Second messengers control a wide range of important cellular functions in eukaryotes and prokaryotes. Here we show that cyclic di-GMP, a global bacterial second messenger, promotes cell cycle progression in Caulobacter crescentus by mediating the specific degradation of the replication initiation inhibitor CtrA. During the G1-to-Sphase transition, both CtrA and its cognate protease ClpXP dynamically localize to the old cell pole, where CtrA is rapidly degraded. Sequestration of CtrA to the cell pole depends on PopA, a newly identified cyclic di-GMP effector protein. PopA itself localizes to the cell pole and directs CtrA to this subcellular site via the direct interaction with a mediator protein, RcdA. We present evidence that c-di-GMP regulates CtrA degradation during the cell cycle by controlling the dynamic sequestration of the PopA recruitment factor to the cell pole. Furthermore, we show that cell cycle timing of CtrA degradation relies on converging pathways responsible for substrate and protease localization to the old cell pole. This is the first report that links cyclic di-GMP to protein dynamics and cell cycle control in bacteria.[Keywords: PopA; cyclic di-GMP; protein degradation; Caulobacter crescentus; cell cycle; second messenger] Supplemental material is available at http://www.genesdev.org.

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer-membrane (OM) ring), YscD (forms the inner-membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside-in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co-immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.

Summary In Caulobacter crescentus, phosphorylation of key regulators is coordinated with the second messenger cyclic di-GMP to drive cell cycle progression and differentiation. The diguanylate cyclase PleD directs pole morphogenesis while the c-di-GMP effector PopA initiates degradation of the replication inhibitor CtrA by the AAA+ protease ClpXP to license S-phase entry. Here we establish a direct link between PleD and PopA reliant on the phosphodiesterase PdeA and the diguanylate cyclase DgcB. PdeA antagonizes DgcB activity until the G1-S transition when PdeA is degraded by the ClpXP protease. The unopposed DgcB activity, together with PleD activation, upshifts c-di-GMP to drive PopA-dependent CtrA degradation and S-phase entry. PdeA degradation requires CpdR, a response regulator that delivers PdeA to the ClpXP protease in a phosphorylation-dependent manner. Thus, CpdR serves as a crucial link between phosphorylation pathways and c-di-GMP metabolism to mediate protein degradation events that irreversibly and coordinately drive bacterial cell cycle progression and development.

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Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.

Transposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions.

Many bacteria mediate important life-style decisions by varying levels of the second messenger c-di-GMP. Behavioral transitions result from the coordination of complex cellular processes such as motility, surface adherence or the production of virulence factors and toxins. While the regulatory mechanisms responsible for these processes have been elucidated in some cases, the global pleiotropic effects of c-di-GMP are poorly understood, primarily because c-di-GMP networks are inherently complex in most bacteria. Moreover, the quantitative relationships between cellular c-di-GMP levels and c-di-GMP dependent phenotypes are largely unknown. Here, we dissect the c-di-GMP network of Caulobacter crescentus to establish a global and quantitative view of c-di-GMP dependent processes in this organism. A genetic approach that gradually reduced the number of diguanylate cyclases identified novel c-di-GMP dependent cellular processes and unraveled c-di-GMP as an essential component of C. crescentus cell polarity and its bimodal life cycle. By varying cellular c-di-GMP concentrations, we determined dose response curves for individual c-di-GMP-dependent processes. Relating these values to c-di-GMP levels modeled for single cells progressing through the cell cycle sets a quantitative frame for the successive activation of c-di-GMP dependent processes during the C. crescentus life cycle. By reconstructing a simplified c-di-GMP network in a strain devoid of c-di-GMP we defined the minimal requirements for the oscillation of c-di-GMP levels during the C. crescentus cell cycle. Finally, we show that although all c-di-GMP dependent cellular processes were qualitatively restored by artificially adjusting c-di-GMP levels with a heterologous diguanylate cyclase, much higher levels of the second messenger are required under these conditions as compared to the contribution of homologous c-di-GMP metabolizing enzymes. These experiments suggest that a common c-di-GMP pool cannot fully explain spatiotemporal regulation by c-di-GMP in C. crescentus and that individual enzymes preferentially regulate specific phenotypes during the cell cycle.