The Role Of Lipoprotein NlpI In Escherichia Coli Cell Division

New lipoprotein I(Nlp I) is a lipoprotein and located in the outer membrane in Escherichia coli(E. coli). It has been found that Nlp I is involved in the cell division and bacterial virulence. Deletion of nlp I in E. coli strain LF82 affected the synthesis of type 1 pili and flagella, leading to changes in extracellular adhesion properties. Moreover, deletion of nlp I decreased the ability of E. coli K1 binding to and invasion of human brain microvascular endothelial cells(HBMEC). The overall process of cell division is a complex process involved in the coordinated roles of numerous proteins. Incubation of the E. coli nlp I::cm insertion mutant at 42℃ resulted in the abnormal cell division and the formation of cell filamentation, suggesting that Nlp I is involved in cell division. However, the underlying role of Nlp I in E. coli cell division is remaining unknown. To further investigate the role of Nlp I in cell division, in-depth understanding of the process of cell division, this subject adopts the means of molecular biology, microbiology and biochemistry research methods to expand the study of the following three parts:First, we found that in E. coli strain MG1655 over-expression of the nlp I gene at 37℃ led to severe inhibition of bacterial growth. We used Gram staining and scanning electron microscopy to identify cellular morphology and detected the increase of width and length of cell, severe adhesion between the bacteria and parts of the bacteria cracked death. DAPI staining showed that the bacterial nucleoids were anomalous after the over-expression of nlp I. We constructed p QE80-nlp I-M(residues 20-294, lacking the signal sequence and Cys19), p QE80-nlp I-282(lacking the C-terminal 12 residues) and p QE80-nlp I-233(lacking fifth TPR). These three plasmids were introduced individually into MG1655 followed by inducing with IPTG at 37℃. We found that the over-expression of nlp I-282 inhibited severely the bacterial growth and nucleoids division similar to the full length Nlp I, but the over-expression of nlp I-M and nlp I-233 did not influence the nucleoids division. The above experiments further suggested that Nlp I was involved in the bacterial division in E. coli, which we named Nlp I-mediated cell division pathway. Both N-terminal and C-terminal sequences were critical to the function of Nlp I. Microarray showed that the nlp I over-expression resulted in the change of transcriptional level of some genes.In the second part, the protein profiling analyzed by SDS-PAGE showed the nlp I over-expression induced the cytoplasm and the outer membrane of the host cells the dramatic changes. One significantly increased band was identified as heat shock protein Ibp A(inclusion body-associated protein A) and another significantly decreased band was outer membrane protein Omp W by mass spectrometry(MS) analysis. The ibp A and ibp B formed an operon, and were regulated by σ32 protein. We constructed the Δibp A, Δibp B, Δibp AB(deletion of ibp A and ibp B), Δomp W isogenic mutant strains and the trans-complementation of Δibp A with plasmid p AC-ibp A, and observed bacterial change after the over-expression of nlp I. Through monitoring the OD600 and nucleoids morphology, quantitative real-time PCR(q RT-PCR) and Western blot, our data confirmed that Ibp A and Ibp B were required for the Nlp I-mediated cell division.Our data showed that the over-expression of nlp I-233 did not increase the transcript levels of ibp A and ibp B and the protein levels of Ibp A and Ibp B, contrary to the over-expression of full length of nlp I by q RT-PCR and Western blot. The results confirmed that C-terminal of Nlp I contributed to Nlp I to induce expression of ibp A and ibp B increased. Nlp I located in the outer membrane(OM) through N-terminal signal sequence. The over-expression of nlp I-M increased the m RNA levels of ibp A and ibp B, and Ibp A/Ibp B proteins were increasing in the CP(cytoplasmic) fraction, but not in the OM. Both co-immunoprecipitation assay and bacterial two-hybrid assay confirmed that Nlp I physically interacts with Ibp B. We speculated that Nlp I induce the transcription of ibp A and ibp B. Ibp B, together with Ibp A, located to the OM through interaction with Nlp I. Then Ibp A and Ibp B,together with Nlp I, inhibited nucleoids division. In this study, we first found that another important role of Ibp A and Ibp B was involved in Nlp I-mediated cell division.Microarray, q RT-PCR and Western blot indicated that the over-expression of nlp I decreased the transcription of omp W and localization of Omp W in the OM. We speculate that the over-expression of nlp I resulted in disintegration of the outer membrane and cell lysis by decreasing expression of Omp W.In the third part, we observed that over-expression of nlp I or nlp I-282 inhibited bacterial nucleoids division and influenced Fts Z localization and assembly, but the over-expression of nlp I-M or nlp I-233 did not. We speculate that the nlp I over-expression resulted in larger nucleoids and abnormal division, then influenced Fts Z localization and assembly. This further confirmed that Nlp I involved in cell division.This project is first in-depth study of the function and mechanism of action of Nlp I, and will enhance our understanding of the important life processes of cell division. Since Nlp I contains 5 TPR which is only conserved in the prokaryotes, our findings will provide theoretical and experimental foundations for the development of new antibiotic targets.