Functional analysis of the methylerythritol phosphate pathway terminal enzymes IspG and IspH from Zymomonas mobilis.

Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions. IMPORTANCE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.

Fe-S cluster homeostasis and beyond: The multifaceted roles of IscR.

The role of IscR in regulating the transcription of genes involved in Fe-S cluster homeostasis has been well established for the model organism Escherichia coli K12. In this bacterium, IscR coordinates expression of the Isc and Suf Fe-S cluster assembly pathways to meet cellular Fe-S cluster demands shaped by a variety of environmental cues. However, since its initial discovery nearly 25 years ago, there has been growing evidence that IscR function extends well beyond Fe-S cluster homeostasis, not only in E. coli, but in bacteria of diverse lifestyles. Notably, pathogenic bacteria have exploited the ability of IscR to respond to changes in oxygen tension, oxidative and nitrosative stress, and iron availability to navigate their trajectory in their respective hosts as changes in these cues are frequently encountered during host infection. In this review, we highlight these broader roles of IscR in different cellular processes and, in particular, discuss the importance of IscR as a virulence factor for many bacterial pathogens.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

Repression by the H-NS/YmoA histone-like protein complex enables IscR dependent regulation of the Yersinia T3SS.

The type III secretion system (T3SS) is an appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive H-NS/YmoA histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. While deletion of iscR or ymoA has been shown to decrease and increase LcrF expression and type III secretion, respectively, the role of H-NS in this system has not been definitively established because hns is an essential gene in Yersinia. Using CRISPRi knockdown of hns, we show that hns depletion causes derepression of lcrF. Furthermore, we find that while YmoA is dispensable for H-NS binding to the lcrF promoter, YmoA binding to H-NS is important for H-NS repressive activity. We bioinformatically identified three H-NS binding regions within the lcrF promoter and demonstrate binding of H-NS to these sites in vivo using chromatin immunoprecipitation. Using promoter truncation and binding site mutation analysis, we show that two of these H-NS binding regions are important for H-NS/YmoA-mediated repression of the lcrF promoter. Surprisingly, we find that IscR is dispensable for lcrF transcription in the absence of H-NS/YmoA. Indeed, IscR-dependent regulation of LcrF and type III secretion in response to changes in oxygen, such as those Yersinia is predicted to experience during host infection, only occurs in the presence of an H-NS/YmoA complex. These data suggest that, in the presence of host tissue cues that drive sufficient IscR expression, IscR can act as a roadblock to H-NS/YmoA-dependent repression of RNA polymerase at the lcrF promoter to turn on T3SS expression.

The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis.

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

Creation of Markerless Genome Modifications in a Nonmodel Bacterium by Fluorescence-Aided Recombineering.

Metabolic engineering of nonmodel bacteria is often challenging because of the paucity of genetic tools for iterative genome modification necessary to equip bacteria with pathways to produce high-value products. Here, we outline a homologous recombination-based method developed to delete or add genes to the genome of a nonmodel bacterium, Zymomonas mobilis, at the desired locus using a suicide plasmid that contains gfp as a fluorescence marker to track its presence in cells. The suicide plasmid is engineered to contain two 500 bp regions homologous to the DNA sequence immediately flanking the target locus. A single crossover event at one of the two homologous regions facilitates insertion of the plasmid into the genome and subsequent homologous recombination events excise the plasmid from the genome, leaving either the original genotype or the desired modified genotype. A key feature of this plasmid is that Green Fluorescent Protein (GFP) expressed from the suicide plasmid allows easy identification and sorting of cells that have lost the plasmid by use of a fluorescence activated cell sorter. Subsequent PCR amplification of genomic DNA from strains lacking GFP allows rapid identification of the desired genotype, which is confirmed by DNA sequencing. This method provides an efficient and flexible platform for improved genetic engineering of Z. mobilis, which can be easily adapted to other nonmodel bacteria.

Minor Alterations in Core Promoter Element Positioning Reveal Functional Plasticity of a Bacterial Transcription Factor.

IscR is a global transcription factor that regulates Fe-S cluster homeostasis and other functions in Escherichia coli by either activating or repressing transcription. While the interaction of IscR with its DNA sites has been studied, less is known about the mechanism of IscR regulation of transcription. Here, we show that IscR recruits RNA polymerase to an activated promoter and that IscR binding compensates for the lack of an optimal RNA polymerase σ70 -35 promoter element. We also find that the position of the -35 promoter element within the IscR DNA site impacts whether IscR activates or represses transcription. RNA polymerase binding at a distally positioned -35 element within the IscR site results in IscR activation. Molecular modeling suggests that this position of the -35 element allows IscR and RNA polymerase to bind to the promoter from opposite faces of the helix. Shifting the -35 element 1 nucleotide upstream within the IscR binding site results in IscR repression and a steric clash of IscR and RNA polymerase binding in the models. We propose that the sequence similarity of the IscR binding site with the -35 element is an important feature in allowing plasticity in the mechanism of IscR regulation. IMPORTANCE Transcription regulation is a key process in all living organisms, involving a myriad of transcription factors. In E. coli, the regulator of the iron-sulfur cluster biogenesis pathway, IscR, acts as a global transcription factor, activating the transcription of some pathways and repressing others. The mechanism by which IscR is able to activate and repress from a similar sequence space within bacterial promoter elements was not known. In this work, we show that subtle changes in the position of the σ70 -35 promoter element within an IscR binding site can switch the role of IscR from an activator to a repressor. Our work provides insights as to how the IscR site might have evolved around the -35 promoter element to allow a single transcription factor to differentially regulate promoters.

Corrigendum: A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

[This corrects the article DOI: 10.3389/fmicb.2019.02216.].

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems.

