Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus.

BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS. CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).

Understanding the Physiology of Liberibacter asiaticus: An Overview of the Demonstrated Molecular Mechanisms.

Citrus greening disease, or huanglongbing, may entirely eradicate all varieties of citrus cultivars worldwide in the near future. This disease is caused by non-cultivable bacteria of the genus Liberibacter; among them, the more pathogenic being Liberibacter asiaticus. The complexity of the host-pathogen relationship, associated with the impossibility of performing research using axenic cultures, has severely hindered the basic research on microbiology. Since its genome sequence was published in 2009, most of the scientific publications in the field were dedicated to in silico analysis and selection of targets to design early detection methods. The knowledge gained with these approaches felt short to articulate effective methods to control the disease progression. There is a critical need to understand the basic biology of bacteria to design effective strategies to inactivate central mechanisms of pathogenesis. In this review, we summarize the scientific progress made by studying L. asiaticus' biology through direct experimentation. The evidence collected thus far is not enough to understand L. -asiaticus' fundamental biology. It is imperiously necessary to increase the basic research to identify relevant biological clues to control citrus greening. The gained knowledge may also help to prevent potential catastrophic diseases in other crops of significant importance caused by other unculturable Liberibacter species.

LdtR is a master regulator of gene expression in Liberibacter asiaticus.

Huanglongbing or citrus greening disease is causing devastation to the citrus industry. Liberibacter asiaticus, an obligate intracellular pathogen of citrus, is one the causative agents of the disease. Most of the knowledge about this bacterium has been deduced from the in silico exploration of its genomic sequence. L. asiaticus differentially expresses genes during its transmission from the psyllid vector, Diaphorina citri, to the plant. However, the regulatory mechanisms for the adaptation of the bacterium into either hosts remain unknown. Here we show that LdtR, a MarR family transcriptional regulator, activates or represses transcription genome-wide. We performed a double approach to identify the components of the LdtR regulon: a transcriptome analysis in both the related bacterium Liberibacter crescens and citrus-infected leaves, strengthened with an in silico prediction of LdtR regulatory sites. Our results demonstrated that LdtR controls the expression of nearly 180 genes in L. asiaticus, distributed in processes such as cell motility, cell wall biogenesis, energy production, and transcription. These results provide new evidence about the regulatory network of L. asiaticus, where the differential expression of genes from these functional categories could be of great importance during the adaptation of the bacterium to either hosts.

Functional characterization of LotP from Liberibacter asiaticus.

Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host-pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N-terminus. Co-immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP-interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two-hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.