京都大学 / 新制・課程博士 / 博士(理学) / 甲第23041号 / 理博第4718号 / 新制||理||1676(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 曽田 貞滋, 教授 木庭 啓介 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The bacteroides group is a subdivision in the <I>Cytophaga-Flavobacterium-Bacteroides</I> phylum. This group is as phylogenetically distinct from other Gram-negative enterics, including <I>Escherichia coli</I>, as they are from Gram-positive organisms. Furthermore, there is no cross expression between genes of <I>E. coli</I> and <I>Bacteroides</I> species. It is thought that this difference in gene expression lies in part at the level of transcription initiation and is due to the sequences within the promoter region itself. A putative consensus sequence for <I>Bacteroides</I> promoters has been published by C. Jeff Smith&#146;s research group based on alignments of the sequences upstream of certain regulated genes. However, this consensus has not been found within all putative <I>Bacteroides</I> promoters. In this study, the promoter structure and function of a strong housekeeping <I>B. thetaiotaomicron</I> 16S rRNA promoter was examined and compared to an <I>E. coli</I> 16S rRNA promoter. Our hypothesis is that there are significant differences between the promoters of these two organisms. Analysis of <I>B. thetaiotaomicron</I> sequence upstream of the 16S rRNA gene has revealed the same overall structure known for <I>E. coli</I> 16S rRNA promoters in that there are two putative promoters separated by approximately 150 bp. However, the <I>B. thetaiotaomicron</I> 16S rRNA promoter contains the proposed <I>Bacteroides</I> &#151;7 and &#151;33 consensus sequences instead of the well known <I>E. coli</I> &#151;10 and &#151;35 consensus sequences. The biological activity of the<I> B. thetaiotaomicron</I> 16S rRNA full-length promoter was confirmed using a <I>Bacteroides lux</I> reporter system. A newly designed <I>Bacteroides lux</I> reporter was used to analyze specific regions of the <I>B. thetaiotaomicron</I> 16S rRNA promoter. In addition, by pairing the <I>B. thetaiotaomicron</I> 16S rRNA promoter with an <I>E. coli</I> ribosomal binding site, and vice-versa, the improved <I>lux</I> reporter was used to further confirm that the difference in gene expression between the two species lies at the level of transcription in <I>E. coli</I>. In <I>Bacteroides</I>, however, transcription and translation may work together to create a barrier to efficient gene expression of foreign genes. </P> / Master of Science

In the present study a character data set suitable for cladistic analysis at the family level was developed. A data matrix consisting of 50 morphological characters from 15 families of tardigrades was analyzed by maximum parsimony. Kinorhynchs, loriciferans, and gastrotrichs were used as outgroups. The results agree with the currently accepted hypothesis that Eutardigrada and Heterotardigrada are distinct monophyletic groups. Among the eutardigrades, Eohypsibiidae was found to be a sister group to Macrobiotidae + Hypsibiidae, while Milnesiidae was the basal eutardigrade family. The basal heterotardigrade family was found to be Oreellidae. Echiniscoideans grouped with some traditional Arthrotardigrada (Renaudarctidae, Coronarctidae + Batillipedidae) suggesting that the arthrotardigrades are not monophyletic. The 18S rRNA gene sequence of Batillipes mirus Richters, 1909 and Calohypsibius schusteri Nelson & McGlothlin, 1996 were obtained and their addition to a previously published dataset supports the monophyly of Heterotardigrada and of Parachela versus Apochela within the Eutardigrada.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19960号 / 理博第4227号 / 新制||理||1607(附属図書館) / 33056 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 木庭 啓介, 教授 沼田 英治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

