bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of a Bacteroides gene, csuF, which encodes an outer membrane protein that is essential for growth on chondroitin sulfate
Authors: Cheng Q , Yu M C , Reeves A R , Salyers A A
Published: November 2016

Bacteroides thetaiotaomicron can utilize a variety of polysaccharides, including charged mucopolysaccharides such as chondroitin sulfate (CS) and hyaluronic acid (HA). Since the enzymes (chondroitin lyases I and II) that catalyze the first step in breakdown of CS and HA are located in the periplasm, we had proposed that the first step in utilization of these polysaccharides was binding to one or more outer membrane proteins followed by translocation into the periplasm, but no such outer membrane proteins had been shown to play a role in CS or HA utilization. Previously we have isolated a transposon-generated mutant, CS4, which was unable to grow on CS or HA but retained the ability to grow on disaccharide components of CS. This phenotype suggested that the mutation in CS4 either blocked the transport of the mucopolysaccharides into the periplasmic space or blocked the depolymerization of the mucopolysaccharides into disaccharides. We have mapped the CS4 mutation to a single gene, csuF, which is capable of encoding a protein of 1,065 amino acids and contains a consensus signal sequence. Although CsuF had a predicted molecular weight and pI similar to those of chondroitin lyases, it did not show significant sequence similarity to the Bacteroides chondroitin lyase II, a Proteus chondroitin ABC lyase, or two hyaluronidases from Clostridium perfringens and Streptococcus pyogenes, nor was any CS-degrading enzyme activity associated with csuF expression in Bacteroides species or Escherichia coli. The deduced amino acid sequence of CsuF exhibited features suggestive of an outer membrane protein. We obtained antibodies to CsuF and demonstrated that the protein is located in the outer membrane. This is the first evidence that a nonenzymatic outer membrane protein is essential for utilization of CS and HA.
Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies
Authors: Derman A I , Beckwith J
Published: November 2016

Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.
Separate mechanisms activate sigma B of Bacillus subtilis in response to environmental and metabolic stresses
Authors: Voelker U , Voelker A , Maul B , Hecker M , Dufour A , Haldenwang W G
Published: November 2016

sigma B is a secondary sigma factor that controls the general stress response of Bacillus subtilis. sigma B-dependent transcription is induced by the activation of sigma B itself, a process that involves release of sigma B from an inhibitory complex with its primary regulator, RsbW. sigma B becomes available to RNA polymerase when RsbW forms a complex with an additional regulatory protein (RsbV) and, because of this, fails to bind sigma B. Using Western blot (immunoblot) analyses, reporter gene fusion assays, and measurements of nucleotide pool sizes, we provide evidence for two independent processes that promote the binding of RsbW to RsbV. The first occurs during carbon limitation or entry into stationary phase. Activation of sigma B under these circumstances correlates with a drop in the intracellular levels of ATP and may be a direct consequence of ATP levels on RsbW's binding preference. The second activation process relies on the product of a third regulatory gene, rsbU. RsbU is dispensable for sigma B activation during carbon limitation or stationary phase but is needed for activation of sigma B in response to any of a number of different environmental insults (ethanol treatment, salt or acid shock, etc.). RsbU, or a process dependent on it, alters RsbW binding without regard for intracellular levels of ATP. In at least some instances, the effects of multiple inducing stimuli are additive. The data are consistent with RsbW being a regulator at which distinct signals from separate effectors can be integrated to modulate sigma B activity.
Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli
Authors: DeShazer D , Wood G E , Friedman R L
Published: November 2016

Transcription of the pertussis toxin operon (ptx) is positively regulated in Bordetella pertussis by the bvgAS locus. However, a ptx-lacZ transcriptional fusion in Escherichia coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of ptx. A gene encoding a Bvg accessory factor (Baf) was identified by its ability to activate an E. coli ptx-lacZ fusion in the presence of bvgAS. The expression of ptx-lacZ was decreased by the addition of 40 mM MgSO4, a compound that also modulates ptx expression in B. pertussis. Baf alone did not activate expression of an E. coli fhaB-lacZ fusion, nor did it increase expression of fhaB-lacZ in trans with bvgAS. The gene encoding Baf was localized, sequenced, and found to produce a novel 28-kDa protein. Sequences homologous to B. pertussis baf were identified in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium. When an additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that produces a low level of pertussis toxin, pertussis toxin production was partially complemented in the cointegrate strain.
Purification and characterization of a 52-kilodalton immunoglobulin G-binding protein from Streptococcus suis capsular type 2
Authors: Serhir B , Dubreuil D , Higgins R , Jacques M
Published: November 2016

