Global Regulatory Systems Research Articles

Manipulation and epigenetic control of silent biosynthetic pathways in actinobacteria.

Most biosynthetic gene clusters (BGCs) of Actinobacteria are either silent or expressed less than the detectable level. The non-genetic approaches including biological interactions, chemical agents, and physical stresses that can be used to awaken silenced pathways are compared in this paper. These non-genetic induction strategies often need screening approaches, including one strain many compounds (OSMAC), reporter-guided mutant selection, and high throughput elicitor screening (HiTES) have been developed. Different types of genetic manipulations applied in the induction of cryptic BGCs of Actinobacteria can be categorized as genome-wide pleiotropic and targeted approaches like manipulation of global regulatory systems, modulation of regulatory genes, ribosome and engineering of RNA polymerase or phosphopantheteine transferases. Targeted approaches including genome editing by CRISPR, mutation in transcription factors and modification of BGCs promoters, inactivation of the highly expressed biosynthetic pathways, deleting the suppressors or awakening the activators, heterologous expression, or refactoring of gene clusters can be applied for activation of pathways which are predicted to synthesize new bioactive structures in genome mining studies of Acinobacteria. In this review, the challenges and advantages of employing these approaches in induction of Actinobacteria BGCs are discussed. Further, novel natural products needed as drug for pharmaceutical industry or as biofertilizers in agricultural industry can be discovered even from known species of Actinobactera by the innovative approaches of metabolite biosynthesis elicitation.

Proteomic analysis reveals the global effect of the BarA/SirA-Csr regulatory cascade in Salmonella Typhimurium grown in conditions that favor the expression of invasion genes

In many bacteria, the BarA/SirA and Csr regulatory systems control expression of genes encoding a wide variety of cellular functions. The BarA/SirA two-component system induces the expression of CsrB and CsrC, two small non-coding RNAs that sequester CsrA, a protein that binds to target mRNAs and thus negatively or positively regulates their expression. BarA/SirA and CsrB/C induce expression of the Salmonella Pathogenicity Island 1 (SPI-1) genes required for Salmonella invasion of host cells. To further investigate the regulatory role of the BarA/SirA and Csr systems in Salmonella, we performed LC-MS/MS proteomic analysis using the WT S. Typhimurium strain and its derived ΔsirA and ΔcsrB ΔcsrC mutants grown in SPI-1-inducing conditions. The expression of 164 proteins with a wide diversity, or unknown, functions was significantly affected positively or negatively by the absence of SirA and/or CsrB/C. Interestingly, 19 proteins were identified as new targets for SirA-CsrB/C. Our results support that SirA and CsrB/C act in a cascade fashion to regulate gene expression in S. Typhimurium in the conditions tested. Notably, our results show that SirA-CsrB/C-CsrA controls expression of proteins required for the replication of Salmonella in the intestinal lumen, in an opposite way to its control exerted on the SPI-1 proteins. SignificanceThe BarA/SirA and Csr global regulatory systems control a wide range of cellular processes, including the expression of virulence genes. For instance, in Salmonella, BarA/SirA and CsrB/C positively regulate expression of the SPI-1 genes, which are required for Salmonella invasion to host cells. In this study, by performing a proteomic analysis, we identified 164 proteins whose expression was positively or negatively controlled by SirA and CsrB/C in SPI-1-inducing conditions, including 19 new possible targets of these systems. Our results support the action of SirA and CsrB/C in a cascade fashion to control different cellular processes in Salmonella. Interestingly, our data indicate that SirA-CsrB/C-CsrA controls inversely the expression of proteins required for invasion of the intestinal epithelium and for replication in the intestinal lumen, which suggests a role for this regulatory cascade as a molecular switch for Salmonella virulence. Thus, our study further expands the insight into the regulatory mechanisms governing the virulence and physiology of an important pathogen.

The acetoin assimilation pathway of Pseudomonas putida KT2440 is regulated by overlapping global regulatory elements that respond to nutritional cues.

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTSNtr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms.

