Growth of microorganisms in an interfacially driven space bioreactor analog.

Fluid bioreactors in microgravity environments may utilize alternative methods of containment and mixing. The ring-sheared drop (RSD) is a containerless mixing device which functions in microgravity using surface tension for containment and mixes through interfacially-driven flow. To assess the feasibility of using interfacially driven flow devices, such as the RSD, as bioreactors, Escherichia coli growth and recombinant protein expression were analyzed in a ground-based analog of the RSD called the knife edge surface viscometer (KEV). Results demonstrated that the KEV can facilitate the growth of E. coli and that growth rate increases logarithmically with increasing knife edge rotation rate, similar to the standard growth method on Earth (orbital shaker). Furthermore, the KEV was shown to be viable for supporting recombinant protein expression in E. coli at levels comparable to those achieved using standard growth methods.

The BvgAS Regulon of Bordetella pertussis.

Nearly all virulence factors in Bordetella pertussis are activated by a master two-component system, BvgAS, composed of the sensor kinase BvgS and the response regulator BvgA. When BvgS is active, BvgA is phosphorylated (BvgA~P), and virulence-activated genes (vags) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (vrgs) are induced [Bvg(-) mode]. Here, we have used transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR) to define the BvgAS-dependent regulon of B. pertussis Tohama I. Our analyses reveal more than 550 BvgA-regulated genes, of which 353 are newly identified. BvgA-activated genes include those encoding two-component systems (such as kdpED), multiple other transcriptional regulators, and the extracytoplasmic function (ECF) sigma factor brpL, which is needed for type 3 secretion system (T3SS) expression, further establishing the importance of BvgA~P as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we demonstrate that the promoter for brpL is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions -41.5 and -63.5 in bprL Most importantly, we show for the first time that genes for multiple and varied metabolic pathways are significantly upregulated in the B. pertussis Bvg(-) mode. These include genes for fatty acid and lipid metabolism, sugar and amino acid transporters, pyruvate dehydrogenase, phenylacetic acid degradation, and the glycolate/glyoxylate utilization pathway. Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new vrgs that can be tested for function.IMPORTANCE Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due, at least in part, to the introduction of a less effective acellular vaccine in the 1990s. It is crucial, then, to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of B. pertussis virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in B. pertussis, revealing that more than 550 genes are regulated by BvgAS. We show that genes for multiple and varied metabolic pathways are highly regulated in the Bvg(-) mode (absence of BvgA phosphorylation). Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence.

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12.

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.

Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase.

Horizontal gene transfer permits rapid dissemination of genetic elements between individuals in bacterial populations. Transmitted DNA sequences may encode favourable traits. However, if the acquired DNA has an atypical base composition, it can reduce host fitness. Consequently, bacteria have evolved strategies to minimize the harmful effects of foreign genes. Most notably, xenogeneic silencing proteins bind incoming DNA that has a higher AT content than the host genome. An enduring question has been why such sequences are deleterious. Here, we showed that the toxicity of AT-rich DNA in Escherichia coli frequently results from constitutive transcription initiation within the coding regions of genes. Left unchecked, this causes titration of RNA polymerase and a global downshift in host gene expression. Accordingly, a mutation in RNA polymerase that diminished the impact of AT-rich DNA on host fitness reduced transcription from constitutive, but not activator-dependent, promoters.

Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

ChIP-seq for genome-scale analysis of bacterial DNA-binding proteins.

Protein-DNA interactions are central to many basic biological processes, including transcription regulation, DNA replication, and DNA repair. Chromatin Immunoprecipitation (ChIP) is used to determine the position and strength of protein-DNA interactions in vivo. Coupling ChIP with microarrays (ChIP-chip), and more recently with deep sequencing (ChIP-seq), has allowed genome-wide profiling of DNA binding events in vivo. In this chapter we outline the steps to generate ChIP-seq libraries from bacterial samples and briefly discuss basic analysis of the data.

Comprehensive mapping of the Escherichia coli flagellar regulatory network.

Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process.

Homing endonucleases: from genetic anomalies to programmable genomic clippers.