We previously reported that group D streptococci exhibited immunoglobulin G (IgG)-binding activity and that a 52-kDa IgG-binding protein was present in all Streptococcus suis strains examined (B. Serhir, R. Higgins, B. Foiry, and M. Jacques, J. Gen. Microbiol. 139:2953-2958, 1993). The objective of the present study was to purify and characterize this protein. Pig IgG were immobilized through their Fab fragments to ECH-Sepharose 4B, and the protein was purified by affinity chromatography. Electron microscopy observations of the purified material showed filamentous structures with a diameter of approximately 4 nm; these structures were not observed when the material was treated with either urea or ethanolamine. Electrophoretic and Western immunoblot analyses showed that the 52-kDa protein constituted the bulk of the recovered material. This protein was stained with either Coomassie brilliant blue or silver nitrate; it reacted with a large variety of mammalian IgG, human IgG (Fc) fragments, human IgA, and other human plasma proteins. The 52-kDa protein exhibited lower IgG-binding affinities than protein A and protein G. However, it was able to compete with protein A and protein G for binding to human IgG. In addition, it bound chicken IgG with high affinity. This last property differentiated the 52-kDa protein of S. suis from the six IgG-binding proteins described to date. The 52-kDa protein displayed similar affinities for untreated and deglycosylated pig IgG. The N-terminal amino acid sequence (SIITDVYAXEVLDSXGNPTLEV) revealed no homology with any bacterial proteins in the Swiss-Prot database. Its isoelectric point of approximately 4.6 and its amino acid composition, rich in aspartic and glutamic acids, showed that it had some similarities with other IgG-binding proteins. In this report, we have purified and characterized a 52-kDa IgG-binding protein from S. suis capsular type 2. Although this protein shares some similarities with other IgG- and/or IgA-binding proteins, it is unique in reacting with chicken IgG.
Cellular localization of the Escherichia coli SpoT protein
Authors: Gentry D R , Cashel M
Published: November 2016

The SpoT protein of Escherichia coli serves as a source of degradation as well as an apparent source of synthesis of (p)ppGpp. Since the subcellular localization of SpoT might be a clue to its function, we have used SpoT-specific antisera to analyze cell extracts fractionated on sucrose gradients. We find that the SpoT protein is not bound to ribosomes or to either inner or outer membrane fractions. Although the SpoT protein is found in large aggregates, its localization is probably cytosolic.
Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated
Authors: Ahmed M , Lyass L , Markham P N , Taylor S S , Vázquez-Laslop N , Neyfakh A A
Published: November 2016

The Bacillus subtilis genome encodes two multidrug efflux transporters sharing 51% sequence identity: Bmr, described previously, and Blt, described here. Overexpression of either transporter in B. subtilis leads to a similar increase in resistance to ethidium bromide, rhodamine and acridine dyes, tetraphenylphosphonium, doxorubicin, and fluoroquinolone antibiotics. However, Blt differs widely from Bmr in its expression pattern. Under standard cultivation conditions, B. subtilis expresses Bmr but Blt expression is undetectable. We have previously shown that Bmr expression is regulated by BmrR, a member of the family of MerR-like transcriptional activators. Here we show that blt transcription is regulated by another member of the same family, BltR. The DNA-binding domains of BmrR and BltR are related, but their putative inducer-binding domains are dissimilar, suggesting that Bmr and Blt are expressed in response to different inducers. Indeed, rhodamine, a substrate of Bmr and Blt and a known inducer of Bmr expression, does not induce Blt expression. Blt expression has been observed only in B. subtilis, carrying mutation acfA, which, as we show here, alters the sequence of the blt gene promoter. Unlike bmr, which is transcribed as a monocistronic mRNA, blt is cotranscribed with a downstream gene encoding a putative acetyltransferase. Overall, the differences in transcriptional control and operon organization between bmr and blt suggest that the transporters encoded by these genes have independent functions involving the transport of distinct physiological compounds.
The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette
Authors: Li L Y , Shoemaker N B , Wang G R , Cole S P , Hashimoto M K , Wang J , Salyers A A
Published: November 2016