Two-Component Systems of Streptomyces coelicolor: An Intricate Network to Be Unraveled

Bacteria of the Streptomyces genus constitute an authentic biotech gold mine thanks to their ability to produce a myriad of compounds and enzymes of great interest at various clinical, agricultural, and industrial levels. Understanding the physiology of these organisms and revealing their regulatory mechanisms is essential for their manipulation and application. Two-component systems (TCSs) constitute the predominant signal transduction mechanism in prokaryotes, and can detect a multitude of external and internal stimuli and trigger the appropriate cellular responses for adapting to diverse environmental conditions. These global regulatory systems usually coordinate various biological processes for the maintenance of homeostasis and proper cell function. Here, we review the multiple TCSs described and characterized in Streptomyces coelicolor, one of the most studied and important model species within this bacterial group. TCSs are involved in all cellular processes; hence, unravelling the complex regulatory network they form is essential for their potential biotechnological application.

Cautionary Notes on the Use of Arabinose- and Rhamnose-Inducible Expression Vectors in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution. IMPORTANCE Heterologous gene expression and complementation constitute a valuable and widely utilized tool in bacterial genetics. The arabinose-inducible ParaBAD (PBAD) and rhamnose-inducible PrhaBAD (PRha) promoter systems are commonly used in P. aeruginosa genetics and prized for the tight control and dynamic expression ranges that can be achieved. In this study, we demonstrate that the activity of both promoters is dependent upon the cAMP-dependent transcription factor Vfr. While this poses an obvious problem for use in a vfr mutant background, the issue is more pervasive, considering that vfr transcription/synthesis and cAMP homeostasis are highly integrated into the cellular physiology of the organism and influenced by numerous global regulatory systems. Fortunately, the synthetic PTac promoter is not subject to Vfr regulatory control.

Regulatory density as a means to refine current regulatory approaches for increasingly complex medicines

The continuous scientific, societal, and technological advancements have shifted drug development toward increasingly complex and ever more targeted treatments. This creates new and unprecedented challenges for global regulatory systems. To address the increased risks and uncertainties of increasingly complex medicine, we advocate for a more tailored and flexible regulatory approach, which is explained here with the concept of ‘regulatory density’. In the context of this paper, ‘regulatory density’ describes the relative amount of obligatory standards, measures and procedures applied to certain medicinal products or product classes and the resources required to meet these requirements. Given that risk and uncertainty are dynamic variables that can change over time, with this paper, we want to stimulate (re)thinking of regulatory approaches for managing the challenges of future complex medicines.

Piggybacking on Niche Adaptation Improves the Maintenance of Multidrug-Resistance Plasmids.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made toward resolving this “plasmid paradox” but mainly in a nonclinical context. Here, we propose an additional explanation for the maintenance of multidrug‐resistance plasmids in clinical Escherichia coli strains. After coevolving two multidrug‐resistance plasmids encoding resistance to last resort carbapenems with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (carbon catabolite repression) and ArcAB (aerobic respiration control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional rewiring during bacterial niche adaptation may facilitate plasmid maintenance.

The Genome of Staphylococcus epidermidis O47.

The skin colonizing coagulase-negative Staphylococcus epidermidis causes nosocomial infections and is an important opportunistic and highly adaptable pathogen. To gain more insight into this species, we sequenced the genome of the biofilm positive, methicillin susceptible S. epidermidis O47 strain (hereafter O47). This strain belongs to the most frequently isolated sequence type 2. In comparison to the RP62A strain, O47 can be transformed, which makes it a preferred strain for molecular studies. S. epidermidis O47’s genome has a single chromosome of about 2.5 million base pairs and no plasmid. Its oriC sequence has the same directionality as S. epidermidis RP62A, S. carnosus, S. haemolyticus, S. saprophyticus and is inverted in comparison to Staphylococcus aureus and S. epidermidis ATCC 12228. A phylogenetic analysis based on all S. epidermidis genomes currently available at GenBank revealed that O47 is closest related to DAR1907. The genome of O47 contains genes for the typical global regulatory systems known in staphylococci. In addition, it contains most of the genes encoding for the typical virulence factors for S. epidermidis but not for S. aureus with the exception of a putative hemolysin III. O47 has the typical S. epidermidis genetic islands and several mobile genetic elements, which include staphylococcal cassette chromosome (SCC) of about 54 kb length and two prophages φO47A and φO47B. However, its genome has no transposons and the smallest number of insertion sequence (IS) elements compared to the other known S. epidermidis genomes. By sequencing and analyzing the genome of O47, we provide the basis for its utilization in genetic and molecular studies of biofilm formation.

