Functions of the DUF1127 protein and ProQ in oxidative stress resistance and extracellular proteinase secretion.
In our previous study, it was showed that expressions of genes encoding a DUF1127 containing protein and ProQ were induced by oxidative stress [37]. The function of DUF1127 protein is still unknown in V. alginolyticus. Bioinformatic analysis revealed there was only one gene encoding DUF1127 protein (66 amino acids) in the genome. The gene encoding the DUF1127 protein is connected to four sRNAs in R. sphaeroides [18]. However, our analysis showed that there were no sRNAs predicted located at downstream of the DUF1127 gene in V. alginolyticus (Fig. 1A). It was showed that DUF1127 protein from R. sphaeroides was similar to Smaug protein from Drosophila melanogaster which is involved in RNA turnover and in the development of fruit flies and mammals [39, 40]. It is predicted that the structure of the DUF1127 protein in V. alginolyticus is also similar to Smaug protein (Figure S1) (https://swissmodel.expasy.org/interactive), the conservated amino acids (isoleucine, leucine and glycine) were identified in the DUF1127 protein from V. alginolyticus, R. sphaeroides and SAM domain (Fig. 1B).
Additionally, ProQ containing the FinO domain is highly conserved in bacteria (Fig. 2A). Red highlights showed that the conserved amino acids of ProQ in V. alginolyticus and E. coli. Furthermore, in both bacterial strains, the proq gene is followed by prc (carboxy terminal-processing peptidase) (Fig. 2B).
In order to demonstrate the function of the DUF1127 protein and ProQ in V. alginolyticus, the DUF1127 and ProQ gene deletion strains were constructed. The two deletion strains were validated by PCR (Figure S2). In previous study, it was showed that expressions of DUF1127 protein and ProQ were induced by oxidative stress. Here, ΔDUF1127 showed no difference on oxidative stress resistance compared to WT (data not shown), however, we observed that ROS (Reactive Oxygen Species) level was decreased after deletion of DUF1127 in V. alginolyticus (Fig. 3A). The level of ROS in the ΔProQ showed no difference compared to the WT strain (data not shown). However, when exposed to H2O2 stress, we observed that ProQ deletion strain showed less resistance to oxidative stress (Fig. 3B). In existing reports, ProQ is also involved in the process of resisting oxidative stress in many bacteria, including Escherichia coli and Salmonella [15, 38].
V. alginolyticus can produce various virulence factors such as extracellular proteases, which can directly damage the host's immune defense system. In this study, the effect of the DUF1127 protein on the extracellular proteinase secreted was investigated using a skim milk agar plate method. The results showed that extracellular proteinase secreted was significantly reduced after deletion of DUF1127 (Fig. 3C), indicating that the DUF1127 protein is involved in regulating the extracellular proteinase secreted in V. alginolyticus. However, deletion of ProQ had no effect.
The DUF1127 protein and ProQ have opposite effects on biofilm formation in Vibrio alginolyticus
Biofilm can help bacteria to resist unfavorable external conditions and maintain their growth and reproduction. To investigate whether the DUF1127 protein and ProQ are involved in biofilm formation, the deletion strains were cultured for 48 hours to analyze biofilm formation. As shown in Fig. 4A, the amount of biofilm formed by the DUF1127 deletion strain was much higher than that of the wild type. In addition, scanning electron microscopy was used to observe the biofilm at the cellular level after 24 hours of cultured. The results showed that the ΔDUF1127 had a higher stacking density than that of the WT, indicating that the DUF1127 protein has a negative regulatory effect on biofilm formation (Fig. 4C). In A. tumefaciens, the biofilm formation of the DUF1127 deletion strain was also increased [39].