Bacteroides conjugative transposons can act in trans to excise, circularize, and transfer unlinked integrated elements called NBUs (for nonreplicating Bacteroides units). Previously, we localized and sequenced the mobilization region of one NBU, NBU1, and showed that this mobilization region was recognized by the IncP plasmids RP4 and R751, as well as by the Bacteroides conjugative transposons. We report here that the single mobilization protein carried by NBU1 appears to be a bifunctional protein that binds to the oriT region and catalyzes the nicking reaction that initiates the transfer process. We have also localized and sequenced the mobilization region of a second NBU, NBU2. The NBU2 mobilization region was 86 to 90% identical at the DNA sequence to the oriT-mob region of NBU1. The high sequence similarity between NBU1 and NBU2 ended abruptly after the stop codon of the mob gene and about 1 kbp upstream of the oriT region, indicating that the oriT-mob regions of NBU1 and NBU2 may be on some sort of cassette. A region on NBU1 and NBU2 which lies immediately upstream of the oriT region had 66% sequence identity to a region upstream of the oriT region on a mobilizable transposon, Tn4399, an element that had previously appeared to be completely unrelated to the NBUs.
Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant
Authors: Revill W P , Bibb M J , Hopwood D A
Published: November 2016

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.
Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose
Authors: Klein W , Horlacher R , Boos W
Published: November 2016

A gene bank of partially Sau3A-digested Escherichia coli DNA ligated in plasmid pBR322 was screened for the ability to complement a mutant unable to metabolize trehalose at low osmolarity. The resulting plasmid was shown to contain the genes encoding transport (treB) and metabolic (treC) functions. The complementing DNA region was sequenced and shown to contain an operon of two genes, with treB as the promoter proximal gene and with treC as the promoter distal gene. The transcriptional start point was determined, and one major transcript was detected. The control region of the operon was found to contain consensus binding motifs for the cyclic AMP-catabolite activator protein complex and for a specific repressor protein whose gene, treR, is located immediately upstream of treB, being transcribed in the same direction as treB treC. The products of both genes could be expressed in minicells in which TreB revealed itself as a protein with an apparent molecular weight of 42,000. The gene product of treB consists of 485 amino acids with a calculated molecular weight of 52,308. It showed high homology to enzymes IIScr of enteric bacteria specific for the uptake of sucrose and encoded by plasmid pUR400 of enteric bacteria. Like enzyme IIScr, enzyme IITre belongs to the EIIBC domain type and lacks a covalently bound EIIA domain. Instead, enzyme IITre-mediated phosphorylation of trehalose requires the activity of enzyme IIAGlc, a component of the major glucose transport system.
Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development
Authors: Laue B E , Gill R E
Published: November 2016

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.
Induction of cold-responsive proteins in Vibrio vulnificus
Authors: McGovern V P , Oliver J D
Published: November 2016

We have studied the response of Vibrio vulnificus to temperature shifts (23 to 13 degrees C) within the organism's permissive growth range. Cold shift induced a diminution in protein synthesis. Following a short lag, cells began growth at a new rate. Forty proteins were induced by this downshift.
Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins
Authors: Sirko A , Zatyka M , Sadowy E , Hulanicka D
Published: November 2016

In Escherichia coli, sulfate and thiosulfate ions are transported by an ABC-type transporter consisting of both the membrane components (the products of cysT, cysW, and cysA genes) and the periplasmic binders (the products of cysP and sbp genes). The single cysP and sbp mutants are able to utilize both sulfate and thiosulfate as a sole sulfur source, while the inactivation of both genes leads to cysteine auxotrophy resulting from the block in the transport of both ions.
Molecular characterization of a deletion-prone region of plasmid pAE1 of Alcaligenes eutrophus H1
Authors: Chow W Y , Wang C K , Lee W L , Kung S S , Wu Y M
Published: November 2016

