ModifiedmarinerTransposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

ABSTRACTTransposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA.marineris a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existingmarinertransposon, TnYLB, such that it can easily be genetically manipulated and introduced intoBacillus subtilis. We generate a series of three newmarinerderivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterlesslacZgene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted toB. subtilisand should be applicable to anymariner-compatible host organism, provided thatin vitromutagenesis or anin vivospecies-specific delivery vector is employed.

Download Full-text

Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

Journal of Bacteriology ◽

10.1128/jb.00257-15 ◽

2015 ◽

Vol 197 (16) ◽

pp. 2675-2684 ◽

Cited By ~ 4

Author(s):

Seram Nganbiton Devi ◽

Brittany Kiehler ◽

Lindsey Haggett ◽

Masaya Fujita

Keyword(s):

Bacillus Subtilis ◽

Mutant Protein ◽

Phosphoryl Group ◽

Atp Binding ◽

Histidine Kinases ◽

Content Type ◽

Point Mutant ◽

Terminal Domain

ABSTRACTEntry into sporulation inBacillus subtilisis governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur intrans. To test this hypothesis, we generated a series ofB. subtilisstrains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in atransfashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressedin vivo. Taken together, thesein vitroandin vivoresults reinforce the evidence that KinA autophosphorylation is able to occur in atransfashion.IMPORTANCEAutophosphorylation of histidine kinases is known to occur by either thecis(one subunit of kinase phosphorylating itself within the multimer) or thetrans(one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided directin vivoandin vitroevidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur intranswithin the homotetramer complex. While the physiological and mechanistic significance of thetransautophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

Download Full-text

Amphotericin B- and Voriconazole-Echinocandin Combinations against Aspergillus spp.: Effect of Serum on Inhibitory and Fungicidal Interactions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00597-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4656-4663 ◽

Cited By ~ 21

Author(s):

Antigoni Elefanti ◽

Johan W. Mouton ◽

Paul E. Verweij ◽

Athanassios Tsakris ◽

Loukia Zerva ◽

...

Keyword(s):

Amphotericin B ◽

Colorimetric Method ◽

Antifungal Drugs ◽

Content Type ◽

Synergistic Interactions ◽

Inhibitory Activities ◽

Additive Interactions ◽

Species Specific

ABSTRACTAntifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15Aspergillus fumigatuscomplex,A. flavuscomplex, andA. terreuscomplex isolates to assess both their growth-inhibitory and fungicidal activities. Thein vitroactivity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P< 0.001). Drug- and species-specific differences were found, with caspofungin and theA. fumigatuscomplex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, andA. flavuscomplex >A. fumigatuscomplex >A. terreuscomplex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for theA. terreuscomplex (median, 0.56). The presentin vitrodata showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observedin vivo.

Download Full-text

Engineering Pseudochelin Production inMyxococcus xanthus

Applied and Environmental Microbiology ◽

10.1128/aem.01789-18 ◽

2018 ◽

Vol 84 (22) ◽

Cited By ~ 7

Author(s):

Juliane Korp ◽

Lea Winand ◽

Angela Sester ◽

Markus Nett

Keyword(s):

Iron Homeostasis ◽

Condensation Reaction ◽

Myxococcus Xanthus ◽

Inducible Promoter ◽

Content Type ◽

Intramolecular Condensation ◽

Amidohydrolase Superfamily ◽

Interesting Alternative

ABSTRACTMyxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores, along with a new myxochelin derivative named pseudochelin A, was reported for the marine bacteriumPseudoalteromonas piscicidaS2040. The latter derivative features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains that produce solely myxochelin A and B with those ofP. piscicidaS2040. This approach revealed a gene exclusive to the myxochelin regulon inP. piscicidaS2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstitutedin vitrousing a hexahistidine-tagged recombinant protein made inEscherichia coli, with myxochelin B as the substrate. To test the production of pseudochelin A underin vivoconditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacteriumMyxococcus xanthusDSM 16526, a native producer of myxochelin A and B. Following induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutivepilApromoter led to increased production levels inM. xanthus, which facilitated the isolation and subsequent structural verification of the heterologously produced compound.IMPORTANCEIn this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained afterin vitroreconstitution. Furthermore, the function of this enzyme was demonstratedin vivoby transferring the corresponding gene into the bacteriumMyxococcus xanthus, which thereby became a producer of pseudochelin A. In addition to clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without chromosomal integration. Due to its metabolic proficiency,M. xanthusrepresents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.

Download Full-text

Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.02625-14 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1952-1962 ◽

Cited By ~ 24

Author(s):

Katherine A. Black ◽

Patricia C. Dos Santos

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Cysteine Desulfurase ◽

Content Type ◽

E Coli ◽

Wobble Position ◽

Lysine Trna ◽

Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

Download Full-text

The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

mSphere ◽

10.1128/msphere.00817-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Sonja Schibany ◽

Rebecca Hinrichs ◽

Rogelio Hernández-Tamayo ◽

Peter L. Graumann

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Single Molecule ◽

High Mobility ◽

Single Molecule Tracking ◽

Content Type ◽

Dna Compaction ◽

Transient Interactions

ABSTRACT Although DNA-compacting proteins have been extensively characterized in vitro, knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis, Smc (structural maintenance of chromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo, avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo, displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo.

