Protein expression levels optimize cell fitness: Too low an expression level of essential proteins will slow growth by compromising essential processes, whereas overexpression slows growth by increasing the metabolic load. This trade-off naïvely predicts that cells maximize their fitness by sufficiency, expressing just enough of each essential protein for function. We test this prediction in the naturally competent bacterium Acinetobacter baylyi by characterizing the proliferation dynamics of essential-gene knockouts at a single-cell scale (by imaging) as well as at a genome-wide scale. In these experiments, cells proliferate for multiple generations as target protein levels are diluted from their endogenous levels. This approach facilitates a proteome-scale analysis of the fitness landscape with respect to protein abundance. We find that most essential proteins are subject to a threshold-like fitness landscape: Growth is independent of protein abundance above a critical threshold and arrests below that threshold. We have recently analyzed the implications of this landscape for growth robustness. Confirming signature predictions of this model, we find that (i) roughly 70% of essential proteins are overabundant, (ii) overabundance increases as the expression level decreases, and (iii) the lowest abundance proteins are in vast excess (>10×) of what is required for growth in the typical cell. These results reveal that robustness plays a fundamental role in determining the expression levels of essential genes and that overabundance is a key mechanism for ensuring robust growth.

ABSTRACT Chromobacterium violaceum is a ubiquitous environmental pathogen. Despite its remarkable adaptability, little is known about the mechanisms of stress resistance in this bacterium. Here, in a screen for iron-susceptible transposon mutants, we identified a cytochrome bd that protects C. violaceum against multiple stresses. The two subunits of this cytochrome bd (CioAB) are encoded by the cioRAB operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A ∆ cioAB mutant strain was sensitive to iron and the iron-requiring antibiotic streptonigrin and showed a decrease in siderophore production. Growth curves and survival assays revealed that the ∆ cioAB strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the cioRAB operon and the transcript levels of the cioAB genes were increased in a ∆ cioR mutant. CioR bound the promoter region of the cio operon in vitro , indicating that CioR is a direct repressor of its own operon. Expression of the cio operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for cio expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in C. violaceum , we suggest that CioAB is a cyanide-insensitive terminal oxidase that allows respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome bd CioAB protects C. violaceum against multiple stress agents that are potentially produced endogenously or during interactions with a host. IMPORTANCE The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd ) to catalyze the reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum , protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.

SlyA and its homologs are conserved transcription factors in enteric bacteria, including Salmonella enterica, in which it upregulates horizontally acquired virulence genes. SlyA is a member of the MarR family of transcription factors, which possess a small molecule binding pocket. Ligand binding causes the protein to undergo a conformational change that abrogates DNA binding. Although the original discovery of MarR was based on its ability to recognize xenobiotic compounds and promote their efflux, the conservation of MarR family members throughout Bacteria and Archaea suggests a more general function. Because SlyA is known to bind xenobiotic aromatic carboxylates, we performed a targeted analysis of aromatic metabolic genes in Salmonella Typhimurium to identify potential endogenous ligands. We found that SlyA is promiscuously inhibited by multiple aromatic carboxylates including 2,3-dihydroxybenzoate, a precursor of iron-scavenging catecholate siderophores, and 4-hydroxybenzoate, a precursor of quinone-based electron-carriers, which allows it to sense changes in iron availability, respiration, and growth on succinate. We suggest that SlyA and other MarR proteins sense metabolic status via the flux of aromatic carboxylates in biosynthetic pathways. This allows SlyA to function as a counter-silencer of horizontally acquired genes that is exquisitely responsive to the metabolic state of the cell.IMPORTANCEMarR proteins comprise an ancient group of transcription factors that emerged before the divergence of Archaea and Bacteria. First identified as regulators of antibiotic efflux, they have also been suggested to sense and regulate concentrations of endogenous intracellular metabolites, but such metabolites have not previously been identified. Here we show that SlyA, a conserved and essential MarR family virulence gene regulator in enteric pathogens, binds to and is inhibited by aromatic metabolites required for many essential cellular processes. Our findings show how SlyA integrates metabolic status into bacterial transcriptional networks that control both molecular efflux and virulence.

Gene duplication facilitates the evolution of biological complexity, as one copy of a gene retains its original function while a duplicate copy can acquire mutations that would otherwise diminish fitness. Duplication has played a particularly important role in the evolution of regulatory networks by permitting novel regulatory interactions and responses to stimuli. The diverse MarR family of transcription factors (MFTFs) illustrate this concept, ranging from highly specific repressors of single operons to pleiotropic global regulators controlling hundreds of genes. MFTFs are often genetically and functionally linked to antimicrobial efflux systems. However, the SlyA MFTF lineage in the Enterobacteriaceae plays little or no role in regulating efflux but rather functions as transcriptional counter-silencers, which alleviate xenogeneic silencing of horizontally acquired genes and facilitate bacterial evolution by horizontal gene transfer. This review will explore recent advances in our understanding of MFTF traits that have contributed to their functional evolution.

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands. IMPORTANCE Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.

ABSTRACT Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands. IMPORTANCE Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.