A 93-kb region (D region) of plasmid pAE1 of Alcaligenes eutrophus H1 has been found to have a high rate of spontaneous deletion. In this study, we constructed a restriction endonuclease map and carried out limited sequencing of an approximately 100-kb region from pAE1 which includes the D region (the deleted region) in order to detect and characterize repetitive sequences. Two types of repetitive sequences, the R1 and R2 sequences, were observed to flank the D region; within the D region are three copies of insertion element ISAE1. The R1 and R2 sequences are arranged in direct and inverted orientations, respectively. Molecular analysis of the end product of the deletion is consistent with the hypothesis that the loss of the D-region DNA is the result of recombination between two copies of the R1 sequence. The R1 sequence encodes a 415-amino-acid protein which exhibits substantial sequence similarity to the lambda integrase family of site-specific recombinases. Its genetic function remains to be determined.
Identification of cutC and cutF (nlpE) genes involved in copper tolerance in Escherichia coli
Authors: Gupta S D , Lee B T , Camakaris J , Wu H C
Published: November 2016

It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.
Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection
Authors: Bayer M , Eferl R , Zellnig G , Teferle K , Dijkstra A , Koraimann G , Högenauer G
Published: November 2016

F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection.
Organization and expression of the Rhodobacter sphaeroides cycFG operon
Authors: Flory J E , Donohue T J
Published: November 2016

The Rhodobacter sphaeroides cycFG operon has been cloned, sequenced, and mapped to approximately coordinate 2500 of chromosome I. The cycF gene encodes cytochrome c554, a member of the class II family of soluble cytochrome c proteins. The cycF open reading frame includes a 20-amino acid extension at its N terminus which has not been detected in cytochrome c554. Antiserum against cytochrome c554 shows that this protein is localized to the periplasm of wild-type cells, which suggests that this N-terminal extension functions as a signal peptide. The predicted cycG gene product is a diheme cytochrome c with a subunit molecular mass of approximately 32 kDa. While a cytochrome with the properties predicted for CycG has not been reported for R. sphaeroides, we have tentatively identified this protein as a heme-staining polypeptide that is associated with membranes. CycG could have an overall structure similar to that of several other electron carriers, since the similarity between the predicted amino acid sequence of CycG and other multiheme cytochrome c proteins extends throughout the polypeptide. The cycFG transcript is approximately 1,500 nucleotides long and has a single 5' end 26 nucleotides upstream of the start of cycF translation. Expression of cycFG is regulated at the level of mRNA accumulation, since approximately fivefold-higher levels of both cycF-specific transcript and cytochrome c554 protein are detected in cell extracts from aerobic cultures in comparison with those from anaerobically grown cells. Although cytochrome c554 was detected under all growth conditions tested, the highest levels of this protein were found when cells generate energy via aerobic respiration.
Escherichia coli genes required for cytochrome c maturation
Authors: Thöny-Meyer L , Fischer F , Künzler P , Ritz D , Hennecke H
Published: November 2016

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.
A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions
Authors: Malakooti J , Wang S P , Ely B
Published: November 2016

Caulobacter crescentus differentiates prior to each cell division to form two different daughter cells: a monoflagellated swarmer cell and a nonmotile stalked cell. Thus, one might expect that developmentally expressed genes would be regulated by mechanisms different from those used to regulate the expression of the biosynthetic genes. To determine a consensus promoter sequence for genes involved in biosynthetic or housekeeping functions, DNA fragments containing the regulatory regions of the ilvD, ilvR, cysC, pleC, and fdxA genes were cloned. S1 nuclease protection mapping and primer extension techniques were used to identify the transcription initiation sites. Comparison of the regulatory regions of these genes with those of the published sequences of the ilvBN, rrnA, trpFBA, dnaA, dnaK, hemE, and rsaA genes has resulted in the identification of a putative promoter consensus sequence. The -35 region contains the sequence TTGACGS, which is similar to the Escherichia coli -35 region, while the -10 region, GCTANAWC, has a more balanced GC content than the corresponding region in E. coli. Oligonucleotide-directed site-specific mutagenesis of both the ilvBN and pleC promoters indicates that mutations that make a promoter more like the consensus result in increased promoter activity, while mutations decreasing similarity to the consensus result in decreased promoter activity.
Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii
Authors: Daniel R , Stuertz K , Gottschalk G
Published: November 2016

