The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus

Abstract

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas. The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus. IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas. We show that the presence of FerR stabilizes and augments the activity of PrbPLas. In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens. Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

PrbP modulates biofilm formation in Liberibacter crescens

“…The selected proteins were fused to two non‐functional but complementing β‐galactosidase truncations (Δα and Δω). The β‐galactosidase activity driven by the protein–protein recognition between both non‐β‐galactosidase parts of the chimaeras allowed analysis of protein–protein interactions (Borloo et al ., 2007; Pan et al ., 2019). The sequences encoding for RpoH, PleD, VisN, VisR, CtrA, MocR, MucR and PrbP were cloned and fused to the β‐galactosidase truncations Δα and Δω on plasmids pB2HΔα and pB2HΔω respectively.…”

“…asiaticus RNA polymerase in a dose‐dependent manner. FerR was also found to improve the affinity of PrbP for rplK , in vitro (Pan et al ., 2019). Using small‐molecule screening, tolfenamic acid (TA) was identified as a molecule that binds PrbP and disrupts interactions with the rplK promoter, in vitro .…”

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

“…In this article, we demonstrate in vivo that CarD activity leads to diverse gene expression outcomes and does not solely act as a transcriptional activator. Importantly, CarD homologs are conserved in many bacterial phyla (8,9), and regulate transcription to mediate diverse processes including stress responses, cell division, and metabolic homeostasis (10)(11)(12)(13). Therefore, these studies shed light on the maintenance of optimal gene expression patterns broadly across bacteria.…”

“…In mycobacteria, carD expression is induced by genotoxic, oxidative, starvation, and antibiotic stresses (8), suggesting that CarD may serve as a stress-responsive transcriptional regulator in addition to its essential function. CarD homologs in other bacteria have also been shown to respond to diverse environmental stimuli (10,11) and regulate critical processes such as metabolic homeostasis and cell division (12,13).…”

Assessment of unconventional antimicrobial compounds for the control of ‘Candidatus Liberibacter asiaticus’, the causative agent of citrus greening disease

“…The genes encoding those proteins were cloned, purified, characterized and successfully screened against the Prestwick chemical library (Prestwick Chemical, France) to identify ligands with high binding affinity. Using this methodology, we successfully identified small molecules that inhibit the activity of specific transcription factors [12][13][14][15][16] . Based on these previously published data, we performed field trials using benzbromarone and tolfenamic acid, the small molecules that were found to inhibit activity of the regulatory proteins LdtR and PrbP.…”