bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants
Authors: Gilmore M S , Segarra R A , Booth M C , Bogie C P , Hall L R , Clewell D B
Published: November 2016

Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.
Characterization of a glucose transport system in Vibrio parahaemolyticus
Authors: Sarker R I , Ogawa W , Tsuda M , Tanaka S , Tsuchiya T
Published: November 2016

Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence.
Sequence of ornithine decarboxylase from Lactobacillus sp. strain 30a
Authors: Hackert M L , Carroll D W , Davidson L , Kim S O , Momany C , Vaaler G L , Zhang L
Published: November 2016

A gene encoding biodegradative ornithine decarboxylase from Lactobacillus sp. strain 30a was isolated from a genomic DNA library and sequenced. Primer extension analysis revealed two transcription initiation sites. The deduced amino acid sequence is compared with the amino acid sequences of five previously reported bacterial decarboxylases, and conserved pyridoxal phosphate motif residues are identified.
Transcriptional and translational analysis of ferredoxin and flavodoxin under iron and nitrogen stress in Anabaena sp. strain PCC 7120
Authors: Razquin P , Schmitz S , Fillat M F , Peleato M L , Böhme H
Published: November 2016

In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nifH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdxH gene product were not detectable.
The mutation DGT1-1 decreases glucose transport and alleviates carbon catabolite repression in Saccharomyces cerevisiae
Authors: Gamo F J , Lafuente M J , Gancedo C
Published: November 2016

Glucose in ethanol-glycerol mixtures inhibits growth of Saccharomyces cerevisiae mutants lacking phosphoglycerate mutase. A suppressor mutation that relieved glucose inhibition was isolated. This mutation, DGT1-1 (decreasing glucose transport), was dominant and produced pleiotropic effects even in an otherwise wild-type background. Growth of the DGT1-1 mutant in glucose was dependent on respiration, and no ethanol was detected in the medium within 7 h of glucose addition. When grown on glucose, the mutant had a reduced glucose uptake and both the low- and high-affinity transport systems were affected. In galactose-grown cells, only the high-affinity glucose transport system was detected. This system had similar kinetic characteristics in the wild type and in the mutant. Catabolite repression of several enzymes was absent in the mutant during growth in glucose but not during growth in galactose. In contrast with the wild type, the mutant grown in glucose had high transcription of the glucose transporter gene SNF3 and no transcription of HXT1 and HXT3. Expression of multicopy plasmids carrying the HXT1, HXT2, or HXT3 gene allowed partial recovery of both fermentative capacity and catabolite repression in the mutant. The results suggest that DGT1 codes for a regulator of the expression of glucose transport genes. They also suggest that glucose flux might determine the levels of molecules implicated as signals in catbolite repression.
The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family
Authors: West S E , Sample A K , Runyen-Janecky L J
Published: November 2016

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.
An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols
Authors: Pavel H , Forsman M , Shingler V
Published: November 2016

The pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 carries the dmp system, which comprises the divergently transcribed dmpR gene and the dmp operon coding for the catabolic enzymes required for growth on (methyl)phenols. The constitutively expressed DmpR transcriptional activator positively controls the expression of the RpoN-dependent dmp operon promoter in the presence of the aromatic effector in the growth medium. However, the magnitude of the transcriptional response differs depending on the position of the methyl substituent on the aromatic ring. Experiments involving an elevated copy number of the dmp system demonstrate that growth on para-substituted methylphenols is limited by the level of the catabolic enzymes. An effector specificity mutant of DmpR, DmpR-E135K, that responded to the presence of 4-ethylphenol, a noneffector of the wild-type protein, was isolated by genetic selection. The single point mutation in DmpR-E135K, which results in a Glu-to-Lys change in residue 135, also results in a regulator with enhanced recognition of para-substituted methylphenols. The DmpR-E135K mutation, when introduced into the wild-type strain, confers enhanced utilization of the para-substituted methylphenols. These experiments demonstrate that the aromatic effector activation of wild-type DmpR by the para-substituted methylphenols is a major factor limiting the catabolism of these compounds.
Multiple N-acyl-L-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri
Authors: Kuo A , Blough N V , Dunlap P V
Published: November 2016