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent Kms of the enzyme for glycerol and NAD+ were 1.27 mM and 57 microM, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent Kms were 30 and 70 microM, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the sigma 54 bacterial enhancer-binding protein family.
Involvement of the "A" isozyme of methyltransferase II and the 29-kilodalton corrinoid protein in methanogenesis from monomethylamine
Authors: Burke S A , Krzycki J A
Published: November 2016

An assay which allowed detection of proteins involved in the trimethylamine- or monomethylamine (MMA)-dependent methylation of coenzyme M (CoM) was developed. The two activities could be separated by anion-exchange chromatography. The unresolved activity responsible for MMA:CoM methyl transfer eluted from a gel permeation column in the molecular mass range of 32 kDa. The activity was purified to two monomeric proteins of 40 and 29 kDa. The preparation contained protein-bound corrinoid in a mixture of Co(II) and Co(III) states, as well as methyl-B12:CoM methyltransferase (MT2) activity. N-terminal sequence analysis demonstrated that the polypeptides were two previously identified proteins of undefined physiological function. The smaller polypeptide was the monomeric 29-kDa corrinoid protein. The larger polypeptide was the "A" isozyme of MT2. Individually purified preparations of both proteins increased the rate of MMA-dependent CoM methylation by approximately 1.7 mumol/min/mg of purified protein above background activity in the extract of methanol-grown cells. These results indicate that the 29-kDa corrinoid protein and the "A" isozyme of MT2 function in methanogenesis from MMA. A likely mechanism is that the 29-kDa corrinoid is methylated by MMA and the methyl group is then transferred by the "A" isozyme of MT2 to CoM.
CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258
Authors: Endo G , Silver S
Published: November 2016

The CadC protein from the cadA cadmium resistance operon of Staphylococcus aureus plasmid pI258 regulates transcription of this system in vitro. The CadC protein was overproduced in Escherichia coli cells and partially purified. Gel shift assays of the proposed cadA operator/promoter region DNA showed specific association with the CadC protein. Control arsenic resistance operator/promoter DNA from the same plasmid was not shifted by the CadC protein. Cd2+, Bi3+, and Pb2+ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions -7 to +14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.
Different internal metabolites trigger the induction of glycolytic gene expression in Saccharomyces cerevisiae
Authors: Müller S , Boles E , May M , Zimmermann F K
Published: November 2016

In the yeast Saccharomyces cerevisiae, the sugar-induced expression of various genes coding for glycolytic enzymes is triggered by increases in the concentrations of different internal metabolites. Here, we show that the induction of the glycolytic isoenzyme enolase 2 is strictly dependent on the abilities of different mutant strains to increase the level of glucose-6-phosphate after the addition of sugars. In contrast, the induction of alcohol dehydrogenase I is dependent on increasing concentrations of metabolites in the late stages of glycolysis.
Effects of spo0 mutations on spo0A promoter switching at the initiation of sporulation in Bacillus subtilis
Authors: Chibazakura T , Kawamura F , Asai K , Takahashi H
Published: November 2016

Transcriptional analyses of the Bacillus subtilis sporulation initiator gene spo0A revealed that promoter switching from the vegetative (Pv) to the sporulation-specific (Ps) promoter did not occur in the spo0A, spo0B, spo0E, spo0F, and spo0H mutants. The sof-1 mutation in spo0A restored the promoter switching in the spo0F mutant. These results strongly suggest that Spo0A plays a central role in the regulation of its own promoter switching.
Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam
Authors: Oppezzo O J , Antón D N
Published: November 2016

cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium. The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations. Most, but not all, cysB and cysE mutations from other origins displayed the same behavior. Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants. It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.
Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes
Authors: Sieberth V , Rigg G P , Roberts I S , Jann K
Published: November 2016