Homing endonucleases are strong drivers of genetic exchange and horizontal transfer of both their own genes and their local genetic environment. The mechanisms that govern the function and evolution of these genetic oddities have been well documented over the past few decades at the genetic, biochemical, and structural levels. This wealth of information has led to the manipulation and reprogramming of the endonucleases and to their exploitation in genome editing for use as therapeutic agents, for insect vector control and in agriculture. In this chapter we summarize the molecular properties of homing endonucleases and discuss their strengths and weaknesses in genome editing as compared to other site-specific nucleases such as zinc finger endonucleases, TALEN, and CRISPR-derived endonucleases.

Mapping homing endonuclease cleavage sites using in vitro generated protein.

Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays.

Widespread suppression of intragenic transcription initiation by H-NS.

Widespread intragenic transcription initiation has been observed in many species. Here we show that the Escherichia coli ehxCABD operon contains numerous intragenic promoters in both sense and antisense orientations. Transcription from these promoters is silenced by the histone-like nucleoid structuring (H-NS) protein. On a genome-wide scale, we show that 46% of H-NS-suppressed transcripts in E. coli are intragenic in origin. Furthermore, many intergenic promoters repressed by H-NS are for noncoding RNAs (ncRNAs). Thus, a major overlooked function of H-NS is to prevent transcription of spurious RNA. Our data provide a molecular description for the toxicity of horizontally acquired DNA and explain how this is counteracted by H-NS.

Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon.

Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.

Non-canonical protein-DNA interactions identified by ChIP are not artifacts.

BACKGROUND: ChIP-chip and ChIP-seq are widely used methods to map protein-DNA interactions on a genomic scale in vivo. Waldminghaus and Skarstad recently reported, in this journal, a modified method for ChIP-chip. Based on a comparison of our previously-published ChIP-chip data for Escherichia coli σ32 with their own data, Waldminghaus and Skarstad concluded that many of the σ32 targets identified in our earlier work are false positives. In particular, we identified many non-canonical σ32 targets that are located inside genes or are associated with genes that show no detectable regulation by σ32. Waldminghaus and Skarstad propose that such non-canonical sites are artifacts, identified due to flaws in the standard ChIP methodology. Waldminghaus and Skarstad suggest specific changes to the standard ChIP procedure that reportedly eliminate the claimed artifacts. RESULTS: We reanalyzed our published ChIP-chip datasets for σ32 and the datasets generated by Waldminghaus and Skarstad to assess data quality and reproducibility. We also performed targeted ChIP/qPCR for σ32 and an unrelated transcription factor, AraC, using the standard ChIP method and the modified ChIP method proposed by Waldminghaus and Skarstad. Furthermore, we determined the association of core RNA polymerase with disputed σ32 promoters, with and without overexpression of σ32. We show that (i) our published σ32 ChIP-chip datasets have a consistently higher dynamic range than those of Waldminghaus and Skarstad, (ii) our published σ32 ChIP-chip datasets are highly reproducible, whereas those of Waldminghaus and Skarstad are not, (iii) non-canonical σ32 target regions are enriched in a σ32 ChIP in a heat shock-dependent manner, regardless of the ChIP method used, (iv) association of core RNA polymerase with some disputed σ32 target genes is induced by overexpression of σ32, (v) σ32 targets disputed by Waldminghaus and Skarstad are predominantly those that are most weakly bound, and (vi) the modifications to the ChIP method proposed by Waldminghaus and Skarstad reduce enrichment of all protein-bound genomic regions. CONCLUSIONS: The modifications to the ChIP-chip method suggested by Waldminghaus and Skarstad reduce rather than increase the quality of ChIP data. Hence, the non-canonical σ32 targets identified in our previous study are likely to be genuine. We propose that the failure of Waldminghaus and Skarstad to identify many of these σ32 targets is due predominantly to the lower data quality in their study. We conclude that surprising ChIP-chip results are not artifacts to be ignored, but rather indications that our understanding of DNA-binding proteins is incomplete.

A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette.

Since its initial description more than two decades ago, the ribosome bypass (or "hop") sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an in-phase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron.