This study was conducted to determine dynamics of bacterial communities during decomposition and to find out whether the occurrence of bacterial communities was affected by soil and residue types. It was hypothesized that there would be a shift in bacterial community structure during decomposition. Also, distinct microbial communities in different two soils associated with two residues would result in colonization by different microbial taxa. The first hypothesis was based on expected changes in the composition of decomposing residues. The second hypothesis was based on the fact that soil microbial diversity is soil-specific and immense with numerous different functionally redundant but phylogeneticaly different microbial types. Residues with different chemical properties were also expected to affect bacterial community composition, however, its impact would be lesser compared to soil. A 2 x 2 x 4 factorial experiment was conducted consisting of switchgrass (Panicum virgatum) and rice (Oryza sativa) straw; 2 soil types (Sharkey and Marietta series); and 4 incubation periods (3, 23, 48 and 110 days). Clone libraries of the bacterial communities were constructed from the detritusphere (residues and adhering soil). Non-metric multidimensional scaling of the detritusphere communities showed distinct separation of the communities at day 3 which coincided with high levels of cellulase enzyme activity and reduction of soluble carbon. style='mso-spacerun:yes'> Availability of labile carbon appeared to be important in driving bacterial community succession at early stage of colonization. During the later stages of decomposition (day 23-110), bacterial communities were segregated into two groups according to soil type. Although important, this segregation was relatively small compared to the community-level similarities observed between the soils and residues. For example, 16 of the 22 most abundant OTU's, dominated by a-,b- and style='fontamily:Symbol'>g- Proteobacteria, Bacilli and Shingobacteria, were shared among all soil and residue treatments indicating that residue decomposition is carried out by few key-player taxa. These results run counter to our hypothesis and suggest that decomposition process may be mediated by certain domineering bacterial taxa which occur at the later stage of decomposition. Further research is needed to determine whether key functional ecosystem processes are dominated by only a few taxa despite taxonomically hyper-diverse soils.

Mjölksyrabakterier tillhörande släkten Lactobacillus och Bifidobacterium har nyligen upptäckts hos bin och i honungen de producerar och innefattar 13 nya arter[1]. Forskarna arbetar med att ta fram nya snabba och mer pålitliga identifierings metoder för att karakterisera dessa bakterier.I detta projekt undersöktes möjligheten att identifiera dessa bakterier med en ny metod som heter denaturing high performance liquid chromatography (DHPLC). Metoden bygger på separation av PCR (polymerase chain reaction) amplifierade 16S rDNA fragment i DHPLC [8]. Vid identifiering av bakterier amplifierades olika variabla regioner från 16S rRNA genen, som påvisade efter sekvensering störst genetisk variation mellan dessa bakterier [1]. Separationen utfördes med ion-pair reverse-phase high presure liquid chromatoghaphy (IP RP HPLC) med delvis denaturering av DNA molekylen. Tidigare studier av identifiering av marina bakterier med DHPLC resulterade i optimal separation [9]. Identifiering av följande mjölksyrabakterier kunde verifieras till en viss grad. Analys i DHPLC visade profiler med urskiljbara toppar som utgjorde separation på artnivå, detta enbart mellan två bakterier tillhörande släktet Lactobacillus. Trots olika justeringar av analys parametrar gällande kolonntemperatur och elueringsbuffert, erhölls inte separation mellan alla 13 arter. Analysen kan ha påverkats av en rad olika funktionsfel i HPLC systemet och felaktig beredning av prov. Metoden kunde eventuellt förbättrats om tiden inte varit en begränsning. / Lactic acid bacteria belonging to genera Lactobacillus and Bifidobacterium has been recently discovered in bees and the honey they produce, and includes 13 new species [1]. Researchers are working to develop new rapid and more reliable detection methods to characterize these bacteria.In this project we investigate the possibility of identifying these bacteria with a new method called denaturing high performance liquid chromatography, DHPLC. The method involves the separation of the PCR (polymerase chain reaction) amplified 16S rDNA fragments in the DHPLC [8].For the identification of bacteria various variable regions of 16S rRNA gene was amplified, sequencing proved great genetic variability between these bacteria [1]. Separation is effected by means of ion-pair reverse-phase high pressure liquid chromatography (IP RP HPLC) with partial denaturation of the DNA molecule.Previous studies of the identification of marine bacteria by DHPLC resulted in optimal separation [9]. Identification of the following lactic acid bacteria was verified to some limited degree. Analysis of the DHPLC demonstrated profiles with distinguishable peaks that represented the separation at the species level, that only between two bacteria of the genus Lactobacillus.Despite numerous adjustments of operating conditions such as existing column temperature and eluent buffer, did not result in separation of all 13 species. The analysis may have been influenced by a variety of malfunctions in the HPLC system and improper sample preparation. The method could possibly be improved if time was not a limitation.

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