디지털 플랫폼에 대한 주요국 규제체계의 비교법 연구 - 독점규제법을 중심으로 -

전 세계적으로 기업의 사회적 책임과 개인정보 보호의무가 중대한 문제인바, 각 국은 디지털 플랫폼에 대한 현행규제체계를 개선하고자 노력하고 있다. 디지털 플랫폼의 양면·다면시장은 완전히 새로운 시장구조가 아니며 기존에도 존재해온 시장이므로 독점규제법의 법적 도구가 충분히 적용될 수 있다고 보기도 한다. 디지털 플랫폼의 경쟁 제한적 효과에 대해서는 이론적·실무적으로 합의된 원칙이 부족하므로 독점규제법 체계에 대한 갑작스런 변동은 지양해야 할 것이다. 그러나 일부 디지털 시장의 진입장벽을 축소하기 위해 경쟁자들 간에 디지털 비즈니스의 핵심 자산인 ‘데이터’ 공유의 중요성이 부각되면서, 각 국은 독점규제법 및 기타의 법령을 통해 디지털 플랫폼에 대한 규제방식을 발전시키고 있다.BR 각국의 독점규제체계는 독점화 시도 내지 시장지배적사업자의 지위남용 금지 및 기업결합 규제영역에서 관련 규정을 마련하고 있다. 비록 독점규제법의 목적이 경쟁과 소비자를 보호하는 것인 반면에 디지털 플랫폼의 무상 서비스 제공은 소비자 후생을 감소시키지는 않지만, 관련시장 획정 시 무상 서비스 제공시장도 관련시장에 포함하는 것이 최근의 경향이다. 독일과 오스트리아의 방식에 따라 기업결합의 사전 신고기준에 기존 자산·매출액과는 별도로 거래금액을 포함시키거나, 일본의 방식에 따라 데이터에 대한 시장지배력의 문제를 심각하게 인식하여 기업결합의 사전통제 및 시장지배적지위의 남용규제를 위한 보다 세부적인 지침을 마련할 필요가 있다. 또한 일부 디지털 시장의 기업결합 계획에 대해서는 공익성 심사도 고려하겠다는 영국 입법부의 논의도 주목할 만하다.BR 디지털 플랫폼에 대한 규제는 독점규제법 이외의 법령을 통해 추진하기도 한다. 프랑스는 기존 소비자보호법을 개정하여, 온라인 플랫폼 운영자가 소비자에게 전문가의 재화·서비스 가격과 특징에 대한 비교 서비스, 비교의 조건에 영향을 미치는 정보 및 자유로운 이용이 가능한 온라인 공간을 제공하도록 하였다. 아울러 세계 각국은 디지털 시대에 플랫폼의 경쟁 제한적 행위를 적합하게 규제하기 위한 법률이나 제도를 마련하고 전문성을 가진 전담기구를 설치했거나 설치하려고 한다는 점을 직시하여, 전 국가적 차원의 디지털 경제를 위한 미래지향적인 법제를 정비하고 이를 운영할 별도의 기관을 설치할지 여부를 고려해야 할 시점이다.

Investigation of the roles of AgrA and σB regulators in Listeria monocytogenes adaptation to roots and soil.

