JOURNAL OF BACTERIOLOGY, Aug. 2010, p. 4083–4085 0021-9193/10/$12.00 doi:10.1128/JB.00641-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 16
COMMENTARIES The Phosphoenolpyruvate Phosphotransferase System: as Important for Biofilm Formation by Vibrio cholerae as It Is for Metabolism in Escherichia coli 䌤 Beth A. Lazazzera* Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, California 90095 indirect readout of the presence of PTS sugars and the metabolic status of the cell. The PTS system has long been known to participate in regulation that impinges on metabolism, including chemotaxis, inducer exclusion, and catabolite repression (7, 25). Recently, it has also been implicated in regulating processes not directly related to metabolism, such as virulence gene expression. In addition to this canonical PTS, many bacteria also posses a “nitrogen” PTS, which is comprised of an EI component, an HPr component (called NPr), and an EIIA component (Fig. 1) (23). This “nitrogen” PTS is unique for the lack of an EIIB/C complex. This has led to the proposal that the “nitrogen” PTS would function solely in a regulatory capacity, but the precise mechanism by which this would work is still unknown. From bioinformatic analyses, V. cholerae would appear to possess both a canonical PTS and a “nitrogen” PTS. For the canonical PTS, there is a single EI component, an HPr and FPr, and eight different EIIA components. These EIIAs work with one of the many different EIIB/C complexes in V. cholerae (11), each of which will transport a specific sugar, including glucose, fructose, mannose, mannitol, and chitobiose among others (2, 11, 12). The V. cholerae “nitrogen” PTS appears to be composed of an EI component, an NPr, and two EIIANtr components. Prior to the work of Houot et al., it was shown that the PTS system is required for intestinal colonization and inhibits biofilm formation (11, 12). Specifically, the phosphorylation of HPr or FPr, which accumulates in the absence of a transportable PTS sugar, was required to inhibit expression of the biofilm matrix genes.
Microbial biofilms are multicellular aggregates embedded in an exopolymeric matrix that contribute to survival in environmental niches such as rock surfaces in streams, plant roots, or the middle ear or teeth in the human body. Intense research effect has gone into identifying molecular mechanisms used by bacteria to build and regulate biofilms. In an earlier issue of the Journal of Bacteriology, Houot et al. (12a) reported on the identification of three different phosphoenolpyruvate phosphotransferase system (PTS) pathways that control biofilm formation by Vibrio cholerae. This study highlights how important the PTS is in controlling biofilm formation. While other studies with different bacterial species have shown that a mutation in a gene for a component of the PTS affects biofilm formation (1, 19, 22), these studies have not yet done a thorough analysis of which components of the PTS affect biofilm formation and whether phosphorylation of these proteins is required for regulation. The PTS can transport a variety of sugars, and this report highlights the importance of studying the role of the PTS in different growth media to reveal the full extent to which the PTS affects biofilm formation. Along with the recent work demonstrating how the PTS system can influence virulence gene expression in some bacterial species (14, 24, 27), the finding that the PTS has a profound effect on biofilm formation may be bringing out a renaissance in research on this sugar transport system that was first described in the 1960s. THE PTS AND VIBRIO CHOLERAE The PTS is used for the transport of sugars and requires phosphate transfer from PEP to enzyme I (EI) to the histidine protein (HPr) or the homologous protein FPr and then to an EII complex (Fig. 1). A membrane-associated EIIB then transfers the phosphate from EIIA to the specific sugar that is transported across the membrane by the EIIC/D complex (7). The nature of the PTS is that, in the absence of transported sugars, phosphate will accumulate on the protein components of the PTS. Thus, the phosphorylation state of the PTS is an
VIBRIO CHOLERAE BIOFILMS Control of biofilm formation by V. cholerae is complex. This complexity largely centers on the transcriptional control of the vps operon, which encodes the V. cholerae exopolysaccharide, also known as VPS. Expression of vps is critical for biofilm formation by V. cholerae (29). A multiple-component signal transduction cascade controls expression of vps and includes the proteins VpsS, VpsR, VpsT, and HapR (3, 6, 10, 16, 26, 28, 30). This protein cascade allows a range of signals to control biofilm formation either negatively or positively. For example, expression of vps is negatively regulated by quorum sensing, Ca2⫹, and cyclic AMP (4, 8, 10, 18, 30). Other molecules, such as indole, bile acids, and norspermidine, stimulate vps expres-
* Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90095. Phone: (310) 794-4804. Fax: (310) 206-5231. E-mail:
[email protected] .edu. 䌤 Published ahead of print on 18 June 2010. 4083
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FIG. 1. Model for how the PTS systems of Vibrio cholerae affects biofilm formation. Shown in yellow is the sugar-dependent PTS and in blue is the nitrogen PTS. Green arrow and “T” lines indicate points at which a PTS component stimulates and inhibits, respectively, biofilm formation through controlling vps expression. The gray boxes indicate that either the unphosphorylated or phosophorylated protein can control vps expression.