Interestingly, later results showed that the ProQ protein had a positive regulatory effect on the biofilm formation (Fig. 4B). By crystal violet staining, it was evident that the ΔProQ strain had a significantly lower biofilm formation capacity compared to the WT strain. Further validation through scanning electron microscopy supported this observation. In panoramic view, it was apparent that the ΔProQ strain with large gaps between bacteria. In contrast, the WT strain with close inter-bacterial connections. Upon closer examination at higher magnification, it becomes evident that the ΔProQ strain, compared to the WT, hardly formed any biofilm, it seems that there was no stacking between cell and cell. This indicates that ProQ can promote biofilm formation in V. alginolyticus (Fig. 4C). Previous study have also showed that ProQ is essential for biofilm formation [40]. This aligns with the function exhibited by ProQ in V. alginolyticus.
The genes and sRNAs affected by the DUF1127 protein and ProQ
To analysis the genes affected by the DUF1127 protein, RNA-seq was performed by using DUF1127 deletion compared to WT. In total, 151 differentially expressed genes were identified. 79 genes (log2 fold change ≥ 0.5 and P value ≤ 0.05) were upregulated and 72 genes (log2 fold change ≤-0.5 and P value ≤ 0.05) were significantly downregulated in the ΔDUF1127 strain (Fig. 5A). Details of all differentially expressed genes are summarized in the Table S3. Additionally, we randomly selected several genes from the transcriptome for validation using RT-qPCR to assess the accuracy of the RNA-seq data. The results, as shown in Figure S4, demonstrated a consistent trend between RT-qPCR and RNA-seq. In the transcriptome of the ΔDUF1127 mutant strain, genes associated with the secretion of extracellular proteases were significantly downregulated, such as serine protease (AT730_RS13155) and cobA. Additionally, genes related to the synthesis of biofilm, such as rbsB, alsS [41, 42], and those associated with the two-component system (AT730_RS18845), also exhibited significant differential expression in the transcriptome. Certain genes involved in drug efflux, such as vmeY (multidrug efflux RND transporter permease subunit VmeF) and AT730_RS07965 (efflux RND transporter periplasmic adaptor subunit), were also influenced by the DUF1127 protein (Fig. 5B).
Moreover, a total of 218 genes were regulated by ProQ, with 74 genes upregulated and 144 genes downregulated (Fig. 6A). The differentially expressed genes are involved in various processes, including biofilm synthesis (flgC, flgE) [43, 44], virulence (betB, hutG), and oxidative stress (sodB, recR) (Fig. 6B). Details of all differentially expressed genes are summarized in the Table S4. RT-qPCR was also performed to validate the RNA-seq data (Figure S4). Additionally, genes related to secretion systems were also influenced by the ProQ protein, including AT730_RS04390 (Hcp family type VI secretion system effector), AT730_RS25955 (type IV secretion system protein), and tssM (type VI secretion system membrane subunit TssM).
The DUF1127 protein was proposed as an RNA binding protein[18]. Here, according to the transcriptome data, the predicted noncoding RNAs were filtered by length, removing those shorter than 20 bp or longer than 500 bp and annotated by Blastx against the Nr database, with unannotated sRNAs selected as candidates for further analysis. Total 523 sRNA candidates were yielded. Further analysis revealed that 32 sRNAs (|log2 fold change|≥0.5 and P value ≤ 0.05) with significant differential expression, including 30 sRNAs located in the antisense to mRNA (AM) and 2 sRNAs located in the intergenic region (IGR) (Table 1). Among these, 13 sRNAs showed upregulation level and 19 sRNAs showed downregulation level.
Using the same method, an analysis of sRNAs in the ΔProQ strain was performed, resulting in a total prediction of 55 sRNAs. Among them, 5 sRNAs showed significant differential expression, all of which located in the antisense to mRNA (AM) (Table 2). The predicted sRNA Candidate_1–005 is significantly downregulated. The prediction of sRNAs regulated by ProQ using the same method yielded limited results, may due to the inappropriate timing of RNA extraction. Certain sRNAs may be expressed during specific growth stages or under particular environmental conditions, while being scarce or inactive under other conditions. Subsequent efforts could involve processing samples from multiple time points to enhance the prediction of sRNAs regulated by ProQ.