Little is known about the regulatory mechanisms that ensure the survival of the food-borne bacterial pathogen Listeria monocytogenes in the telluric environment and on roots. Earlier studies have suggested a regulatory overlap between the Agr cell-cell communication system and the general stress response regulator σB. Here, we investigated the contribution of these two systems to root colonisation and survival in sterilised and biotic soil. The ability to colonise the roots of the grass Festuca arundinacea was significantly compromised in the double mutant (∆agrA∆sigB). In sterile soil at 25°C, a significant defect was observed in the double mutant, suggesting some synergy between these systems. However, growth was observed and similar population dynamics were shown in the parental strain, ΔagrA and ΔsigB mutants. In biotic soil at 25°C, viability of the parental strain declined steadily over a two-week period highlighting the challenging nature of live soil environments. Inactivation of the two systems further decreased survival. The synergistic effect of Agr and σB was stronger in biotic soil. Transcriptional analysis confirmed the expected effects of the mutations on known Agr- and σB-dependent genes. Data highlight the important role that these global regulatory systems play in the natural ecology of this pathogen.

Inactivation of the Agrobacterium tumefaciens ActSR system affects resistance to multiple stresses with increased H2O2 sensitivity due to reduced expression of hemH.

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl3), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H2O2) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH, encoding ferrochelatase, restored wild-type catalase activity and H2O2 resistance levels. Gel mobility shift and hemH promoter-lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H2O2 resulting from a reduction in catalase activity due to reduced expression of hemH.

H-NS Family Members MvaT and MvaU Regulate the Pseudomonas aeruginosa Type III Secretion System.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of causing severe disease in immunocompromised individuals. A major P. aeruginosa virulence factor is the type III secretion system (T3SS). The T3SS is used to translocate effector proteins into host cells, causing cytotoxicity. The T3SS is under the transcriptional control of the master regulator ExsA. ExsA is encoded in the exsCEBA operon and autoregulates transcription via the P exsC promoter. There is also a Vfr-dependent promoter (P exsA ) located in the intergenic region between exsB and exsA A previous chromatin immunoprecipitation (ChIP)-on-chip experiment identified strong binding signatures for MvaT and MvaU in the intergenic region containing the P exsA promoter. MvaT and MvaU are DNA-binding histone-like nucleoid-structuring proteins that can repress gene expression. As predicted from the previous ChIP data, purified MvaT specifically bound to the P exsA promoter region in electrophoretic mobility shift assays. Whereas disruption of mvaT or mvaU by either transposon insertion or clustered regularly interspaced short palindromic repeat interference (CRISPRi) derepressed P exsA promoter activity and T3SS gene expression, overexpression of MvaT or MvaU inhibited P exsA promoter activity. Disruption of mvaT, however, did not suppress the Vfr requirement for P exsA promoter activity. Mutated MvaT/MvaU defective in transcriptional silencing exhibited dominant negative activity, resulting in a significant increase in P exsA promoter activity. Because no effect of MvaT or MvaU on Vfr expression was detected, we propose a model in which the primary effect of MvaT/MvaU on T3SS gene expression is through direct silencing of the P exsA promoter.IMPORTANCE Global regulatory systems play a prominent role in controlling the P. aeruginosa T3SS and include the Gac/RsmA, c-di-GMP, and Vfr-cAMP signaling pathways. Many of these pathways appear to directly or indirectly influence exsA transcription or translation. In this study, the histone-like proteins MvaT and MvaU are added to the growing list of global regulators that control the T3SS. MvaT and MvaU bind AT-rich regions in the genome and silence xenogeneic genes, including pathogenicity islands. The T3SS gene cluster has been horizontally transmitted among many Gram-negative pathogens. Control by MvaT/MvaU may reflect a residual effect that has persisted since the initial acquisition of the gene cluster, subsequently imposing a requirement for active regulatory mechanisms to override MvaT/MvaU-mediated silencing.

The biocide triclosan triggers multiple regulatory systems in Staphylococcus aureus to induce antibiotic tolerance

The biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.