sion (13, 15, 21). To this complexity can be added three independent pathways for PTS to control vps expression. How all these signals are integrated to control biofilm formation by V. cholerae is unknown. THE THREE PTS PATHWAYS CONTROLLING BIOFILM FORMATION Consider that all PTS proteins required to transport glucose in Escherichia coli regulate a metabolic function. EI interacts with CheA to control chemotaxis, HPr interacts with glycogen phosphorylase to control recycling of glycogen, EIIAGlc interacts with non-PTS transporters to prevent transport of other sugars and the adenylate cyclase that makes cyclic AMP, and EIIBGlc interacts with the transcription factor Mlc to control expression of the genes involved in uptake of glucose, just to list a few (7). The multifunctionality of the PTS system indicates that the mechanism of PTS control of biofilm formation cannot readily be predicted from a finding that any single mutation in a gene for the PTS system affects biofilm formation. Furthermore, by studying which component of the PTS system is responsible for regulation, one can reveal, as the study of Houot et al. did (12a), that PTS control of biofilm formation may be almost as complex as the PTS control of metabolism in E. coli. The first pathway described for controlling vps expression in V. cholerae is active in cells grown on either a glucose minimal medium or a rich medium like LB. In this pathway, PEPdependent, phosphorylated HPr or FPr inhibits vps expression (Fig. 1). PEP-dependent, phosphorylated HPr controls the activity of a group of antiterminator proteins and transcription factors in Gram-positive bacteria (9). The vps operon is not known to be controlled by transcription antitermination. Could the V. cholerae HPr/FPr be phosphorylating a component of the signal transduction cascade that controls vps expression? Phosphorylated PTS components accumulate in the absence of a transportable sugar, indicating that lack of a PTS sugar inhibits biofilm formation by V. cholerae. The second pathway involves the EIIAGlc and EIIBCGlc components of the glucose-specific PTS and is active only in cells grown on LB medium (Fig. 1). EIIBCGlc inhibits vps transcription, and acts presumably as it does in E. coli (5),
where unphosphorylated EIIBCGlc inhibits the activity of the transcriptional repressor Mlc. However, Mlc does not appear to control vps gene expression directly but instead largely appears to work by potentiating EIIAGlc activity. How Mlc affects EIIAGlc activity is unknown, but the interesting question here is how EIIAGlc stimulates vps expression. Unphosphorylated EIIAGlc of E. coli interacts with non-PTS transporters to inhibit their activity (7). What new type of protein might future V. cholerae work reveal about the types of proteins that can interact with EIIAGlc proteins? The third pathway diverges from components of the canonical PTS components to components of the far-less-understood PTSNtr. V. cholerae possesses two EIIANtr homologs, and both contribute to inhibit vps expression (Fig. 1). It is interesting that whether these EIIANtr homologs are unphosphorylated or phosphorylated, they can still inhibit vps expression. Is there something upstream of the EIIANtr homolog pathway that affects homlogs’ ability to inhibit vps expression? Also, what is downstream of the EIIANtr homologs that allows these proteins to control vps transcription in V. cholerae? It will be interesting to know whether inhibiting vps expression occurs through interaction of EIIANtr with the K⫹ transporter, as it does in E. coli (17). Alternatively, EIIANtr could interact with one of the two-component regulatory systems that are known to control vps expression, similar to how EIIANtr binds and inhibits the activity of the KdpD histidine-protein kinase of E. coli (20). The cumulative studies of the PTS tell us how well suited the PTS is to monitoring the metabolic capability of the cells and control relevant pathways. It has long been known that the PTS could affect related metabolic pathways, chemotaxis, inducer exclusion, and catabolite repression. The finding that the PTS affects processes such as virulence and biofilm formation takes the centrality of the PTS to the physiology of the cell in a new direction and suggests new avenues for the development of antimicrobials. REFERENCES 1. Abranches, J., M. M. Candella, Z. T. Wen, H. V. Baker, and R. A. Burne. 2006. Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. J. Bacteriol. 188:3748–3756. 2. Berg, T., S. Schild, and J. Reidl. 2007. Regulation of the chitobiose-phosphotransferase system in Vibrio cholerae. Arch. Microbiol. 187:433–439. 3. Beyhan, S., K. Bilecen, S. R. Salama, C. Casper-Lindley, and F. H. Yildiz. 2007. Regulation of rugosity and biofilm formation in Vibrio cholerae: comparison of VpsT and VpsR regulons and epistasis analysis of vpsT, vpsR, and hapR. J. Bacteriol. 189:388–402. 4. Bilecen, K., and F. H. Yildiz. 2009. Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation. Environ. Microbiol. 11:2015–2029. 5. Bohm, A., and W. Boos. 2004. Gene regulation in prokaryotes by subcellular relocalization of transcription factors. Curr. Opin. Microbiol. 7:151–156. 6. Casper-Lindley, C., and F. H. Yildiz. 2004. VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor. J. Bacteriol. 186:1574–1578. 7. Deutscher, J., C. Francke, and P. W. Postma. 2006. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol. Mol. Biol. Rev. 70:939–1031. 8. Fong, J. C., and F. H. Yildiz. 2008. Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation. J. Bacteriol. 190:6646–6659. 9. Fujita, Y. 2009. Carbon catabolite control of the metabolic network in Bacillus subtilis. Biosci. Biotechnol. Biochem. 73:245–259. 10. Hammer, B. K., and B. L. Bassler. 2003. Quorum sensing controls biofilm formation in Vibrio cholerae. Mol. Microbiol. 50:101–104. 11. Houot, L., S. Chang, C. Absalon, and P. I. Watnick. 2010. Vibrio cholerae
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