Hfq-mediated B. subtilis survival advantages in the stationary phase
The results of the growth curve are similar to those observed by Hermann and Rochat et al., that is, the existence of Hfq benefits B. subtilis survival (Figure 1) (Hermann et al., 2014; Rochat et al., 2015). Compared with those in LB medium, the growth defects caused by Hfq knockout are more intense in Na-CMC medium (Figure 1). One possible explanation is that Hfq is more important for the survival of B. subtilis in a nutrient-poor environment than in a nutrient-rich environment. In addition, the decrease in survival rate after 48 h may be related to the lack of nutrients.
Aid of Hfq in B. subtilis adaptation to cellulose stress
The positive regulation of cellulase activity by Hfq further explains the advantages given by this protein in cellulose hydrolysis ability and stationary phase survival under cellulose stress, since the cellulase activity of B. subtilis is closely related to the rate of obtaining nutrients regardless of being in Na-CMC medium or on Congo red plates (Figure 1-Figure 3 and Table S3). High-fiber conditions constitute a stress environment for B. subtilis (Ziyao et al., 2015). The possible role of HfqBS in stress adaptation has been emphasized by this study; nevertheless, it remains to be confirmed.
In B. subtilis, the two promoters that initiate Hfq transcription are regulated by σB and σH (encoded by sigH), respectively (Jagtap et al., 2016). σH-dependent Hfq transcription results in the synthesis of monocistronic transcripts, while σB-dependent transcription results in the synthesis of polycistronic transcripts (ymaF-miaA-Hfq) (Jagtap et al., 2016). The monocistronic transcription driven by σH may be closely related to the role of Hfq in general life activities such as DNA replication and DNA compaction because a large number of monocistronic transcripts are also detected in a variety of non-stress environments (Antoine et al., 2018; Dambach et al., 2013; Irnov et al., 2010). Contrarily, in B. subtilis, σB-dependent genes are generally expressed under stressors, such as antibiotics, temperature, salt, and ethanol (Bingyao and Jörg, 2018). Induction of σB-dependent genes is also observed during the stationary phase as bacteria experience stress due to nutrient limitation (Benson and Haldenwang, 1992; Jagtap et al., 2016). In the medium with Na-CMC as the only carbon source, the decomposition ability of cellulose determines the number of available carbon sources obtained by B. subtilis for survival. Therefore, our results also proved the positive regulatory role of Hfq in the cellulose stress adaptation of B. subtilis. The rapid response of σB to stress regulation may contribute to the rapid expression of Hfq under cellulose stress (Haldenwang, 1995; Jagtap et al., 2016).
A hypothesis: Indirect regulation of eglS expression by Hfq through weakening stability of 6S-1 RNA
The positive regulation of HfqBS on the expression of the cellulase gene further explains the usefulness of this protein in cellulose stress adaptation. Combined with the results of the growth curve, cellulose hydrolysis ability, cellulase activity, and RT-qPCR, the possible regulatory network of Hfq in cellulose decomposition can be proposed (Figure 5). Nevertheless, the accuracy of this view needs to be confirmed by further research.
