L. plantarum is a versatile species that can grow on numerous types of carbohydrates. Notably, this bacterial species can utilize FOS as efficiently as glucose, and harbors two gene clusters that participate in FOS metabolism . Specifically, CcpA, a GalR-LacI family protein, is a vital regulator of FOS metabolism in L. plantarum . Previously, we predicted that two local regulators, SacR1 and SacR2, are also involved in the regulation of these two FOS metabolism-related clusters , suggesting that the utilization of FOS in L. plantarum may involve the double effects of global and local regulation. However, the specific manner by which SacR1 and SacR2 control local regulation had not been determined. In this report, we evaluated the regulation of FOS metabolism by local regulatory elements in L. plantarum both in vitro and in vivo.
The growth curves of wild-type and mutant L. plantarum strains cultured in media containing glucose and FOS did not differ significantly. The CCR in response to glucose may have been predominant in the context of dual regulation, whereas the effects of local regulators could not be observed . As the global regulation mechanism of CCR is generally triggered by glucose , we used ribose, a non-preferred substrate, as an alternative carbon source to verify the roles of SacR1 and SacR2 through RT-qPCR experiments. Notably, both FOS metabolism-related clusters were significantly activated by FOS (versus ribose), whereas this effect vanished after SacR1 and SacR2 inactivation. These results demonstrated that SacR1 and SacR2 inhibit FOS metabolism in L. plantarum, and that FOS can induce or derepress these effects.
The regulation of locally regulated gene transcription involves the binding of specific regulators to binding sites on the target genes [25, 26]. However, potential SacR1 and SacR2 binding sequences had not previously been clarified in the two FOS metabolism-related clusters in L. plantarum. In this study, we identified four putative TFBSs in the promoter regions of SacR1 and SacR2. These sites were located in the FOS-related clusters based on the consensus motif generated from a Regulatory Sequence Analysis Tools (RSAT) analysis. These TFBSs of SacR1 and SacR2 deviated by only one or two nucleotides from published binding consensus sequences , and specific binding interactions in vitro and in vivo were verified in this study by EMSA and ChIP-qPCR, respectively. Although both regulators showed a low level of sequence identity (28%), they both belong to the GalR-LacI family of CcpA-like proteins and are expected to have similar DNA-binding features [11, 26]. When combining the predicted TFBSs of SacR1 and SacR2 with our present results, we deduced a consensus sequence for the SacR1 and SacR2 binding sites, WNNNNNAACGNNTTNNNNNW, which is also similar to the consensus sequence of cre sites [19, 26]. These results provide a new insight into the structures of local regulator recognition sites in Gram-positive bacteria. Related foot-printing and CHIP-seq experiments to confirm the binding of SacR1 and SacR2 to the target sites are ongoing.
Many studies revealed a double effect of global and local regulation on carbohydrate metabolism in LAB [39, 40]. In contrast to these global regulators, local regulators regulate only one or a few genes that are often linked genetically to the gene encoding the regulator itself . For instance, Tamara et al.  identified a novel RpiR-family transcription activator, GlaR, positioned directly upstream of the gal-lac gene cluster in Lactococcus lactis IL1403. GlaR was identified as a transcriptional activator of galactose and lactose utilization genes, the expression of which can be induced by galactose. Moreover, six LacI-family local transcriptional factors and a TetR-family regulator were identified as presumptive local repressors of arabino-oligosaccharide (AOS) utilization in Bifidobacterium species . According to our previous studies and the present work, FOS metabolism is regulated both globally and locally in L. plantarum [11, 12]. Regulation can be divided into four conditions based on the available carbon source, as follows: only glucose, only FOS, both glucose and FOS, and neither glucose nor FOS. These conditions enable the deduction of the possible regulatory mode. If only glucose is present (Fig. 6a), the binding of CcpA to cre sites would block the transcription of FOS-related genes in L. plantarum. If only FOS is present (Fig. 6c), FOS would bind to repressor proteins (SacR1 and SacR2) to reverse the inhibition induced by the binding of SacR1 and SacR2 to TFBSs in the promoter regions of FOS-related clusters. If neither source is present (Fig. 6d), SacR1 and SacR2 act as repressors and inhibit the expression of FOS-related clusters. If both sources are present (Fig. 6b), FOS acts as an inducer, thus rendering the repressor proteins allosteric and releasing inhibition; however, the global regulator CcpA binds to cre sites and thus remains capable of eliciting CcpA-mediated CCR. This latter process is also the cause of the diauxic growth phenomenon, in which cells resume growing and enter a second growth phase fueled by FOS as the carbon source once glucose is depleted . However, the actual mechanism of regulation may be more complex, as these local regulators are also activated or repressed by CcpA [10, 12]. Furthermore, SacR1 and SacR2 are co-transcribed with other FOS-related genes, suggesting that both proteins act as self-regulators to maintain their own expression . In summary, FOS metabolism is an extremely complex network in which the combined actions of global and local regulators orchestrate the transcription of various units that enable bacteria to adjust sugar utilization to their metabolic capacities.