scholarly journals Novel Tripartite Aromatic Acid Transporter Essential for Terephthalate Uptake in Comamonas sp. Strain E6

2013 ◽  
Vol 79 (19) ◽  
pp. 6148-6155 ◽  
Author(s):  
Masaru Hosaka ◽  
Naofumi Kamimura ◽  
Shotaro Toribami ◽  
Kosuke Mori ◽  
Daisuke Kasai ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Substrate Binding ◽  
Aromatic Acid ◽  
Resting Cells ◽  
Uptake System ◽  
Content Type ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Acid Transporter ◽  
Membrane Components

ABSTRACTIt has been suggested that a novel type of aromatic acid transporter, which is similar to the tripartite tricarboxylate transporter (TTT), is involved in terephthalate (TPA) uptake byComamonassp. strain E6. This suggestion was based on the presence of the putative TPA-binding protein gene,tphC, in the TPA catabolic operon. ThetphCgene is essential for growth on TPA and is similar to the genes encoding TTT-like substrate-binding proteins. Here we identified two sets of E6 genes,tctBAandtpiBA, which encode TTT-like cytoplasmic transmembrane proteins. Disruption oftctAshowed no influence on TPA uptake but resulted in a complete loss of the uptake of citrate. This loss suggests thattctAis involved in citrate uptake. On the other hand, disruption oftpiAortpiBdemonstrated that both genes are essential for TPA uptake. Only when bothtphCandtpiBAwere introduced with the TPA catabolic genes into cells of a non-TPA-degradingPseudomonasstrain did the resting cells of the transformant acquire the ability to convert TPA. From all these results, it was concluded that the TPA uptake system consists of the TpiA-TpiB membrane components and TPA-binding TphC. Interestingly, not only was thetpiAmutant of E6 unable to grow on TPA or isophthalate, it also showed significant growth delays ono-phthalate and protocatechuate. These results suggested that the TpiA-TpiB membrane components are able to interact with multiple substrate-binding proteins. ThetpiBAgenes were constitutively transcribed as a single operon in E6 cells, whereas the transcription oftphCwas positively regulated by TphR. TPA uptake by E6 cells was completely inhibited by a protonophore, carbonyl cyanidem-chlorophenyl hydrazone, indicating that the TPA uptake system requires a proton motive force.

scholarly journals Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

2015 ◽  
Vol 197 (9) ◽  
pp. 1582-1591 ◽  
Author(s):  
Lauren M. Sheehan ◽  
James A. Budnick ◽  
R. Martin Roop ◽  
Clayton C. Caswell
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Zinc Uptake ◽  
Zinc Homeostasis ◽  
Zinc Toxicity ◽  
Uptake System ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

scholarly journals Bacterial Catabolism of β-Hydroxypropiovanillone and β-Hydroxypropiosyringone Produced in the Reductive Cleavage of Arylglycerol-β-Aryl Ether in Lignin

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Yudai Higuchi ◽  
Shogo Aoki ◽  
Hiroki Takenami ◽  
Naofumi Kamimura ◽  
Kenji Takahashi ◽  
...  
Keyword(s):  
Value Added ◽  
Lignin Biodegradation ◽  
Gene Products ◽  
Aryl Ether ◽  
Ether Linkage ◽  
Content Type ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Stereospecific Reaction