Bacteriophage T4 MotA activator and the ß-flap tip of RNA polymerase target the same set of σ70 carboxyl-terminal residues.

Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, σ(70). MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of σ(70), H5, a region that is normally bound within RNA polymerase by its interaction with the ß-flap tip. To identify the specific σ(70) residues responsible for interacting with MotA and the ß-flap tip, we generated single substitutions throughout the C terminus of σ(70). We find that MotA targets H5 residues that are normally engaged by the ß-flap. In two-hybrid assays, the interaction of σ(70) with either the ß-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/ß-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.

A likely pathway for formation of mobile group I introns.

Mobile group I introns are RNA splicing elements that have been invaded by endonuclease genes. These endonucleases facilitate intron mobility by a unidirectional, duplicative gene-conversion process known as homing [1]. Survival of the invading endonuclease depends upon its ability to promote intron mobility. Therefore, the endonuclease must either quickly change its cleavage specificity to match the site of intron insertion, or it must already be preadapted to cleave this sequence. Here we show that the group I intron in the DNA polymerase gene of T7-like bacteriophage PhiI is mobile, dependent upon its intronic HNH homing endonuclease gene, I-TslI. We also show that gene 5.3 of phage T3, located adjacent to its intronless DNA polymerase gene, is a homologous homing endonuclease gene whose protein product initiates efficient spread of gene 5.3 into empty sites in related phages. Both of these endonucleases cleave intronless DNA polymerase genes at identical positions. This shared feature between an intronic and free-standing endonuclease is unprecedented. Based on this evidence, we propose that introns and their homing endonucleases evolve separately to target the same highly conserved sequences, uniting afterwards to create a composite mobile element.

A free-standing homing endonuclease targets an intron insertion site in the psbA gene of cyanophages.

Homing endonuclease genes are mobile elements that promote their duplication into cognate sites that lack the endonuclease gene [1, 2]. The homing endonuclease initiates this event through site-specific DNA cleavage. Copying of the endonuclease gene follows as a consequence of DNA repair. A genome containing a homing endonuclease gene is subject to self-cleavage. Protection is accomplished through DNA sequence polymorphisms, as is the case in intronless homing of free-standing endonuclease genes [3, 4], or by disruption of the recognition site by a group I intron (or intein) into which the endonuclease ORF is embedded. We describe here a novel free-standing homing endonuclease from cyanobacteriophage S-PM2, which is similar to the DNA resolvase of bacteriophage T4 and is encoded adjacent to an intron-containing psbA gene [5, 6]. The endonuclease makes a specific double-strand cut near the intron insertion site (IIS), its DNA recognition site spans the IIS, and it is unable to cleave intron-containing psbA genes. This interdependence of a free-standing endonuclease gene and a group I intron, which we denote "collaborative homing," has not been reported previously and gives support to a hypothesis of formation of composite mobile introns by independent convergence of an intron and an endonuclease gene on the same target sequence.

A basic/hydrophobic cleft of the T4 activator MotA interacts with the C-terminus of E.coli sigma70 to activate middle gene transcription.

Transcriptional activation often employs a direct interaction between an activator and RNA polymerase. For activation of its middle genes, bacteriophage T4 appropriates Escherichia coli RNA polymerase through the action of two phage-encoded proteins, MotA and AsiA. Alone, AsiA inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of E. coli RNA polymerase. MotA interacts both with sigma(70) region 4 and with a DNA element present in T4 middle promoters. AsiA-induced remodelling is proposed to make the far C-terminus of sigma(70) region 4 accessible for MotA binding. Here, NMR chemical shift analysis indicates that MotA uses a 'basic/hydrophobic' cleft to interact with the C-terminus of AsiA-remodelled sigma(70), but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28 and Q76, both impair the interaction of MotA with sigma(70) region 4 and MotA-dependent activation. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target sigma(70) directly use acidic residues to engage a basic surface of region 4. Our work supports accumulated evidence indicating that 'sigma appropriation' by MotA and AsiA uses a fundamentally different mechanism to activate transcription.

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif.

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.