Serdar Turkarslan

Cytochrome c biogenesis: the Ccm system

Cytochromes of c-type contain covalently attached hemes that are formed via thioether bonds between the vinyls of heme b and cysteines within C(1)XXC(2)H motifs of apocytochromes. In diverse organisms this post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation (Ccm) systems. A highly complex version of these systems, Ccm or System I, is found in Gram-negative bacteria, archaea and plant mitochondria. We describe emerging functional interactions between the Ccm components categorized into three conserved modules, and present a mechanistic view of the molecular basis of ubiquitous vinyl-2 approximately Cys(1) and vinyl-4 approximately Cys(2) heme b-apocytochrome thioether bonds in c-type cytochromes.

The DNA-binding network of Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.

Mapping and manipulating the Mycobacterium tuberculosis transcriptome using a transcription factor overexpression-derived regulatory network

BackgroundMycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter.ResultsWe cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcription factor overexpressing (TFOE) strains, we predicted and validated the phenotype of a regulator that reduces susceptibility to a first line anti-tubercular drug, isoniazid. We also combined the TFOE data with an existing model of M. tuberculosis metabolism to predict the growth rates of individual TFOE strains with high fidelity.ConclusionThis work has led to a systems-level framework describing the transcriptome of a devastating bacterial pathogen, characterized the transcriptional influence of nearly all individual transcription factors in M. tuberculosis, and demonstrated the utility of this resource. These results will stimulate additional systems-level and hypothesis-driven efforts to understand M. tuberculosis adaptations that promote disease.

Compensatory thio–redox interactions between DsbA, CcdA and CcmG unveil the apocytochrome c holdase role of CcmG during cytochrome c maturation

During cytochrome c maturation (Ccm), the DsbA‐dependent thio‐oxidative protein‐folding pathway is thought to introduce a disulphide bond into the haem‐binding motif of apocytochromes c. This disulphide bond is believed to be reduced through a thio‐reductive pathway involving the Ccm components CcdA (DsbD), CcmG and CcmH. Here, we show in Rhodobacter capsulatus that in the absence of DsbA cytochrome c levels were decreased and CcdA or CcmG or the putative glutathione transporter CydDC was not needed for Ccm. This decrease was not due to overproduction of the periplasmic protease DegP as a secondary effect of DsbA absence. In contrast, CcmH was absolutely necessary regardless of DsbA, indicating that compensatory thio–redox interactions excluded it. Remarkably, the double (DsbA–CcmG) and triple (DsbA–CcmG–CcdA) mutants produced cytochromes c at lower levels than the DsbA‐null mutants, unless they contained a CcmG derivative (CcmG*) lacking its thio‐reductive activity. Purified CcmG* can bind apocytochrome c in vitro, revealing for the first time a thiol‐independent, direct interaction between apocytochrome c and CcmG. Furthermore, elimination of the thio–redox components does not abolish cytochrome c production, restricting the number of Ccm components essential for haem–apocyt c ligation per se during Ccm.

The cytochrome c maturation components CcmF, CcmH and CcmI form a membrane-integral multisubunit heme ligation complex

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

The Dithiol:Disulfide Oxidoreductases DsbA and DsbB of Rhodobacter capsulatus Are Not Directly Involved in Cytochrome c Biogenesis, but Their Inactivation Restores the Cytochrome c Biogenesis Defect of CcdA-Null Mutants

The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of Escherichia coli, are thought to supply the reducing equivalents required for the biogenesis of c-type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA-null mutants of the facultative phototroph Rhodobacter capsulatus are unable to grow under photosynthetic conditions (Ps(-)) and do not produce any active cytochrome c oxidase (Nadi(-)) due to a pleiotropic cytochrome c deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps(+) Nadi(+) colonies that produce c-type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps(+) growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the dsbA allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a dsbA ccdA double mutant was shown to be Ps(+) Nadi(+), establishing that in R. capsulatus the inactivation of dsbA suppresses the c-type cytochrome deficiency due to the absence of ccdA. Next, the ability of the wild-type dsbA allele to suppress the Ps(+) growth phenotype of the dsbA ccdA double mutant was exploited to isolate dsbA-independent ccdA revertants. Sequence analysis revealed that these revertants carried mutations in dsbB and that their Ps(+) phenotypes could be suppressed by the wild-type allele of dsbB. As with dsbA, a dsbB ccdA double mutant was also Ps(+) Nadi(+) and produced c-type cytochromes. Therefore, the absence of either DsbA or DsbB restores c-type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of R. capsulatus are Ps(+) and produce c-type cytochromes, unlike their E. coli counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in R. capsulatus the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome c biogenesis even though they are important for respiration under certain conditions.

