Filamentation initiated by Cas2 and its association with the acquisition process in cells

Abstract

Cas1-and-Cas2-mediated new spacer acquisition is an essential process for bacterial adaptive immunity. The process is critical for the ecology of the oral microflora and oral health. Although molecular mechanisms for spacer acquisition are known, it has never been established if this process is associated with the morphological changes of bacteria. In this study, we demonstrated a novel Cas2-induced filamentation phenotype in E. coli that was regulated by co-expression of the Cas1 protein. A 30 amino acid motif at the carboxyl terminus of Cas2 is necessary for this function. By imaging analysis, we provided evidence to argue that Cas-induced filamentation is a step coupled with new spacer acquisition during which filaments are characterised by polyploidy with asymmetric cell division. This work may open new opportunities to investigate the adaptive immune response and microbial balance for oral health.

Fig. 1

The C-terminal 30 amino acids are necessary and sufficient for Cas2Em-initiated filamentation in BL21(DE3). a Representative images of BL21(DE3) cells with expression of the vector control, Cas1Em, Cas2Em and Cas1Em-Cas2Em (scale bar, 25 μm). b Fluorescent images of Cas2Em-initiated filamentation labelled by SYTO 9 (green) and FM 4–64 (magenta) to indicate the DNA and membrane, respectively (scale bar, 5 μm). c The presence (+) and absence (-) of the filamentation phenotype with Cas2Em constructs, including four N-terminal and four C-terminal deletions. d Representative images of BL21(DE3) cells with expression of the vector control, Cas2Em_71-101 and Cas2Nm

Fig. 2

Cas1Ec-Cas2Ec-initiated filamentation in BL21-AI. a Representative images of BL21-AI cells with the vector control, Cas1Ec, Cas2Ec and Cas1Ec-Cas2Ec at 2 h, 4 h, 6 h and 12 h after induction (scale bar, 25 μm). b Quantitative analysis of the length (n = 4 per group, mean with S.D.) of bacterial cells. Significant differences between the control and treatment groups are indicated by *P < 0.05 and ***P < 0.000 1. c The results of the in vivo new spacer acquisition assay for the tested cells. The parental and expanded bands are indicated. New spacer acquisition is defined by the appearance of the expanded band. Representative data from at least three independent experiments are presented

Fig. 3

Cas1Ec-Cas2Ec-initiated filamentation is associated with new spacer acquisition. a The filamentation phenotypes after the separation of filaments and rod cells by the filter-based method: Top: cells retained by the filter; Bottom: cells filtered through the membrane (scale bar, 25 μm). b New spacer acquisition of unfiltered cell and filter-separated fractions at 6 h after induction of Cas1Ec-Cas2Ec expression. c Sequence comparison of Cas2 proteins from E. coli, E. meningoseptica and N. meningitidis generated by Clustal Omega. Identical amino acids are indicated by “*”; similar amino acids in the same group are indicated by “:”; and amino acids from different groups are indicated by “.”. d Quantitative analysis of the average length (n = 4 per group, mean with S.D.) of cells induced by Cas1Ec-Cas2Ec and/or the Cas2Ec mutant in BL21-AI compared with that induced by the vector control. Differences between the groups are indicated by “ns”: not significant; **P < 0.001 and ***P < 0.000 1. e The results of an in vivo new spacer acquisition assay for Cas2Ec mutants. In both b and e, the parental and expanded bands are indicated by arrows. Representative data from at least three independent experiments are presented

Fig. 4

A polyploidy-like structure was detected in Cas1Ec-Cas2Ec-initiated filaments. Fluorescent images of Cas1Ec-Cas2Ec-initiated filamentation labelled by SYTO 9 (green) and FM 4–64 (red) to indicate the DNA and membrane, respectively (scale bar, 5 μm). The membrane junctions are indicated by white arrows

Fig. 5

Asymmetric cell division was detected at the distal end of Cas1Ec-Cas2Ec-initiated filament. The process of asymmetric cell division detected by SEM (scale bar, 1 μm). The sites where the division would occur are indicated by yellow arrows