應用groESL於孿生球菌之快速鑑定、新菌種分類及groE操作組分析

Abstract

我們解開營養變異性鏈球菌(含Abiotrophia defectiva、Granulicatella adiacens及Granulicatella elegans)及孿生菌屬(含Ge. morbillorum、Ge. haemolysans及G. sanguinis)之groESL基因序列。在不同屬間，其groES與groEL之核苷酸序列相似度分別為41.7到85.9%與63.7到84.3%。而臨床分離株其與標準菌株的種內核苷酸序列相似度則為94.4到97.8% (groES基因)與94.0到98.2% (groEL基因)。以groESL基因為標第，我們發展了一套多重聚合酶連鎖反應方法，可快速且正確將營養變異性鏈球菌與孿生菌屬鑑定到屬名。這套方法也對臨床實驗室在鑑定營養挑剔或生長緩慢之細菌時有所助益。 我們曾一度因16S rRNA基因序列之高相似度(大於99.6%)而將四株臨床菌株NTUH_1465、NTUH_2196、NTUH_4957與NTUH_5572鑑定為Ge. haemolysans。由於觀察到這四株臨床菌株在groESL基因序列上的高變異度，我們暫將之命名為「類Ge. haemolysans」並進一步分析。由生化反應特性與多重基因座序列分析法之結果指出這四株臨床菌株可進一步區分成兩群：第一群含菌種NTUH_1465；第二群則含菌種NTUH_2196、NTUH_4957與NTUH_5572。菌種NTUH_1465、NTUH_5572與Ge. haemolysans之典型菌株ATCC 10379三者之間的DNA-DNA雜合值均小於新菌種定義的閥值70%。綜合生化表型、親源分析與DNA-DNA雜合實驗，菌株NTUH_1465 與NTUH_5572可被歸類為新菌種，在此並提出新菌種名稱為Gemella parahaemolysans sp. nov. (典型菌株為 NTUH_1465)與Gemella taiwanensis sp. nov. (典型菌株為NTUH_5572)。 對孿生球菌屬的groESL基因序列基因序列進行分析之後，發現其groE操作組由groES、groEL、trxA三個基因組成一特殊結構。北方拓墨法與反轉錄聚合酶連鎖反應則顯示除了自己轉錄之外，trxA也可與groES與groEL共同轉錄。在groE操作組的上游則沒有CIRCE序列，取而代之的則是一個結構類似CtsR binding site的序列圍繞在啟動子周圍。我們再定出dnaK周圍序列，發現CIRCE存在於dnaK操作組之上游，其基因組成乃是hrcA-grpE-dnaK-dnaJ。因此，我們在麻疹孿生球菌上發現了一個過去未曾報導過的第一型熱刺激蛋白基因系統：groE操作組不接受HrcA之調控但dnaK操作組則含有CIRCE序列。從北方拓墨法亦可觀察到groE操作組可有效對熱刺激與硫醇刺激做出反應，應該是因為此操作組只受CtsR調控的緣故。

We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Ge. morbillorum, Ge. haemolysans, and Ge. sanguinis). The nucleotide sequence similarities between the groES and the groEL genes from the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarity of groESL sequences in clinical isolates was 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid and accurate identification of Abiotrophia, Granulicatella and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories. We presumptively identified the four strains (NTUH_1465, NTUH_2196, NTUH_4957, and NTUH_5572) as Ge. haemolysans by 16S rRNA gene analysis with >99.6% sequence similarities. Since higher intraspecies heterogeneity in groESL was observed, we tentatively named them “Ge. haemolysans-like” and further examined. Data from phenotypic characterizations and multiple locus sequence analysis indicated that these isolates could be divided into two groups, I (strain NTUH_1465) and II (strains NTUH_2196, NTUH_4957, and, NTUH_5572). The DNA-DNA hybridization values between strains NTUH_1465 and NTUH_5572 and between the two strains and the type strain of G. haemolysans were all lower than the recommended threshold point of 70% for the delineation of bacterial species. The polyphasic data indicated that strains NTUH_1465 and NTUH_5572 could be classified as representatives of two novel genospecies in the genus Gemella, for which the names Gemella parahaemolysans sp. nov. (type strain is NTUH_1465) and Gemella taiwanensis sp. nov. (type strain is NTUH_5572) are proposed. Sequence analysis of the groE locus in Gemella species revealed an unusual organization of the groE operon as groES-groEL-trxA. Northern blot hybridization and RT-PCR revealed that trxA could be co-transcribed with groES-groEL, in addition to its own transcription. No CIRCE sequence could be found in the upstream region of the groE operon. Instead, we found a putative CtsR binding site located in the promoter region. We subsequently sequenced the dnaK locus and found that CIRCE was present in the dnaK locus. Thus, the lack of CIRCE in groES and the presence of CIRCE in dnaK in G. morbillorum indicate another regulatory model for the class I heat shock genes. From Northern blot analysis, the groE operon could efficiently respond to heat shock and thiol stress, which might due to single regulation of CtsR.