Expression and regulatory networks of Mycobacterium tuberculosis PE/PPE family antigens

PE/PPE family antigens are distributed mainly in pathogenic mycobacteria and serve as potential antituberculosis (TB) vaccine components. Some PE/PPE family antigens can regulate the host innate immune response, interfere with macrophage activation and phagolysosome fusion, and serve as major sources of antigenic variation. PE/PPE antigens have been associated with mycobacteria pathogenesis; pe/ppe genes are mainly found in pathogenic mycobacteria and are differentially expressed between Mtb and Mycobacterium bovis. PE/PPE proteins were essential for the growth of Mtb, and PE/PPE proteins were differentially expressed under a variety of conditions. Multiple mycobacterial-virulence-related transcription factors, sigma factors, the global transcriptional regulation factor Lsr2, MprAB, and PhoPR two-component regulatory systems, and cyclic adenine monophosphate-dependent regulators, regulate the expression of PE/PPE family antigens. Multiple-scale integrative analysis revealed the expression and regulatory networks of PE/PPE family antigens underlying the virulence and pathogenesis of Mtb, providing important clues for the discovery of new anti-TB measures.

Global Regulation by CsrA and Its RNA Antagonists.

The sequence-specific RNA binding protein CsrA is employed by diverse bacteria in the posttranscriptional regulation of gene expression. Its binding interactions with RNA have been documented at atomic resolution and shown to alter RNA secondary structure, RNA stability, translation, and/or Rho-mediated transcription termination through a growing number of molecular mechanisms. In Gammaproteobacteria, small regulatory RNAs (sRNAs) that contain multiple CsrA binding sites compete with mRNA for binding to CsrA, thereby sequestering and antagonizing this protein. Both the synthesis and turnover of these sRNAs are regulated, allowing CsrA activity to be rapidly and efficiently adjusted in response to nutritional conditions and stresses. Feedback loops between the Csr regulatory components improve the dynamics of signal response by the Csr system. The Csr system of Escherichia coli is intimately interconnected with other global regulatory systems, permitting it to contribute to regulation by those systems. In some species, a protein antagonist of CsrA functions as part of a checkpoint for flagellum biosynthesis. In other species, a protein antagonist participates in a mechanism in which a type III secretion system is used for sensing interactions with host cells. Recent transcriptomics studies reveal vast effects of CsrA on gene expression through direct binding to hundreds of mRNAs, and indirectly through its effects on the expression of dozens of transcription factors. CsrA binding to base-pairing sRNAs and novel mRNA segments, such as the 3' untranslated region and deep within coding regions, predict its participation in yet-to-be-discovered regulatory mechanisms.

A semi-synthetic regulon enables rapid growth of yeast on xylose

Nutrient assimilation is the first step that allows biological systems to proliferate and produce value-added products. Yet, implementation of heterologous catabolic pathways has so far relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. In contrast, natural systems prefer nutrient-responsive gene regulation (called regulons) that control multiple cellular functions necessary for cell survival and growth. Here, in Saccharomyces cerevisiae, by partially- and fully uncoupling galactose (GAL)-responsive regulation and metabolism, we demonstrate the significant growth benefits conferred by the GAL regulon. Next, by adapting the various aspects of the GAL regulon for a non-native nutrient, xylose, we build a semi-synthetic regulon that exhibits higher growth rate, better nutrient consumption, and improved growth fitness compared to the traditional and ubiquitous constitutive expression strategy. This work provides an elegant paradigm to integrate non-native nutrient catabolism with native, global cellular responses to support fast growth.

Differential expression of the three Alcanivorax borkumensis SK2 genes coding for the P450 cytochromes involved in the assimilation of hydrocarbons.

Alcanivorax borkumensis, a marine bacterium highly specialized in degrading linear and branched alkanes, plays a key ecological role in the removal of marine oil spills. It contains several alternative enzyme systems for terminal hydroxylation of alkanes, including three P450 cytochromes (P450-1, P450-2 and P450-3). The present work shows cytochrome P450-1 to be expressed from the promoter of the upstream gene fdx. Promoter Pfdx was more active when C8 -C18 n-alkanes or pristane were assimilated than when pyruvate was available. The product of ABO_0199 (named CypR) was identified as a transcriptional activator of Pfdx . The inactivation of cypR impaired growth on tetradecane, showing the importance of the fdx-P450-1 and/or cypR genes. P450-2 expression was low-level and constitutive under all conditions tested, while that of P450-3 from promoter P450-3 was much higher when cells assimilated pristane than when n-alkanes or pyruvate were available. However, the inactivation of P450-3 had no visible impact on pristane assimilation. Cyo terminal oxidase, a component of the electron transport chain, was found to stimulate promoter PP450-3 activity, but it did not affect promoters Pfdx or PP450-2 . A. borkumensis, therefore, appears to carefully coordinate the expression of its multiple hydrocarbon degradation genes using both specific and global regulatory systems.

Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems.

CsrA of Escherichia coli is an RNA-binding protein that globally regulates a wide variety of cellular processes and behaviors, including carbon metabolism, motility, biofilm formation, and the stringent response. CsrB and CsrC are small RNAs (sRNAs) that sequester CsrA, thereby preventing CsrA-mRNA interaction. RpoE (σE) is the extracytoplasmic stress response sigma factor of E. coli Previous RNA sequencing (RNA-seq) studies identified rpoE mRNA as a CsrA target. Here, we explored the regulation of rpoE by CsrA and found that CsrA represses rpoE translation. Gel mobility shift, footprint, and toeprint studies identified three CsrA binding sites in the rpoE leader transcript, one of which overlaps the rpoE Shine-Dalgarno (SD) sequence, while another overlaps the rpoE translation initiation codon. Coupled in vitro transcription-translation experiments showed that CsrA represses rpoE translation by binding to these sites. We further demonstrate that σE indirectly activates the transcription of csrB and csrC, leading to increased sequestration of CsrA, such that repression of rpoE by CsrA is reduced. We propose that the Csr system fine-tunes the σE-dependent cell envelope stress response. We also identified a 51-amino-acid coding sequence whose stop codon overlaps the rpoE start codon and demonstrate that rpoE is translationally coupled with this upstream open reading frame (ORF51). The loss of coupling reduces rpoE translation by more than 50%. Identification of a translationally coupled ORF upstream of rpoE suggests that this previously unannotated protein may participate in the cell envelope stress response. In keeping with existing nomenclature, we named ORF51 rseD, resulting in an operon arrangement of rseD-rpoE-rseA-rseB-rseC IMPORTANCE CsrA posttranscriptionally represses genes required for bacterial stress responses, including the stringent response, catabolite repression, and the RpoS (σS)-mediated general stress response. We show that CsrA represses the translation of rpoE, encoding the extracytoplasmic stress response sigma factor, and that σE indirectly activates the transcription of csrB and csrC, resulting in reciprocal regulation of these two global regulatory systems. These findings suggest that extracytoplasmic stress leads to derepression of rpoE translation by CsrA, and CsrA-mediated repression helps reset RpoE abundance to prestress levels once envelope damage is repaired. The discovery of an ORF, rseD, translationally coupled with rpoE adds further complexity to translational control of rpoE.

The Impact of 18 Ancestral and Horizontally-Acquired Regulatory Proteins upon the Transcriptome and sRNA Landscape of Salmonella enterica serovar Typhimurium.

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli.

Csr (Rsm) noncoding small RNAs (sRNAs) CsrB and CsrC of Escherichia coli use molecular mimicry to sequester the RNA binding protein CsrA (RsmA) away from lower-affinity mRNA targets, thus eliciting major shifts in the bacterial lifestyle. CsrB/C transcription and turnover are activated by carbon metabolism products (e.g., formate and acetate) and by a preferred carbon source (glucose), respectively. We show that cAMP-CRP, a mediator of classical catabolite repression, inhibits csrC transcription by binding to the upstream region of this gene and also inhibits csrB transcription, apparently indirectly. We propose that glucose availability activates pathways for both synthesis and turnover of CsrB/C, thus shaping the dynamics of global signaling in response to the nutritional environment by poising CsrB/C sRNA levels for rapid response.