The bacterial histone-like protein H-NS silences AT-rich DNA, binding DNA as 'stiffened' filaments or 'bridged' intrastrand loops. The switch between these modes has been suggested to depend on the concentration of divalent cations, in particular Mg2+, with elevated Mg2+ concentrations associated with a transition to bridging. Here we demonstrate that the observed binding mode is a function of the local concentration of H-NS and its cognate binding sites, as well as the affinity of the interactions between them. Mg2+ does not control a binary switch between these two modes but rather modulates the affinity of this interaction, inhibiting the DNA-binding and silencing activity of H-NS in a continuous linear fashion. The direct relationship between conditions that favor stiffening and transcriptional silencing activity suggests that although both modes can occur in the cell, stiffening is the predominant mode of binding at silenced genes.

SummaryIntegration of horizontally acquired genes into transcriptional networks is essential for the regulated expression of virulence in bacterial pathogens. In Salmonella enterica, expression of such genes is repressed by the nucleoid‐associated protein H‐NS, which recognizes and binds to AT‐rich DNA. H‐NS‐mediated silencing must be countered by other DNA‐binding proteins to allow expression under appropriate conditions. Some genes that can be transcribed by RNA polymerase (RNAP) associated with the alternative sigma factor σS or the housekeeping sigma factor σ70 in vitro appear to be preferentially transcribed by σS in the presence of H‐NS, suggesting that σS may act as a counter‐silencer. To determine whether σS directly counters H‐NS‐mediated silencing and whether co‐regulation by H‐NS accounts for the σS selectivity of certain promoters, we examined the csgBA operon, which is required for curli fimbriae expression and is known to be regulated by both H‐NS and σS. Using genetics and in vitro biochemical analyses, we found that σS is not directly required for csgBA transcription, but rather up‐regulates csgBA via an indirect upstream mechanism. Instead, the biofilm master regulator CsgD directly counter‐silences the csgBA promoter by altering the DNA‐protein complex structure to disrupt H‐NS‐mediated silencing in addition to directing the binding of RNAP.

Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally-responsive and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks.

Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP upregulates conserved and horizontally acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

ABSTRACT The transfer ( tra ) operon of the conjugative F plasmid of Escherichia coli is a polycistronic 33-kb operon which encodes most of the proteins necessary for F-plasmid transfer. Here, we report that transcription from P Y , the tra operon promoter, is repressed by the host nucleoid-associated protein, H-NS. Electrophoretic mobility shift assays indicate that H-NS binds preferentially to the tra promoter region, while Northern blot and transcriptional fusion analyses indicate that transcription of traY , the first gene in the tra operon, is derepressed in an hns mutant throughout growth. The plasmid-encoded regulatory protein TraJ is essential for transcription of the tra operon in wild-type Escherichia coli ; however, TraJ is not necessary for plasmid transfer or traY operon transcription in an hns mutant. This indicates that H-NS represses transcription from P Y directly and not indirectly via its effects on TraJ levels. These results suggest that TraJ functions to disrupt H-NS silencing at P Y , allowing transcription of the tra operon.

ABSTRACT The F plasmid of Escherichia coli allows horizontal DNA transfer between an F + donor cell and an F − recipient. Expression of the transfer genes is tightly controlled by a number of factors, including the following plasmid-encoded regulatory proteins: TraJ, the primary activator of the 33-kb tra operon, and the autoregulators TraM and TraY. Here, we demonstrate that the host RNA binding protein, Hfq, represses TraJ and TraM synthesis by destabilizing their respective mRNAs. Mating assays and immunoblot analyses for TraM and TraJ showed that transfer efficiency and protein levels increased in host cells containing a disruption in hfq compared to wild-type cells in stationary phase. The stability of transcripts containing a putative Hfq binding site located in the intergenic untranslated region between traM and traJ was increased in hfq mutant donor cells, suggesting that Hfq destabilizes these transcripts. Electrophoretic mobility shift assays demonstrated that Hfq specifically binds this region but not the antisense RNA, FinP, encoded on the opposite strand. Together, these findings indicate that Hfq regulates traM and traJ transcript stability by a mechanism separate from FinOP-mediated repression.

The F factor is often associated with Escherichia coli and appears to have been adapted by the bacterial host to act as an agent of genetic exchange and evolution. F encodes a type IV secretion system (T4SS) that enables bacterial conjugation, the transfer of DNA from a donor F+ to a recipient F− cell. The delivery of DNA containing either host or foreign genes has important consequences for the bacterium, allowing it to enlarge or modify its genetic content and rapidly adapt to an environmental niche. Unlike other plasmids, the conjugative functions of F and F-like plasmids appear to be controlled by a complex regulatory network that involves many host proteins resulting in a symbiotic relation between F and its host. This chapter outlines the predicted and known functions for all the genes on the F plasmid and its close relatives, and describes our current knowledge about the regulation of F conjugation.

The conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase. Transfer is dependent on the expression of the plasmid transfer (tra) genes, which are controlled by three plasmid-encoded regulatory proteins: TraJ, TraY and TraM. Here, we show that the nucleoid-associated host protein, H-NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels. Sequence analysis identified regions of predicted intrinsic curvature, to which H-NS preferentially binds, at the promoters of both traM and traJ. H-NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays. Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain. These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild-type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle. Transcriptional fusion studies of the individual promoters demonstrated that H-NS-mediated repression was observed when the promoters of both traM and traJ were present in cis to each other. This suggests that H-NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM.

ABSTRACT The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm. Detection of stress is mediated by the membrane-bound histidine kinase, CpxA. Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress. CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ. It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression. In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE. Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.