Download Full-text

Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽

10.1128/mbio.03438-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Manuel Hörl ◽

Tobias Fuhrer ◽

Nicola Zamboni

Keyword(s):

Bacillus Subtilis ◽

Metabolic Flux ◽

Central Carbon Metabolism ◽

Flux Analysis ◽

Content Type ◽

Malolactic Enzyme ◽

First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

Download Full-text

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

Journal of Bacteriology ◽

10.1128/jb.00842-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4933-4940 ◽

Cited By ~ 25

Author(s):

Lauren J. Rajakovich ◽

John Tomlinson ◽

Patricia C. Dos Santos

Keyword(s):

Functional Analysis ◽

Bacillus Subtilis ◽

Gene Encoding ◽

Cysteine Desulfurase ◽

Content Type ◽

Sulfur Transfer ◽

Essential Enzyme ◽

Sulfur Trafficking

ABSTRACTThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. InEscherichia coliandSalmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, inBacillus subtilisand most species from theFirmicutesphylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis ofB. subtilisthiIand the adjacent gene,nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments inB. subtilisindicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine.In vitrosynthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain.In vivocomplementation studies inE. coliIscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal thatB. subtilisNifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

Download Full-text

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation

Journal of Bacteriology ◽

10.1128/jb.00625-16 ◽

2016 ◽

Vol 198 (24) ◽

pp. 3345-3354 ◽

Cited By ~ 5

Author(s):

Víctor M. Ayala-García ◽

Luz I. Valenzuela-García ◽

Peter Setlow ◽

Mario Pedraza-Reyes

Keyword(s):

Bacillus Subtilis ◽

Developmental Process ◽

Specific Pattern ◽

Dna Glycosylase ◽

Regulatory Sequences ◽

Regulation Of Expression ◽

Content Type ◽

Mutagenic Effects

ABSTRACTAag fromBacillus subtilishas been implicated inin vitroremoval of hypoxanthine and alkylated bases from DNA. The regulation of expression ofaaginB. subtilisand the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptionalaag-lacZfusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably,aag-lacZexpression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in asigG-deficient background, suggesting a forespore-specific mechanism ofaagtranscription. Two additional findings supported this suggestion: (i) expression of anaag-yfpfusion was observed in the forespore, and (ii)in vivomapping of theaagtranscription start site revealed the existence of upstream regulatory sequences possessing homology to σG-dependent promoters. In comparison with the wild-type strain, disruption ofaagsignificantly reduced survival of sporulatingB. subtiliscells following nitrous acid or methyl methanesulfonate treatments, and the Rifrmutation frequency was significantly increased in anaagstrain. These results suggest that Aag protects the genome of developingB. subtilissporangia from the cytotoxic and genotoxic effects of base deamination and alkylation.IMPORTANCEIn this study, evidence is presented revealing thataag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression duringB. subtilissporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

Download Full-text

Regulation of therhaEWRBMAOperon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00856-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 830-845 ◽

Cited By ~ 9

Author(s):

Kazutake Hirooka ◽

Yusuke Kodoi ◽

Takenori Satomura ◽

Yasutaro Fujita

Keyword(s):

Bacillus Subtilis ◽

Plant Pathogens ◽

Bacterial Species ◽

Regulatory Region ◽

Regulatory System ◽

Direct Repeat ◽

Plant Interactions ◽

Content Type

ABSTRACTTheBacillus subtilisrhaEWRBMA(formerlyyuxG-yulBCDE) operon consists of four genes encoding enzymes forl-rhamnose catabolism and therhaRgene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of therhaEWgene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited byl-rhamnulose-1-phosphate, an intermediate ofl-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control inB. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site.In vivoanalysis of therhaEWpromoter-lacZfusion in the background ofccpAdeletion showed that thel-rhamnose-responsive induction of therhaEWpromoter was negated by the disruption ofrhaAorrhaBbut notrhaEWorrhaM, whereasrhaRdisruption resulted in constitutiverhaEWpromoter activity. Thesein vitroandin vivoresults clearly indicate that RhaR represses the operon by binding to the operator site, which is detached byl-rhamnulose-1-phosphate formed froml-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, thelacZreporter analysis using the strains with or without theccpAdeletion under the background ofrhaRdisruption supported the involvement of CcpA in the carbon catabolite repression of the operon.IMPORTANCESincel-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested thatl-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance ofl-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system ofEscherichia coli. In this study, we show that, inBacillus subtilis, one of the plant growth-promoting rhizobacteria, therhaEWRBMAoperon forl-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms ofl-rhamnose catabolism in other bacterial species.

Download Full-text

An Improved Recombination-BasedIn VivoExpression Technology-Like Reporter System Reveals DifferentialcyaAGene Activation in Bordetella Species

Infection and Immunity ◽

10.1128/iai.01445-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1295-1305 ◽

Cited By ~ 8

Author(s):

Matthew S. Byrd ◽

Eliza Mason ◽

Michael W. Henderson ◽

Erich V. Scheller ◽

Peggy A. Cotter

Keyword(s):

Virulence Factors ◽

Gene Activation ◽

Regulatory System ◽

Reporter System ◽

Fluorescent Reporter ◽

Content Type ◽

Genes Encoding ◽

Species Specific

ABSTRACTBordetella pertussisandBordetella bronchisepticarely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg−phase, expression ofvrggenes, including those required for motility inB. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg+phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg+phase during respiratory infection, Bvg-regulated gene activation inB. bronchisepticahas not been investigatedin vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation bothin vitroandin vivo. Using pGFLIP, we demonstrated thatcyaA, considered to be a “late” Bvg+phase gene, is activated substantially earlier inB. bronchisepticathanB. pertussisfollowing a switch from Bvg−to Bvg+phase conditions. We show that the altered activation ofcyaAis not due to differences in thecyaApromoter or in thebvgASalleles ofB. bronchisepticacompared toB. pertussis, but appears to be species specific. Finally, we used pGFLIP to show thatflaAremains repressed during infection, confirming thatB. bronchisepticadoes not modulate to the Bvg−phasein vivo.

Download Full-text