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Chapter 10 : Hierarchical Control of rdar Morphotype Development of Salmonella enterica by Cyclic Di-GMP
Category: Microbial Genetics and Molecular Biology
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This chapter describes in detail the role of bis-(3',5')-cyclic di-GMP (c-di-GMP) signaling in the expression of the rdar morphotype, one of the major targets of c-di-GMP signaling in Salmonella enterica serovar Typhimurium. It describes the impact of this multicellular morphotype and the complex molecular mechanisms by which it is regulated. Further, the chapter discusses the role of c-di-GMP in motility and characterizes the upcoming molecular mechanisms that tightly control the switch between the sedentary state and motility at various levels in the respective regulatory cascades. Initial characterization of the growth of S. enterica serovar Typhimurium in rdar morphotype colonies on agar plates demonstrated that the formation of this morphotype represents a specific form of multicellular behavior (biofilm) in bacteria. An intrinsic characteristic of biofilm formation, the formation of a self-produced extracellular matrix, is required to construct the complex three dimensional architecture of the biofilm. Expression of the rdar morphotype is usually highly controlled in response to environmental conditions. With few exceptions, under laboratory conditions, the rdar morphotype is expressed on Luria broth agar plates without salt at temperatures below 30№C. Experiments in which adrA, encoding a highly potent diguanylate cyclase (DGC), and STM3611, encoding a highly potent phosphodiesterase (PDE), were overexpressed first indicated c-di-GMP-dependent regulation of CsgD expression.
Appearance of the rdar morphotype of S. enterica serovar Typhimurium ATCC 14028. (A) rdar morphotype colonies among smooth colonies on Congo red agar plates. Streak-outs from the original S. enterica serovar Typhimurium ATCC 14028 culture were grown for 24 h at 37°C on Congo red agar plates using Luria broth (LB) without salt as growth medium. The extracellular matrix produced by rdar colonies bind the dye Congo red, which leads to a red-colored colony with extended dimensions. (B) Scanning electron microscopic analysis of the extracellular matrix structure and cell organization of rdar type S. enterica serovar Typhimurium ATCC 14028 (a ΔfliC ΔfljB mutant). The extracellular matrix surrounds the individual cell resembling a honeycomb-like structure. Magnification, X 15,000; bar, 2 µm. Picture taken by Manfred Rohde, Helmholtz Center for Infection Biology, Braunschweig, Germany. (C) rdar morphotype expression in liquid culture leads to cell clumping. Magnification, X∼473. (D) Differential expression of CsgD-GFP in rdar morphotype formation. GFP was fused to the csgD open reading frame on its native chromosomal location (Grantcharova and Römling, unpublished). Cell clumping is associated with the expression of the master regulator of rdar morphotype formation, CsgD, in the cytoplasm, while single cells usually do not express CsgD. Cells were grown for 24 h at 28°C on LB without salt agar plates. Cells were stained with FM4-64 membrane stain. Bar, 2 μm.
rdar morphotype expression in S. enterica serovar Typhimurium ATCC 14028. (A) Morphotypes of S. enterica serovar Typhimurium expressing the extracellular matrix components curli fimbriae and cellulose. For pronounced visualization, strain MAE52, an ATCC 14028 derivative with semiconstitutive expression of the rdar morphotype, and respective mutants were used. MAE52 has a single point mutation in the csgD promoter that confers threefold-enhanced transcription of csgD, which is RpoS and temperature independent ( 83 ). (B) The transcriptional regulator CsgD positively regulates the expression of at least four extracellular matrix components in S. enterica serovar Typhimurium, proteinaceous curli fimbriae, BapA, exopolysaccharide cellulose, and a capsule ( 18 , 32 , 56 , 77 , 82 , 90 ). Thereby, CsgD activates the transcription of the target operons csgBAC, adrA, and bapABCD. The csgBAC operon encodes CsgA and CsgB, major and minor structural components of curli fimbriae, respectively. The bapABCD operon encodes the large surface protein BapA and its type I protein secretion apparatus. Expression of the DGC AdrA produces c-di-GMP required for posttranscriptional activation of cellulose biosynthesis. On the other hand, CsgD represses transcription of the yihVW operon, which leads to the activation of the divergently transcribed yihU-OyshA operon and expression of an O-antigen capsule.