Endoglucanase (encoded by eglS) is the main cellulase secreted by B. subtilis, and σA activity directly determines the transcription level of eglS (Bingyao and Jörg, 2018; Robson and Chambliss, 1987). As expected, both eglS and sigA were downregulated after the knockout of Hfq (Figure 4). 6S RNA is an antagonist of σA (Trotochaud and Wassarman, 2005). Some bacterial species can transcribe two or more 6S RNAs, such as the 6S-1 RNA (encoded by bsrA) and 6S-2 RNA (encoded by bsrB) in B. subtilis (Hoch et al., 2015). However, the transcriptional levels of the two 6S RNA-coding genes (bsrA and bsrB) are diametrically opposed in B. subtilis (Figure 4). The functional and structural properties of Hfq and 6S RNA are conserved in most bacteria; 6S RNA encoded by the E. colissrS gene is a kind of Hfq-associated sRNA (Barrick et al., 2005; Sun et al., 2002; Zhang et al., 2003). Therefore, some interactions may also occur between HfqBS and 6S RNA. Previous studies have shown that 6S-1RNA is not expressed in the early exponential phase, and the expression of 6S-1 RNA is four-fold higher than that of 6S-2RNA from the late exponential to stationary phase (Beckmann et al., 2011). Similarly, our results also show that the knockout of HfqBS mainly leads to an increase in the overall transcriptional level of 6S RNA in the stationary phase (among them, the transcriptional level of 6S-1 RNA is also nearly four-fold higher than that of 6S-2RNA) (Figure 4). The negative regulation of Hfq on the stability of 6S RNA may be due to the formation of the Hfq-6S RNA complex, which induces the degradation of 6S RNA by ribonuclease E (RNase E). It is well known that the binding of Hfq to some Hfq-associated sRNA causes the sRNA to be degraded (degradation by RNase E recruited by Hfq-sRNA complex) or protected, while the Hfq itself is unaffected and re-released into the cell (Vogel and Luisi, 2011; Zhang et al., 2003). Compared with 6S-2 RNA, 6S-1 RNA was more likely to be degraded by Hfq. In B. subtilisΔHfq, the level of 6S-1 RNA was nearly three-fold higher than that of wild strains, while 6S-2 RNA was downregulated by 84.9% ± 3.1% (Figure 4). On the other hand, 6S-1 RNA is the ortholog of E. coli 6S RNA (Barrick et al., 2005). In addition, 6S-2 RNA may be protected by Hfq during the stationary phase, but the significance of this protection needs to be further studied.
Increase in the compensatory activity of the phosphotransferase system from Hfq knockout
In B. subtilis, the combination of the complex formed by ccpA (catabolite control protein A) and P-Ser-HPr or P-Ser-Crh with the catabolite responsive elements (Cre) of the target operons can lead to carbon catabolite activation (CCA) or carbon catabolite repression (CCR) (Fujjta, 2009). The ccpA transcript levels also requires the assistance of a σA-dependent promoter (Bingyao and Jörg, 2018). After the knockout of Hfq, the ccpA transcript levels decreased by 45.5. ± 3.1% (Figure 4B). The downregulation of ccpA may be related to the decrease of functional σA (Bingyao and Jörg, 2018). When the preferred carbon source is limited (such as Na-CMC medium), CCR usually does not exist (Fujjta, 2009). However, four phosphotransferase system (PTS) related genes (such as bglP, licA, licB, and licC) that are inhibited by ccpA (only in the presence of CCR) are upregulated (Figure 4B) (Fujjta, 2009). In Hfq mutants, the transcript levels of the four genes were more than two-fold higher than those in the wild strains (Figure 4B). The PTS is generally composed of non-specific enzyme I (EI), histidine-containing phosphocarrier protein (HPr), and sugar-specific enzyme II (EII), of which, the latter is mainly responsible for the transmembrane transport and phosphorylation of PTS-sugar (Fujjta, 2009). Therefore, the upregulation of these four PTS-EII genes might partly explains the residual cellulose decomposition and utilization ability after Hfq knockout because secondary metabolites produced by extracellular cellulase decomposition cellulose need to be transported through the PTS to enter the cell (Fujjta, 2009). Another important reason is that the expression of the cellulase gene itself can be directly induced by some small molecular soluble sugars produced by secondary metabolites, although the amount of cellulase produced by this induction effect is relatively small (Aro et al., 2001; Fujjta, 2009).
Despite these findings, the regulatory network related to the downregulation of two β-glucosidase genes (bglA and bglC) in Hfq mutants is not clear. One possible hypothesis is that the decrease in the transcription level of β-glucosidase may be due to the weakening of CCA mediated by ccpA. For example, several enzymes (such as degS, degU, and serA) involved in carbohydrate metabolism have been confirmed to be positively regulated by CCA (Bingyao and Jörg, 2018). Identification of Cre sequences of bglA and bglC will help to further understand the regulatory network of these two genes.
The positive regulatory effect of HfqBS on cellulose hydrolysis ability, cellulase activity, cellulase gene expression, and stationary phase survival was confirmed. Therefore, Hfq gives B. subtilis a survival advantage under cellulose stress. Additional targets and functional identification could further consolidate the important position of Hfq as a core regulator of multiple metabolic pathways in bacteria, and contribute to a better understanding of the overall function of the protein in bacteria.