scaron;to odgovara<br />trisaharidima. Konstituenti ovog jedinjenja su glukoza i galaktoza koje su povezane<br />najverovatnije a-tipom veze.</p> / <p><strong>Abstract was processed by technology for Optical character recognition (OCR).</strong></p><p>Analyzing the media from standardized physiological test for raffinose fermentation<br />by means of HPTLC method, the appearance of a compound was registered<br />with retention higher than that of raffinose or its constituents indicating also<br />smaller retentions. No literature data were found for the formation of such a<br />compound during fermentation tests for raffinose and its constituents.<br />It was investigated which yeast will indicate formation of the investigated<br />compound in the course of fermentation as well as which carbohydrate during its<br />fermentation will indicate the presence of this compound. Further investigation<br />included correlation of particular parameters known as significant for the process<br />of fermentation, with the appearance of the noticed compound. Moreover, some<br />structural elements were also determined for the investigated compound, after<br />isolation from fermentation media and purification in order to obtain pure<br />substance.<br />It was proved that the investigated compound appeared just with the yeasts<br />physiologically characterized by fermentation of entire molecule of raffinose as<br />well as melibiose in the course of fermentation. Ibis compound proved to be an<br />intermediate compound of metabolism. Parameters significant for the process of<br />fermentation - inoculum (size and media for its growth), media composition<br />(content of raffinose and melibiose as different nitrogen source) and the condition<br />of incubation, exchanged from the standardized physiological test of fermentation,<br />didn&#39;t change qualitatively the compound composition in the media during<br />raffinose and melibiose fermentation. Therefore, it is obvious that the investigated<br />intermediate compound was present constantly in the media. Its content is<br />effected by the addition of galactose in the course of raffinose fermentation. The<br />highest concentration (0.87 mg/ml) of the investigated compound was registered<br />on the third day of melibiose fermentation, i.e. the second day of raffinose<br />fermentation (1.48 mg/ml). Content of the investigated intermediate compound<br />at the end of raffinose fermentation was six limes higher than of melibiose (1.04<br />in relation to 0.17 mg/ml). The investigated intermediate compound proved to be<br />the reduced carbohydrate with molecular mass of 504 corresponding to trisaccharides.<br />Constituents of this compound, glucose and galactose are probably connected<br />by a-type of bonds.</p>

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12880号 / 論農博第2807号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4879(農学部図書室) / 31598 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 谷 史人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM