scholarly journals The Role of Fnr Paralogs in Controlling Anaerobic Metabolism in the Diazotroph Paenibacillus polymyxa WLY78

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Haowen Shi ◽  
Yongbin Li ◽  
Tianyi Hao ◽  
Xiaomeng Liu ◽  
Xiyun Zhao ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Paenibacillus Polymyxa ◽  
Transcription Analysis ◽  
Content Type ◽  
Double Deletion ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Promoter Deletion Analysis ◽  
Single Deletion

ABSTRACT Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes. IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

scholarly journals The role of Fnr paralogs for controlling anaerobic metabolism in diazotroph Paenibacillus polymyxa WLY78

2020 ◽  
Author(s):  
Haowen Shi ◽  
Yongbin Li ◽  
Tianyi Hao ◽  
Xiaomeng Liu ◽  
Xiyun Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Bacillus Cereus ◽  
Anaerobic Condition ◽  
Anaerobic Metabolism ◽  
Nitrogenase Activity ◽  
Paenibacillus Polymyxa ◽  
Transcription Analysis ◽  
Double Deletion ◽  
Expression Of Genes

ABSTRACTFnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5 and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on the growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to 50% reduction in nitrogenase activity and double deletion of fnr1 and fnr3 resulted to 90% reduction in activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic condition. Fnr1 and Fnr3 inhibited expression of the genes involved in aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.IMPORTANCEPaenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. The members of nitrogen-fixing Paenibacillus have great potential use as a bacterial fertilizer in agriculture. However, the functions of fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we revealed that copy numbers vary largely among different Paenibacillus species and strains. Deletion and complementation analysis demonstrated that fnr1 and fnr3 have significant impacts on the growth and nitrogenase activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 purified in Escherichia coli could bind to the specific DNA promoter as Bacillus cereus Fnr did. Fnr1 and Fnr3 indirectly activated nif expression under anaerobic condition. Fnr1 and Fnr3 directly or indirectly activated or inhibited expression of many important genes involved in respiration, energy metabolism, Fe uptake and potentially specific electron transport for nitrogenase under anaerobic condition. This study not only reveals the roles of fnr genes in nitrogen fixation and anaerobic metabolism, but also provides insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

scholarly journals Transcriptomic and Phenotypic Analyses of the Sigma B-Dependent Characteristics and the Synergism between Sigma B and Sigma L in Listeria monocytogenes EGD-e

2020 ◽  
Vol 8 (11) ◽  
pp. 1644
Author(s):  
Mirjami Mattila ◽  
Panu Somervuo ◽  
Hannu Korkeala ◽  
Roger Stephan ◽  
Taurai Tasara
Keyword(s):  
Cold Stress ◽  
Acid Stress ◽  
Cellular Functions ◽  
Phenotypic Analysis ◽  
Double Deletion ◽  
New Genes ◽  
Genome Wide ◽  
Sigma B ◽  
Single Deletion ◽  
Increased Sensitivity

Numerous gene expression and stress adaptation responses in L. monocytogenes are regulated through alternative sigma factors σB and σL. Stress response phenotypes and transcriptomes were compared between L. monocytogenes EGD-e and its ΔsigB and ΔsigBL mutants. Targeted growth phenotypic analysis revealed that the ΔsigB and ΔsigBL mutants are impaired during growth under cold and organic-acid stress conditions. Phenotypic microarrays revealed increased sensitivity in both mutants to various antimicrobial compounds. Genes de-regulated in these two mutants were identified by genome-wide transcriptome analysis during exponential growth in BHI. The ΔsigB and ΔsigBL strains repressed 198 and 254 genes, respectively, compared to the parent EGD-e strain at 3 °C, whereas 86 and 139 genes, respectively, were repressed in these mutants during growth at 37 °C. Genes repressed in these mutants are involved in various cellular functions including transcription regulation, energy metabolism and nutrient transport functions, and viral-associated processes. Exposure to cold stress induced a significant increase in σB and σL co-dependent genes of L. monocytogenes EGD-e since most (62%) of the down-regulated genes uncovered at 3 °C were detected in the ΔsigBL double-deletion mutant but not in ΔsigB or ΔsigL single-deletion mutants. Overall, the current study provides an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. Besides previously known σB- and σL-dependent genes, the transcriptomes defined in ΔsigB and ΔsigBL mutants reveal several new genes that are positively regulated by σB alone, as well as those co-regulated through σB- and σL-dependent mechanisms during L. monocytogenes growth under optimal and cold-stress temperature conditions.

scholarly journals Exposure to Solute Stress Affects Genome-Wide Expression but Not the Polycyclic Aromatic Hydrocarbon-Degrading Activity of Sphingomonas sp. Strain LH128 in Biofilms

2012 ◽  
Vol 78 (23) ◽  
pp. 8311-8320 ◽  
Author(s):  
Tekle Tafese Fida ◽  
Philip Breugelmans ◽  
Rob Lavigne ◽  
Edith Coronado ◽  
David R. Johnson ◽  
...  
Keyword(s):  
Chronic Stress ◽  
Nacl Stress ◽  
Stress Factors ◽  
Content Type ◽  
Transcriptomic Data ◽  
Acute And Chronic Stress ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Polycyclic Aromatic ◽  
Genome Wide Expression

ABSTRACTMembers of the genusSphingomonasare important catalysts for removal of polycyclic aromatic hydrocarbons (PAHs) in soil, but their activity can be affected by various stress factors. This study examines the physiological and genome-wide transcription response of the phenanthrene-degradingSphingomonassp. strain LH128 in biofilms to solute stress (invoked by 450 mM NaCl solution), either as an acute (4-h) or a chronic (3-day) exposure. The degree of membrane fatty acid saturation was increased as a response to chronic stress. Oxygen consumption in the biofilms and phenanthrene mineralization activities of biofilm cells were, however, not significantly affected after imposing either acute or chronic stress. This finding was in agreement with the transcriptomic data, since genes involved in PAH degradation were not differentially expressed in stressed conditions compared to nonstressed conditions. The transcriptomic data suggest that LH128 adapts to NaCl stress by (i) increasing the expression of genes coping with osmolytic and ionic stress such as biosynthesis of compatible solutes and regulation of ion homeostasis, (ii) increasing the expression of genes involved in general stress response, (iii) changing the expression of general and specific regulatory functions, and (iv) decreasing the expression of protein synthesis such as proteins involved in motility. Differences in gene expression between cells under acute and chronic stress suggest that LH128 goes through changes in genome-wide expression to fully adapt to NaCl stress, without significantly changing phenanthrene degrading activity.

scholarly journals Genome-Wide Transcriptional Responses to Carbon Starvation in Nongrowing Lactococcus lactis

2015 ◽  
Vol 81 (7) ◽  
pp. 2554-2561 ◽  
Author(s):  
Onur Ercan ◽  
Michiel Wels ◽  
Eddy J. Smid ◽  
Michiel Kleerebezem
Keyword(s):  
Lactococcus Lactis ◽  
Natural Transformation ◽  
Recovery Period ◽  
Transcriptional Responses ◽  
Content Type ◽  
Gene Set ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Elevated Expression ◽  
Branched Chain

ABSTRACTThis paper describes the transcriptional adaptations of nongrowing, retentostat cultures ofLactococcus lactisto starvation. Near-zero-growth cultures (μ = 0.0001 h−1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence inL. lactisKF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conservedcis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation inL. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells.

scholarly journals Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

2015 ◽  
Vol 198 (4) ◽  
pp. 633-643 ◽  
Author(s):  
Marie-Christine Hoffmann ◽  
Eva Wagner ◽  
Sina Langklotz ◽  
Yvonne Pfänder ◽  
Sina Hött ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Central Process ◽  
Content Type ◽  
Proteome Profiling ◽  
Biological Nitrogen ◽  
Diazotrophic Growth

ABSTRACTRhodobacter capsulatusis capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, andlacZreporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed.IMPORTANCEBiological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, includingRhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome inR. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

scholarly journals Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

2012 ◽  
Vol 78 (22) ◽  
pp. 8056-8061 ◽  
Author(s):  
Ji Xu ◽  
Xiao-Lin Li ◽  
Li Luo
Keyword(s):  
Nitrogen Fixation ◽  
Drought Stress ◽  
Sinorhizobium Meliloti ◽  
Nitrogenase Activity ◽  
Root Nodules ◽  
Severe Drought ◽  
Control Strain ◽  
Free Living ◽  
Content Type ◽  
Host Tolerance

ABSTRACTCytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineeredSinorhizobiumstrains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed theAgrobacterium iptgene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although theiptgene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineeredSinorhizobiumstrains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.

scholarly journals Roles ofStaphylococcus aureusMnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis

2017 ◽  
Vol 200 (5) ◽  
Author(s):  
Manisha Vaish ◽  
Alexa Price-Whelan ◽  
Tamara Reyes-Robles ◽  
Jun Liu ◽  
Amyeo Jereen ◽  
...  
Keyword(s):  
Growth Rate ◽  
Catalytic Properties ◽  
Infection Model ◽  
Hydrophobic Membrane ◽  
Content Type ◽  
Double Deletion ◽  
Single Deletion ◽  
Major Loss ◽  
Type 3