Regulatory network of rdar morphotype expression in S. enterica serovar Typhimurium with CsgD as central regulator. Regulation by c-di-GMP metabolic enzymes is shown in Fig. 5 . Expression of CsgD is positively (gray arrows) and negatively (black arrows) regulated by two-component systems (EnvZ/OmpR, CpxA/CpxR, and RcsCD/RcsBA), sigma factors and associated proteins (RpoS and Crl), transcriptional regulators (MlrA), and nucleoid-binding proteins (H-NS and IHF).
Panel of GGDEF-EAL domain proteins present in S. enterica serovar Typhimurium. The domain structures of the 5 GGDEF, 7 EAL, and 7 GGDEF-EAL domain proteins are shown. The GGDEF domain proteins STM2410, STM2503, STM3375, and STM3615 do not show a complete signature of conserved amino acids in the GGDEF motif (as indicated in the figure by pale letters) and other signature motifs indicative for enzymatic activity, suggesting that DGC activity is missing. The EAL domain proteins STM1344, STM1697, STM2123, and STM3375 do not show a complete signature of conserved amino acids in the EAL motif (as indicated in the figure by pale letters) and other signature motifs indicative for enzymatic activity, suggesting that c-di-GMP-specific PDE activity is missing. Although STM3611 shows several deviations from the PDE signature motif, considerable c-di-GMP-specific PDE activity was observed ( 69 , 90 , 91 ). Besides STM1344, STM1697, STM1703, and STM3611, the proteins are predicted to be integral membrane proteins, suggesting that the c-di-GMP signaling network responds mainly to external signals in S. enterica serovar Typhimurium.
c-di-GMP metabolic network affecting expression of CsgD, the master regulator of rdar morphotype formation. At least eight GGDEF and/or EAL domain proteins are involved in the regulation of the rdar morphotype in Salmonella serovar Typhimurium growing on agar plates ( 49 , 82 , 89 ). STM2123, STM3388, and other unidentified DGCs produce c-di-GMP (indicated by dots). STM1703, STM4264, STM1827, and STM3611 lead to c-di-GMP degradation. STM4264 and STM1703 have the most drastic effect on CsgD expression (indicated by thick lines). STM4264 acts upstream of STM1703, since STM1703 can complement an STM4264 defect by downregulation of CsgD but not vice versa ( 89 ). STM1827 and STM3611 have a less dramatic effect on CsgD expression. adrA regulated on the transcriptional level by CsgD is necessary for cellulose biosynthesis and is partially involved in the production of curli fimbriae ( 49 ). The EAL-like protein STM1344 does not have PDE activity but represses expression of the PDEs STM1703 and STM3611 ( 91 ). STM1344 and STM3611 are direct targets of the carbon storage regulatory protein CsrA (42a). Thereby, CsrA represses expression of STM1344 and activates expression of STM3611.
c-di-GMP binding proteins identified in S. enterica serovar Typhimurium. The PilZ domain is a c-di-GMP binding domain. The PilZ domain containing proteins STM1798 (YcgR) and BcsA have been identified in S. enterica serovar Typhimurium, which regulate motility and perform cellulose biosynthesis, respectively. The PilZ domain containing protein YcgR regulates motility. Upon high c-di-GMP levels, YcgR binds c-di-GMP ( 85 ) and presumably inhibits the flagellar motor function ( 52 ). c-di-GMP is required for cellulose biosynthesis in S. enterica serovar Typhimurium ( 90 , 114 ). BcsA, the catalytic subunit of the cellulose synthase, contains a PilZ domain at the C terminus. As the PilZ domain of the cellulose synthase of G. xylinus has been demonstrated to bind c-di-GMP ( 85 ), it is hypothesized that PilZ binds c-di-GMP, which allosterically activates cellulose biosynthesis in S. enterica serovar Typhimurium.
Direct control of regulation of GGDEF-EAL domain proteins by the carbon storage regulator CsrA in S. enterica serovar Typhimurium ATCC 14028 controls biofilm formation, motility, and invasion. CsrA directly represses the expression of the GGDEF-EAL protein STM3375 and the EAL-like domain proteins STM1344 and STM1697, whereas the PDE STM3611 is activated through CsrA. CsrA is predicted to inversely control rdar morphotype expression and motility through STM1344 and STM3611. CsrA is suggested to control invasion by regulating STM1697 and STM1987 by an unknown mechanism. CsrA regulates the expression of invasion genes ( 2 ). CsrA controls its own activity by regulating STM3375, which in turn destabilizes the CsrB and CsrC sRNAs, the antagonists of CsrA activity ( 94 ).
Characteristics of GGDEF-EAL domain proteins affecting CsgD expression