Adaptation of cells to new environments

The evolutionary success of an organism is a testament to its inherent capacity to keep pace with environmental conditions that change over short and long periods. Mechanisms underlying adaptive processes are being investigated with renewed interest and excitement. This revival is partly fueled by powerful technologies that can probe molecular phenomena at a systems scale. Such studies provide spectacular insight into the mechanisms of adaptation, including rewiring of regulatory networks via natural selection of horizontal gene transfers, gene duplication, deletion, readjustment of kinetic parameters, and myriad other genetic reorganizational events. Here, we discuss advances in prokaryotic systems biology from the perspective of evolutionary principles that have shaped regulatory networks for dynamic adaptation to environmental change. WIREs Syst Biol Med 2011 3 544–561 DOI: 10.1002/wsbm.136

A high-resolution network model for global gene regulation in Mycobacterium tuberculosis

The resilience of Mycobacterium tuberculosis (MTB) is largely due to its ability to effectively counteract and even take advantage of the hostile environments of a host. In order to accelerate the discovery and characterization of these adaptive mechanisms, we have mined a compendium of 2325 publicly available transcriptome profiles of MTB to decipher a predictive, systems-scale gene regulatory network model. The resulting modular organization of 98% of all MTB genes within this regulatory network was rigorously tested using two independently generated datasets: a genome-wide map of 7248 DNA-binding locations for 143 transcription factors (TFs) and global transcriptional consequences of overexpressing 206 TFs. This analysis has discovered specific TFs that mediate conditional co-regulation of genes within 240 modules across 14 distinct environmental contexts. In addition to recapitulating previously characterized regulons, we discovered 454 novel mechanisms for gene regulation during stress, cholesterol utilization and dormancy. Significantly, 183 of these mechanisms act uniquely under conditions experienced during the infection cycle to regulate diverse functions including 23 genes that are essential to host-pathogen interactions. These and other insights underscore the power of a rational, model-driven approach to unearth novel MTB biology that operates under some but not all phases of infection.

Niche adaptation by expansion and reprogramming of general transcription factors

Numerous lineage‐specific expansions of the transcription factor B (TFB) family in archaea suggests an important role for expanded TFBs in encoding environment‐specific gene regulatory programs. Given the characteristics of hypersaline lakes, the unusually large numbers of TFBs in halophilic archaea further suggests that they might be especially important in rapid adaptation to the challenges of a dynamically changing environment. Motivated by these observations, we have investigated the implications of TFB expansions by correlating sequence variations, regulation, and physical interactions of all seven TFBs in Halobacterium salinarum NRC‐1 to their fitness landscapes, functional hierarchies, and genetic interactions across 2488 experiments covering combinatorial variations in salt, pH, temperature, and Cu stress. This systems analysis has revealed an elegant scheme in which completely novel fitness landscapes are generated by gene conversion events that introduce subtle changes to the regulation or physical interactions of duplicated TFBs. Based on these insights, we have introduced a synthetically redesigned TFB and altered the regulation of existing TFBs to illustrate how archaea can rapidly generate novel phenotypes by simply reprogramming their TFB regulatory network.

Extracytoplasmic prosthetic group ligation to apoproteins: maturation of c‐type cytochromes

In all organisms, haem is post‐translationally and covalently attached to c apocytochromes to produce c holocytochromes via a process called c‐type cytochromes maturation, which involves numerous components. In bacteria it was not clear which of these components catalyses the extracytoplasmic haem–apocytochrome ligation per se. In this issue of Molecular Microbiology, Feissner and colleagues report that a single polypeptide from Helicobacter pylori, corresponding to the fusion of two proteins found in other organisms, performs haem ligation to a coexpressed Bordetella pertussis apocytochrome c in an Escherichia coli mutant lacking its own cytochrome c maturation proteins. This simple experimental system pinpoints the components catalysing extracytoplasmic covalent haem ligation and raises intriguing issues about the requirements for delivery of haem and apocytochrome c substrates to produce c holocytochromes.