ABSTRACTSphingobiumsp. strain SYK-6 converts four stereoisomers of arylglycerol-β-guaiacyl ether into achiral β-hydroxypropiovanillone (HPV) via three stereospecific reaction steps. Here, we determined the HPV catabolic pathway and characterized the HPV catabolic genes involved in the first two steps of the pathway. In SYK-6 cells, HPV was oxidized to vanilloyl acetic acid (VAA) via vanilloyl acetaldehyde (VAL). The resulting VAA was further converted into vanillate through the activation of VAA by coenzyme A. A syringyl-type HPV analog, β-hydroxypropiosyringone (HPS), was also catabolized via the same pathway. SLG_12830 (hpvZ), which belongs to the glucose-methanol-choline oxidoreductase family, was isolated as the HPV-converting enzyme gene. AnhpvZmutant completely lost the ability to convert HPV and HPS, indicating thathpvZis essential for the conversion of both the substrates. HpvZ produced inEscherichia colioxidized both HPV and HPS and other 3-phenyl-1-propanol derivatives. HpvZ localized to both the cytoplasm and membrane of SYK-6 and used ubiquinone derivatives as electron acceptors. Thirteen gene products of the 23 aldehyde dehydrogenase (ALDH) genes in SYK-6 were able to oxidize VAL into VAA. Mutant analyses suggested that multiple ALDH genes, including SLG_20400, contribute to the conversion of VAL. We examined whether the genes encoding feruloyl-CoA synthetase (ferA) and feruloyl-CoA hydratase/lyase (ferBandferB2) are involved in the conversion of VAA. Only FerA exhibited activity toward VAA; however, disruption offerAdid not affect VAA conversion. These results indicate that another enzyme system is involved in VAA conversion.IMPORTANCECleavage of the β-aryl ether linkage is the most essential process in lignin biodegradation. Although the bacterial β-aryl ether cleavage pathway and catabolic genes have been well documented, there have been no reports regarding the catabolism of HPV or HPS, the products of cleavage of β-aryl ether compounds. HPV and HPS have also been found to be obtained from lignin by chemoselective catalytic oxidation by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of the β-aryl ether with zinc. Therefore, value-added chemicals are expected to be produced from these compounds. In this study, we determined the SYK-6 catabolic pathways for HPV and HPS and identified the catabolic genes involved in the first two steps of the pathways. Since SYK-6 catabolizes HPV through 2-pyrone-4,6-dicarboxylate, which is a building block for functional polymers, characterization of HPV catabolism is important not only for understanding the bacterial lignin catabolic system but also for lignin utilization.

scholarly journals Off-Target Gene Regulation Mediated by Transcriptional Repressors of Antimicrobial Efflux Pump Genes in Neisseria gonorrhoeae

2011 ◽  
Vol 55 (6) ◽  
pp. 2559-2565 ◽  
Author(s):  
Paul J. T. Johnson ◽  
Virginia A. Stringer ◽  
William M. Shafer
Keyword(s):  
Dna Binding ◽  
Neisseria Gonorrhoeae ◽  
Binding Proteins ◽  
Efflux Pump ◽  
Dna Binding Proteins ◽  
Transcriptional Repressors ◽  
Content Type ◽  
Genes Encoding ◽  
Encoding Gene ◽  
Microarray Studies

ABSTRACTDNA-binding proteins that control expression of drug efflux pump genes have been termed “local regulators” as their encoding gene is often located adjacent to the gene(s) that they regulate. However, results from recent studies indicate that they can control genes outside efflux pump-encoding loci, which we term as being “off target.” For example, the MtrR repressor was initially recognized for its ability to repress transcription of themtrCDE-encoded efflux pump operon in the strict human pathogenNeisseria gonorrhoeae, but recent results from genetic and microarray studies have shown that it can control expression of nearly 70 genes scattered throughout the chromosome. One of the off-target MtrR-repressed genes isglnA, which encodes glutamine synthetase. Herein, we confirm the capacity of MtrR to repressglnAexpression and provide evidence that such repression is due to its ability to negatively influence the binding of a second DNA-binding protein (FarR), which activatesglnA. FarR was previously recognized as a transcriptional repressor of thefarAB-encoded efflux pump operon. Thus, two DNA-binding proteins previously characterized as repressors of genes encoding efflux pumps that contribute to gonococcal resistance to antimicrobials can act in an opposing manner to modulate expression of a gene involved in basic metabolism.

scholarly journals Comparative Analysis ofkdpandktrMutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