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.
Phosphorylation of Mycoplasma pneumoniae cytadherence-accessory proteins in cell extracts
Authors: Krebes K A , Dirksen L B , Krause D C
Published: November 2016

A cell-free system was used to characterize the phosphorylation of Mycoplasma pneumoniae proteins HMW1 and HMW2, which are involved in the adherence of this organism to human tracheal epithelium during infection. The pH and cation requirements for phosphorylation of HMW1 and HMW2 were determined, and the effects of glycolytic intermediates, cyclic AMP, and eukaryotic kinase-phosphatase inhibitors and stimulators on this process were examined. Phosphoamino acid analysis identified serine as the major phosphate acceptor for both HMW1 and HMW2 in this system.
appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1
Authors: Gomelsky M , Kaplan S
Published: November 2016

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.
An early A-signal-dependent gene in Myxococcus xanthus has a sigma 54-like promoter
Authors: Keseler I M , Kaiser D
Published: November 2016

A-signaling plays an essential role in the early stages of Myxococcus xanthus fruiting body development. Expression of the 452I gene, which is regulated at the level of RNA accumulation, depends on starvation and on A-signaling. To identify the cis-acting regulatory elements which allow gene 4521 to respond to the nutritional and A-factor signals, the 4521 transcription start site was mapped. The region just upstream of the start site showed sequence similarity to the sigma 54 family of promoters and to the developmentally regulated mbhA promoter of M. xanthus. A mutational analysis of this region established that the bases which were conserved between the sigma 54 consensus, mbhA, and 4521 promoters were also important for 4521 promoter activity. Changes which altered the spacing between two conserved regions centered around positions -14 and -24 abolished promoter activity. In contrast, mutations in a putative -10 region for a sigma 70-like promoter had little effect on expression of 4521. Despite their similar promoter regions, the regulation of the 4521 and mbhA genes was shown to differ with respect to timing of expression and requirement for a solid surface and extracellular signals. This suggests a model in which different activator proteins may be responsible for regulating expression of these two genes.
Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides
Authors: Foucaud C , Kunji E R , Hagting A , Richard J , Konings W N , Desmazeaud M , Poolman B
Published: November 2016

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.
Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus
Authors: Grewal S I , Han B , Johnstone K
Published: November 2016

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii.
Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities
Authors: Zakrzewska-Czerwińska J , Majka J , Schrempf H
Published: November 2016

Deletion analysis of a previously constructed minichromosome revealed that a stretch of DNA which is longer than 623 bp but shorter than 837 bp is required for autonomous replication of the Streptomyces lividans chromosome. Each of the dnaA and dnaN genes flanking the oriC region is individually transcribed from two promoters. Within the intergenic, nontranslatable region between the dnaA and dnaN genes, five main transcripts and several less abundant transcripts of various lengths as well as one of the promoters were identified. The introduction of additional DnaA boxes in S. lividans led to a significant increase in dnaA gene transcripts and to an enhanced level of the DnaA (73-kDa) protein. In summary, the data suggest that dnaA gene transcription is autoregulated and that initiation of the S. lividans chromosome is tightly controlled.
Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex
Authors: Haase J , Lurz R , Grahn A M , Bamford D H , Lanka E
Published: November 2016

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.
Bacillus subtilis gnt repressor mutants that diminish gluconate-binding ability
Authors: Yoshida K , Ohmori H , Miwa Y , Fujita Y
Published: November 2016

The Bacillus subtilis gnt operon is negatively regulated by GntR, which is antagonized by gluconate. Three GntR mutants with diminished gluconate-binding ability were obtained. Two were missense mutants (Met-209 to Ile and Ser-230 to Leu), whereas the third had a deletion of the C-terminal 23 amino acids. The mutant GntR proteins were unable to become properly detached from the gnt operator even in the presence of gluconate.