Investigation of the Role of the CusS-CusR Two-Component System in the Transition to VBNC Form of Escherichia coli at Alkaline pH

Öz

The Viable but non-culturable (VBNC) state is a process whose molecular mechanism has not yet been resolved. In this study, it was studied whether CusS-R, one of the two-component phosphorylating systems, has a role in the transition to VBNC form in an alkaline pH (8.5) environment. According to the results obtained in the study, while knocking out both cusS and cusR genes did not have an effect at pH 7.2, it limited the survival of E. coli in an alkaline environment. The t99 value of wild type E. coli W3110 was 57 according to the plaque count, while this value was 31 days when the cusS gene was knockout and 34 days when the cusR gene was knockout. When the knocked out genes were completed with the plasmid, the t99 value was determined as 62 days for the E. coli cusS mutant and 61 days for the E. coli cusR mutant, which is similar to that of wild-type E. coli. To determine whether this decrease in alkaline pH in mutants is due to VBNC formation, live cell counts were obtained by Live Dead count, CTC count and DVC count methods. According to these counting results, it was determined that the decrease in the plaque counts of the mutants was actually caused by bacteria entering the VBNC form. Therefore, it was determined that the cusS and cusR genes do not have a role in the transition to the VBNC form.

Anahtar Kelimeler

• Escherichia coli
• CusS/R
• Alkaline pH
• VBNC
• Two component phosphorelay

Proje Numarası

113T003 nolu TÜBİTAK projesi ile, bir kısmı ise 2018- 02.BŞEÜ.25-01 numaralı Bilecik Şeyh Edebali Üniversitesi BAP projesi

Alkali pH’da Escherichia coli’nin VBNC Forma Geçişinde CusS-CusR İki Bileşikli Sisteminin Rolünün Araştırılması

Öz

Canlı fakat kültürü yapılamayan (VBNC) durum henüz moleküler mekanizması çözülememiş bir süreçtir. Bu çalışmada iki bileşikli fosforlama sistemlerinden birisi olan CusS-R’nin alkalin pH (8.5) ortamında VBNC forma geçişte rollerinin olup olmadığı araştırılmıştır. Çalışmada elde edilen sonuçlara göre hem cusS hem de cusR genlerinin nakavt edilmesi pH 7.2 de bir etki göstermezken, E. coli‘nin alkali ortamda yaşamını sınırlamıştır. Yabani tip E. coli W3110’un plak sayımına göre t99 değeri 57 gün iken, bu değer cusS geni nakavt edildiğinde 31, cusR geni nakavt edildiğinde ise 34 gün olduğu tespit edilmiştir. Nakavt edilmiş olan genler plazmit ile tamamlandığında t99 değerinin E. coli cusS mutantı için 62 gün, E. coli cusR mutantı için 61 gün olarak tespit edilmiş olup bu değerler yabani tip E. coli ile benzerdir. Mutantlarda alkali pH’da görülen bu azalmanın VBNC oluşumundan kaynaklanıp kaynaklanmadığını belirlemek için canlı hücre sayımları Live Dead sayımı, CTC sayımı ve DVC sayım metotları ile elde edilmiştir. Bu sayım sonuçlarına göre mutantların plak sayımlarında görülen azalmanın aslında VBNC forma giren bakterilerden kaynaklandığı tespit edilmiştir. Dolayısı ile cusS ve cusR genlerinin VBNC forma geçişte bir rolleri olmadığı tespit edilmiştir.

Anahtar Kelimeler

• Escherichia coli
• cusS/R
• Alkalin pH
• VBNC
• İki Bileşikli Fosforlama

Destekleyen Kurum

TÜBİTAK ve Bilecik Şeyh Edebali Üniversitesi BAP

Proje Numarası

113T003 nolu TÜBİTAK projesi ile, bir kısmı ise 2018- 02.BŞEÜ.25-01 numaralı Bilecik Şeyh Edebali Üniversitesi BAP projesi

Teşekkür

Bu çalışmanın bir kısmı 113T003 nolu TÜBİTAK projesi ile, bir kısmı ise 2018-02.BŞEÜ.25-01 numaralı Bilecik Şeyh Edebali Üniversitesi BAP projesi ile desteklenmiştir. Bu destek için hem TÜBİTAK hem de Bilecik Şeyh Edebali Üniversitesine teşekkür ederiz.

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