2014 ◽  
Vol 197 (4) ◽  
pp. 676-687 ◽  
Author(s):  
Kei Nanatani ◽  
Toshiaki Shijuku ◽  
Yousuke Takano ◽  
Lalu Zulkifli ◽  
Tomoko Yamazaki ◽  
...  
Keyword(s):  
Recovery Phase ◽  
Transport Systems ◽  
Natural Environments ◽  
Uptake System ◽  
Content Type ◽  
Circadian Expression ◽  
Genes Encoding ◽  
External Potassium ◽  
On Chip ◽  
Pcc 6803

Photoautotrophic bacteria have developed mechanisms to maintain K+homeostasis under conditions of changing ionic concentrations in the environment.Synechocystissp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K+uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K+(Kdp). The contributions of each of these K+transport systems to cellular K+homeostasis have not yet been defined conclusively. To verify the functionality of Kdp,kdpgenes were expressed inEscherichia coli, where Kdp conferred K+uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of singleSynechocystiscells after hyperosmotic shock. Here, Ktr functioned as the primary K+uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of thekdpoperon inSynechocystiswas induced by extracellular K+depletion. Correspondingly, Kdp-mediated K+uptake supportedSynechocystiscell growth with trace amounts of external potassium. This induction ofkdpexpression depended on two adjacent genes,hik20andrre19, encoding a putative two-component system. The circadian expression ofkdpandktrpeaked at subjective dawn, which may support the acquisition of K+required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K+concentration under conditions of limited K+in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K+availability. Through their distinct activities, both Ktr and Kdp coordinate the responses ofSynechocystisto changes in K+levels under fluctuating environmental conditions.

scholarly journals Polyamine transport in bacteria and yeast

1999 ◽  
Vol 344 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Kazuei IGARASHI ◽  
Keiko KASHIWAGI
Keyword(s):  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Transport Systems ◽  
Uptake System ◽  
Polyamine Transport ◽  
Polyamine Content ◽  
Methylene Groups ◽  
Preferential Uptake

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

scholarly journals Mutations in Genes Encoding Penicillin-Binding Proteins and Efflux Pumps Play a Role in β-Lactam Resistance in Helicobacter cinaedi

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Emiko Rimbara ◽  
Shigetarou Mori ◽  
Hyun Kim ◽  
Masato Suzuki ◽  
Keigo Shibayama
Keyword(s):  
Binding Proteins ◽  
Efflux Pump ◽  
Whole Genome Analysis ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Helicobacter Cinaedi ◽  
Genes Encoding ◽  
Resistance Nodulation Division ◽  
The Difference ◽  
H Pylori

ABSTRACTβ-Lactams are often used to treatHelicobacter cinaediinfections; however, the mechanism underlying β-lactam resistance is unknown. In this study, we investigated β-lactam resistance in anH. cinaedistrain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 μg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptibleH. cinaedistrain MRY08-1234 (MICs of AMX and CRO, 1 and 4 μg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two inpbpA, one inpbp2, and two inftsI. Transformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations inpbpA; mutations inftsIalso led to increased resistance to AMX. Knocking outcmeBandcmeD, which encode resistance-nodulation-division-type efflux pump components, inH. cinaeditype strain CCUG18818 (AMX MIC, 4 to 8 μg/ml) resulted in 8- and 64-fold decreases, respectively, in the AMX MIC. Hence, MICs of AMX inH. cinaedibecome similar to those ofHelicobacter pyloriisolates in the absence ofcmeD. In conclusion, the difference in susceptibility to β-lactams betweenH. pyloriandH. cinaediis explained by differences in efflux pump components. Mutations inpbpAare the primary determinant of high resistance to β-lactams inH. cinaedi.

scholarly journals Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Matthew J. Bush ◽  
Maureen J. Bibb ◽  
Govind Chandra ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Regulatory Networks ◽  
Liquid Culture ◽  
Biological Processes ◽  
Streptomyces Venezuelae ◽  
Content Type ◽  
Master Regulators ◽  
Aerial Growth ◽  
Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

Sign in / Sign up

Export Citation Format

Share Document