Zymomonas mobilis has emerged as a promising candidate for production of high-value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr-like type IV restriction modification (RM) system, a type I-F CRISPR system, a chromosomal type I RM system (hsdMSc), and a previously uncharacterized type I RM system, located on an endogenous plasmid (hsdRMSp). The DNA recognition motif of HsdRMSp was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMSc and hsdRMSp systems to that of the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMSc and HsdRMSp recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction-negative strains that we constructed and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. IMPORTANCE Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study, we developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identifying a previously unknown type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis.

Genome Scale Analysis Reveals IscR Directly and Indirectly Regulates Virulence Factor Genes in Pathogenic Yersinia.

The iron-sulfur cluster coordinating transcription factor IscR is important for the virulence of Yersinia pseudotuberculosis and a number of other bacterial pathogens. However, the IscR regulon has not yet been defined in any organism. To determine the Yersinia IscR regulon and identify IscR-dependent functions important for virulence, we employed chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of Y. pseudotuberculosis expressing or lacking iscR following iron starvation conditions, such as those encountered during infection. We found that IscR binds to the promoters of genes involved in iron homeostasis, reactive oxygen species metabolism, and cell envelope remodeling and regulates expression of these genes in response to iron depletion. Consistent with our previous work, we also found that IscR binds in vivo to the promoter of the Ysc type III secretion system (T3SS) master regulator LcrF, leading to regulation of T3SS genes. Interestingly, comparative genomic analysis suggested over 93% of IscR binding sites were conserved between Y. pseudotuberculosis and the related plague agent Yersinia pestis. Surprisingly, we found that the IscR positively regulated sufABCDSE Fe-S cluster biogenesis pathway was required for T3SS activity. These data suggest that IscR regulates the T3SS in Yersinia through maturation of an Fe-S cluster protein critical for type III secretion, in addition to its known role in activating T3SS genes through LcrF. Altogether, our study shows that iron starvation triggers IscR to coregulate multiple, distinct pathways relevant to promoting bacterial survival during infection. IMPORTANCE How bacteria adapt to the changing environment within the host is critical for their ability to survive and cause disease. For example, the mammalian host severely restricts iron availability to limit bacterial growth, referred to as nutritional immunity. Here, we show that pathogenic Yersinia use the iron-sulfur (Fe-S) cluster regulator IscR, a factor critical for pathogenesis, to sense iron availability and regulate multiple pathways known or predicted to contribute to virulence. Under low iron conditions that mimic those Yersinia encounter during infection, IscR levels increase, leading to modulation of genes involved in iron metabolism, stress resistance, cell envelope remodeling, and subversion of host defenses. These data suggest that IscR senses nutritional immunity to coordinate processes important for bacterial survival within the mammalian host.

Model-driven analysis of mutant fitness experiments improves genome-scale metabolic models of Zymomonas mobilis ZM4.

Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies-highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z. mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, iZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z. mobilis ZM4 to date. The methods of analysis that we applied here with the Z. mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.

Tailoring a Global Iron Regulon to a Uropathogen.

Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin.IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein.

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered "SufFeScient" derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

Iron availability and oxygen tension regulate the Yersinia Ysc type III secretion system to enable disseminated infection.

The enteropathogen Yersinia pseudotuberculosis and the related plague agent Y. pestis require the Ysc type III secretion system (T3SS) to subvert phagocyte defense mechanisms and cause disease. Yet type III secretion (T3S) in Yersinia induces growth arrest and innate immune recognition, necessitating tight regulation of the T3SS. Here we show that Y. pseudotuberculosis T3SS expression is kept low under anaerobic, iron-rich conditions, such as those found in the intestinal lumen where the Yersinia T3SS is not required for growth. In contrast, the Yersinia T3SS is expressed under aerobic or anaerobic, iron-poor conditions, such as those encountered by Yersinia once they cross the epithelial barrier and encounter phagocytic cells. We further show that the [2Fe-2S] containing transcription factor, IscR, mediates this oxygen and iron regulation of the T3SS by controlling transcription of the T3SS master regulator LcrF. IscR binds directly to the lcrF promoter and, importantly, a mutation that prevents this binding leads to decreased disseminated infection of Y. pseudotuberculosis but does not perturb intestinal colonization. Similar to E. coli, Y. pseudotuberculosis uses the Fe-S cluster occupancy of IscR as a readout of oxygen and iron conditions that impact cellular Fe-S cluster homeostasis. We propose that Y. pseudotuberculosis has coopted this system to sense entry into deeper tissues and induce T3S where it is required for virulence. The IscR binding site in the lcrF promoter is completely conserved between Y. pseudotuberculosis and Y. pestis. Deletion of iscR in Y. pestis leads to drastic disruption of T3S, suggesting that IscR control of the T3SS evolved before Y. pestis split from Y. pseudotuberculosis.

A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis. This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase (ldh) and the operon for cellulose synthase (bcsABC). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis.

Phage integration alters the respiratory strategy of its host.

Temperate bacteriophages are viruses that can incorporate their genomes into their bacterial hosts, existing there as prophages that refrain from killing the host cell until induced. Prophages are largely quiescent, but they can alter host phenotype through factors encoded in their genomes (often virulence factors) or by disrupting host genes as a result of integration. Here we describe another mechanism by which a prophage can modulate host phenotype. We show that a temperate phage that integrates in Escherichia coli reprograms host regulation of an anaerobic respiratory system, thereby inhibiting a bet hedging strategy. The phage exerts this effect by upregulating a host-encoded signal transduction protein through transcription initiated from a phage-encoded promoter. We further show that this phenomenon occurs not only in a laboratory strain of E. coli, but also in a natural isolate that contains a prophage at this site.

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose-Derived Sugars.

Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations' involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next-generation biofuels.

Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.