Identification of an Extracellular Endoglucanase That Is Required for Full Virulence in Xanthomonas citri subsp. citri.
Xia Tian,Li Yanjiao,Sun Dongling,Zhuo Tao,Fan Xiaojing,Zou Huasong
Xanthomonas citri subsp. citri causes citrus canker disease, which is characterized by the formation of water-soaked lesions, white or yellow spongy pustules and brown corky canker. In this work, we report the contribution of extracellular endoglucanase to canker development during infection. The ectopic expression of nine putative cellulases in Escherichia coli indicated that two endoglucanases, BglC3 and EngXCA, show carboxymethyl cellulase activity. Both bglC3 and engXCA genes were transcribed in X. citri subsp. citri, however, only BglC3 protein was detected outside the cell in western blot analysis. The deletion of bglC3 gene resulted in complete loss of extracellular carboxymethyl cellulase activity and delayed the onset of canker symptoms in both infiltration- and wound-inoculation assays. When growing in plant tissue, the cell density of bglC3 mutant was lower than that of the wild type. Our data demonstrated that BglC3 is an extracellular endoglucanase required for the full virulence of X. citri subsp. citri.
Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris.
Gough C L,Dow J M,Keen J,Henrissat B,Daniels M J
The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.
Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production.
Schröter K,Flaschel E,Pühler A,Becker A
Applied microbiology and biotechnology
Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53-59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.
Competitive control of endoglucanase gene engXCA expression in the plant pathogen Xanthomonas campestris by the global transcriptional regulators HpaR1 and Clp.
Liu Guo-Fang,Su Hui-Zhao,Sun Han-Yang,Lu Guang-Tao,Tang Ji-Liang
Molecular plant pathology
Transcriptional regulators are key players in pathways that allow bacteria to alter gene expression in response to environmental conditions. However, work to understand how such transcriptional regulatory networks interact in bacterial plant pathogens is limited. Here, in the phytopathogen Xanthomonas campestris, we demonstrate that the global transcriptional regulator HpaR1 influences many of the same genes as another global regulator Clp, including the engXCA gene that encodes extracellular endoglucanase. We demonstrate that HpaR1 facilitates the binding of RNA polymerase to the engXCA promoter. In addition, we show that HpaR1 binds directly to the engXCA promoter. Furthermore, our in vitro tests characterize two binding sites for Clp within the engXCA promoter. Interestingly, one of these sites overlaps with the HpaR1 binding site. Mobility shift assays reveal that HpaR1 has greater affinity for binding to the engXCA promoter. This observation is supported by promoter activity assays, which show that the engXCA expression level is lower when both HpaR1 and Clp are present together, rather than alone. The data also reveal that HpaR1 and Clp activate engXCA gene expression by binding directly to its promoter. This transcriptional activation is modulated as both regulators compete to bind to overlapping sites on the engXCA promoter. Bioinformatics analysis suggests that this mechanism may be used broadly in Xanthomonas campestris pv. campestris (Xcc) and is probably widespread in Xanthomonads and, potentially, other bacteria. Taken together, these data support a novel mechanism of competitive activation by two global regulators of virulence gene expression in Xcc which is probably widespread in Xanthomonads and, potentially, other bacteria.
Interference With Quorum-Sensing Signal Biosynthesis as a Promising Therapeutic Strategy Against Multidrug-Resistant Pathogens.
Fleitas Martínez Osmel,Rigueiras Pietra Orlandi,Pires Állan da Silva,Porto William Farias,Silva Osmar Nascimento,de la Fuente-Nunez Cesar,Franco Octavio Luiz
Frontiers in cellular and infection microbiology
Faced with the global health threat of increasing resistance to antibiotics, researchers are exploring interventions that target bacterial virulence factors. Quorum sensing is a particularly attractive target because several bacterial virulence factors are controlled by this mechanism. Furthermore, attacking the quorum-sensing signaling network is less likely to select for resistant strains than using conventional antibiotics. Strategies that focus on the inhibition of quorum-sensing signal production are especially attractive because the enzymes involved are expressed in bacterial cells but are not present in their mammalian counterparts. We review here various approaches that are being taken to interfere with quorum-sensing signal production via the inhibition of autoinducer-2 synthesis, PQS synthesis, peptide autoinducer synthesis, and N-acyl-homoserine lactone synthesis. We expect these approaches will lead to the discovery of new quorum-sensing inhibitors that can help to stem the tide of antibiotic resistance.
Quorum-Sensing Systems as Targets for Antivirulence Therapy.
Trends in microbiology
The development of novel therapies to control diseases caused by antibiotic-resistant pathogens is one of the major challenges we are currently facing. Many important plant, animal, and human pathogens regulate virulence by quorum sensing, bacterial cell-to-cell communication with small signal molecules. Consequently, a significant research effort is being undertaken to identify and use quorum-sensing-interfering agents in order to control diseases caused by these pathogens. In this review, an overview of our current knowledge of quorum-sensing systems of Gram-negative model pathogens is presented as well as the link with virulence of these pathogens, and recent advances and challenges in the development of quorum-sensing-interfering therapies are discussed.
Bacterial Quorum Sensing and Microbial Community Interactions.
Abisado Rhea G,Benomar Saida,Klaus Jennifer R,Dandekar Ajai A,Chandler Josephine R
Many bacteria use a cell-cell communication system called quorum sensing to coordinate population density-dependent changes in behavior. Quorum sensing involves production of and response to diffusible or secreted signals, which can vary substantially across different types of bacteria. In many species, quorum sensing modulates virulence functions and is important for pathogenesis. Over the past half-century, there has been a significant accumulation of knowledge of the molecular mechanisms, signal structures, gene regulons, and behavioral responses associated with quorum-sensing systems in diverse bacteria. More recent studies have focused on understanding quorum sensing in the context of bacterial sociality. Studies of the role of quorum sensing in cooperative and competitive microbial interactions have revealed how quorum sensing coordinates interactions both within a species and between species. Such studies of quorum sensing as a social behavior have relied on the development of "synthetic ecological" models that use nonclonal bacterial populations. In this review, we discuss some of these models and recent advances in understanding how microbes might interact with one another using quorum sensing. The knowledge gained from these lines of investigation has the potential to guide studies of microbial sociality in natural settings and the design of new medicines and therapies to treat bacterial infections.