ABSTRACTStaphylococcus aureushas three types of cation/proton antiporters. The type 3 family includes twomultisubunitNa+/H+(Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 inS. aureus. Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabcEscherichia colistrain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+and K+/H+cations, especially at pH 8.5. Under elevated salt conditions, deletion of themnhA1gene resulted in a significant reduction in the growth rate ofS. aureusin the range of pH 7.5 to 9. Deletion ofmnhA2had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion ofmnhA1andmnhA2led to a severe reduction in theS. aureusgrowth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters inS. aureuswere also assessed via their support of virulence in a mousein vivoinfection model. Deletion of themnhA1gene led to a major loss ofS. aureusvirulence in mice, while deletion ofmnh2led to no change in virulence.IMPORTANCEThis study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters inS. aureusand their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+and K+/H+that have roles in both osmotolerance and halotolerance inS. aureus. Study ofS. aureuswith a single deletion of eithermnhA1ormnhA2was assessed in an infection model of mice. The result shows thatmnhA1, but notmnhA2, plays a major role inS. aureusvirulence.

scholarly journals NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Yuhua Zhan ◽  
Zhiping Deng ◽  
Yongliang Yan ◽  
Hongyang Zhang ◽  
Chao Lu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stress Responses ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Nitrogenase Activity ◽  
Pseudomonas Stutzeri ◽  
Fine Tuning ◽  
Coding Region ◽  
Content Type ◽  
Nitrogenase Genes

ABSTRACT Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level. IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

scholarly journals The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch

2021 ◽  
Vol 22 (6) ◽  
pp. 3033
Author(s):  
Haowei Zhang ◽  
Qin Li ◽  
Yongbin Li ◽  
Sanfeng Chen
Keyword(s):  
Nitrogen Fixation ◽  
Noncoding Rna ◽  
Nitrogenase Activity ◽  
Constitutive Expression ◽  
Paenibacillus Polymyxa ◽  
Leader Region ◽  
Coding Region ◽  
Transcriptional Terminator ◽  
Stem Loop ◽  
T Box

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

scholarly journals Chromosome Segregation Proteins of Vibrio cholerae as Transcription Regulators

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Jong Hwan Baek ◽  
Seesandra V. Rajagopala ◽  
Dhruba K. Chattoraj
Keyword(s):  
Vibrio Cholerae ◽  
Chromosome Segregation ◽  
Transcription Analysis ◽  
Par Proteins ◽  
Content Type ◽  
Cell Functions ◽  
Genome Wide ◽  
Preferential Binding ◽  
Promoter Dna ◽  
Division Cell

ABSTRACT Bacterial ParA and ParB proteins are best known for their contribution to plasmid and chromosome segregation, but they may also contribute to other cell functions. In segregation, ParA interacts with ParB, which binds to parS centromere-analogous sites. In transcription, plasmid Par proteins can serve as repressors by specifically binding to their own promoters and, additionally, in the case of ParB, by spreading from a parS site to nearby promoters. Here, we have asked whether chromosomal Par proteins can likewise control transcription. Analysis of genome-wide ParB1 binding in Vibrio cholerae revealed preferential binding to the three known parS1 sites and limited spreading of ParB1 beyond the parS1 sites. Comparison of wild-type transcriptomes with those of ΔparA1, ΔparB1, and ΔparAB1 mutants revealed that two out of 20 genes (VC0067 and VC0069) covered by ParB1 spreading are repressed by both ParB1 and ParA1. A third gene (VC0076) at the outskirts of the spreading area and a few genes further away were also repressed, particularly the gene for an outer membrane protein, ompU (VC0633). Since ParA1 or ParB1 binding was not evident near VC0076 and ompU genes, the repression may require participation of additional factors. Indeed, both ParA1 and ParB1 proteins were found to interact with several V. cholerae proteins in bacterial and yeast two-hybrid screens. These studies demonstrate that chromosomal Par proteins can repress genes unlinked to parS and can do so without direct binding to the cognate promoter DNA. IMPORTANCE Directed segregation of chromosomes is essential for their maintenance in dividing cells. Many bacteria have genes (par) that were thought to be dedicated to segregation based on analogy to their roles in plasmid maintenance. It is becoming clear that chromosomal par genes are pleiotropic and that they contribute to diverse processes such as DNA replication, cell division, cell growth, and motility. One way to explain the pleiotropy is to suggest that Par proteins serve as or control other transcription factors. We tested this model by determining how Par proteins affect genome-wide transcription activity. We found that genes implicated in drug resistance, stress response, and pathogenesis were repressed by Par. Unexpectedly, the repression did not involve direct Par binding to cognate promoter DNA, indicating that the repression may involve Par interactions with other regulators. This pleiotropy highlights the degree of integration of chromosomal Par proteins into cellular control circuitries.

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