臺灣博碩士論文加值系統

Bacillus subtillis DB1005是一株對溫度敏感sigA突變株，它的σ蛋白和起動子-10
DNA結合區域具有Ile-198-Ala及Ile-202-Ala的雙重取代。研究結果顯示，這兩個胺
基酸取代不但促使σ蛋白構造不穩，容易被分解，也促使σ蛋白在高溫下之轉錄活性
對溫度敏感。為了進一步瞭解這兩個Ile胺基酸在B.subtilis σ蛋白和起子-10 DNA
結合區域的構造及功能上之重要性，我們進一步尋找 B.subtilis DB1005的回復突變
種（revertants）。利用適應性突變（adaptive mutation）原理，我們篩選到兩類
能在高溫下生長的B.subtilis回復突變菌株，分別命名為B.subtilis DB1005R1及
B.subtilis DB1005R3。這兩個回復突變株雖然均比B.subtilis DB1005R1及
B.subtilis DB1005R3。這兩個回復突變株雖然均比B.subtilis DB1005耐高溫，但它
們於高溫生長的能力仍不如B.subtilis DB2。利用染色體DNA同時轉化（
cotransformation）及基因置換（gene conversion）的方法，證實這兩類回復突變
株均屬sigA同基因性（intragenic）的壓抑性突變種，它們分別具有Ser-291-Phe及
Ala-198-Val胺基酸的取代。這兩個回復突變種σ蛋白半生期分析結果顯示，在37℃
下B.subtilis DB1005R3 σ蛋白的半生期（158 分鐘）比B.subtilis DB1005 σ蛋白
者（59 分鐘）為長；然而B.subtilis DB1005R1 σ蛋白的半生期（74 分鐘）並沒有
顯著增長。 在49℃下，不論野生型、溫度敏感型或回復突變型σ蛋構造均不穩定，
容易被分解。利用?-galactosidase 為報導基因之活性分析（37℃）或groEL基因誘
導能力之分析（49℃），比較各種σ蛋白轉錄活性。結果顯示，不論在37℃或49℃下
，B.subtilis DB2、DB1005R1和DB1005R3 σ蛋白均比B.subtilis DB1005R1之Ser-
291-Phe 取代，主要造成σ蛋白活性之回復，而B.subtilis DB1005R3之Ala-198-Val
取代，除了活性回復外，亦兼具穩定σ蛋白，使其免於被分解之功效。由於細胞內大
量σ蛋白的生產未能加強B.subtilis DB1005或各回復突變株在高溫下之生長勢，可
見造成B.subtilis DB1005R1及DB1005R3回復高溫下之生長勢，可見造成B.subtilis
DB1005R1及DB1005R3回復高溫生長特性的原因，並不在細胞中σ蛋白農度的高低，應
取決於σ蛋白的活性是否回復。

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA
mutant which contains two-amino-acid substitutions (Ile-198-Ala
and Ile-202-Ala) in the promoter -10 binding region of sigA
factor. Studies on the structural and functional properties of
this factor have revealed that it was structurally unstable and
easily degraded even at the permissive temperature, besides the
sensitivity to temperature elevation in transcription reaction.
In order to realize more about the importance of these two
isoleucine residues to the functional structure of the promoter
-10 binding region of sigA factor, we looked for suppressor
mutants of B. subtilis DB1005. Two types of revertants which
were able to partially suppress the Ts phenotype of B. subtilis
DB1005 were isolated from the culture exposed to heat stress.
They were named B. subtilis DB1005R1 and B. subtilis DB1005R3,
respectively. Results of genetic analyses and DNA sequencing
showed that they were intragenic suppressors with a Ser-291-Phe
or an Ala-198-Val substitution in the sigA factor of B.
subtilis DB1005R1 and DB1005R3, respectively. The structural
stability of both revertant sigA factors were improved to
different extents; the half-life of B. subtilis DB1005R3 sigA
(t1/2 = 158 min) was about 2.7-fold longer than that of B.
subtilis DB1005 sigA (t1/2 = 59 min)；however, no significant
difference in the degradation rate was observed between B.
subtilis DB1005R1 sigA (t1/2 = 74 min) and DB1005 sigA at 37 ℃.
The sigA factors, regardless of wild-type, Ts or revertant, were
unstable and rapidly degraded at 49 ℃. The transcription
activities of different sigA factors at both 37 ℃ and 49 ℃
were also compared. We found that the sigA factors of B.
subtilis DB1005R1 and DB1005R3 were more active than that of B.
subtilis DB1005, but were less active than that of B. subtilis
DB2 at both temperatures. Taken together, our results
demonstrated that the phenylalanine residue at position 291 of
sigA rescued the Ts phenotype of B. subtilis DB1005 mainly by
improving the transcription activity of sigA, while Val-198
improved both the transcription activity and structural
stability of sigA. Since overexpression of the mutant sigA
proteins in B. subtilis DB1005R1 and DB1005R8 failed to restore
their growth potential to the level of B. subtilis DB2, we
thought that the reversion was mainly attributed to the
improvement of sigA activity, rather than sigA concentration, in
the two revertants.