In Vibrio fischeri, the synthesis of N-3-oxohexanoyl-L-homoserine lactone, the autoinducer for population density-responsive induction of the luminescence operon (the lux operon, luxICDABEG), is dependent on the autoinducer synthase gene luxI. Gene replacement mutants of V. fischeri defective in luxI, which had been expected to produce no autoinducer, nonetheless exhibited lux operon transcriptional activation. Mutants released into the medium a compound that, like N-3-oxohexanoyl-L-homoserine lactone, activated expression of the lux system in a dose-dependent manner and was both extractable with ethyl acetate and labile to base. The luxI-independent compound, also like N-3-oxohexanoyl-L-homoserine lactone, was produced by V. fischeri cells in a regulated, population density-responsive manner and required the transcriptional activator LuxR for activity in the lux system. The luxI-independent compound was identified as N-octanoyl-L-homoserine lactone by coelution with the synthetic compound in reversed-phase high-pressure liquid chromatography, by derivatization treatment with 2,4-dinitrophenylhydrazine, by mass spectrometry, and by nuclear magnetic resonance spectroscopy. A locus, ain, necessary and sufficient for Escherichia coli to synthesize N-octanoyl-L-homoserine lactone was cloned from the V. fischeri genome and found to be distinct from luxI by restriction mapping and Southern hybridization. N-Octanoyl-L-homoserine lactone and ain constitute a second, novel autoinduction system for population density-responsive signalling and regulation of lux gene expression, and possibly other genes, in V. fischeri. A third V. fischeri autoinducer, N-hexanoyl-L-homoserine lactone, dependent on luxI for its synthesis, was also identified. The presence of multiple chemically and genetically distinct but cross-acting autoinduction systems in V. fischeri indicates unexpected complexity for autoinduction as a regulatory mechanism in this bacterium.
The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases
Authors: Ullrich M , Bender C L
Published: November 2016

Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases. Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(II) enzymes which catalyze oxidative cyclizations. The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature.
Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6
Authors: Iwai A , Ito H , Mizuno T , Mori H , Matsui H , Honma M , Okada G , Chiba S
Published: November 2016

The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself.
A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance
Authors: Aagaard C , Phan H , Trevisanato S , Garrett R A
Published: November 2016

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.
Organization and evolution of naphthalene catabolic pathways: sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NAH7 plasmid
Authors: Eaton R W
Published: November 2016

The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmid NAH7 was determined. This segment contains three large open reading frames designated nahQ', nahE, and nahD. The first of these is the 3' end of an open reading frame that has no known function, the second (993 bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), and the third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031). This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segment from the dox (dibenzothiophene oxidation) operon of Pseudomonas sp. strain C18, which encodes a pathway analogous to that encoded by NAH7. However, 84 bp downstream from nahD, the last gene in the nah operon, this homology ends. This 84-bp sequence at the downstream end of nah and dox homology has 76% homology to a sequence that occurs just upstream of the nah promoter in NAH7. These directly repeated 84-bp sequences thus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.
Transcriptional control of dacB, which encodes a major sporulation-specific penicillin-binding protein
Authors: Simpson E B , Hancock T W , Buchanan C E
Published: November 2016

Sporulation-specific sigma factor E (sigma E) of Bacillus subtilis is both necessary and sufficient for transcription of the dacB gene, which encodes penicillin-binding protein 5*. Evidence in support of this conclusion was obtained by primer extension analysis of dacB transcripts and the induction of active sigma E with subsequent synthesis of PBP 5* in vegetative cells.
Division inhibition gene dicF of Escherichia coli reveals a widespread group of prophage sequences in bacterial genomes
Authors: Faubladier M , Bouché J P
Published: November 2016

The genomes of various eubacteria were analyzed by Southern blot hybridization to detect sequences related to the segment of the defective lambdoid prophage Kim which encodes DicF RNA, an antisense inhibitor of cell division gene ftsZ in Escherichia coli K-12. Among the homologous sequences found, one fragment from E. coli B, similar to a piece of Rac prophage, and two fragments from Shigella flexneri were cloned and sequenced. dicF-like elements similar to transcriptional terminators were found in each sequence, but unlike dicF these had no effect on division in E. coli K-12. Like dicF, these sequences are flanked by secondary structures which form potential sites for RNase III recognition. Coding sequences located upstream from the dicF-like feature in E. coli B are related to gene sieB of bacteriophage lambda, while sequences downstream of the S. flexneri elements are similar to the immunity region of satellite bacteriophage P4. Under hybridization conditions in which only strong sequence homologies were detected in E. coli B and S. flexneri, the genomes of a large variety of microorganisms, including some gram-positive bacteria, hybridized to the dicF probe. Our results suggest that dicF and its flanking regions are markers of a widespread family of prophage-like elements of different origins.
Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids
Authors: Prüss B M , Nelms J M , Park C , Wolfe A J
Published: November 2016

We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1. Cells carrying any of these three mutations exhibited identical phenotypes. Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium. As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate. The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate. We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth. During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate. As they began the transition to stationary phase, both cell types consumed L-tryptophan. Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.
Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins
Authors: Belisle J T , Brandt M E , Radolf J D , Norgard M V
Published: November 2016

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.
Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816
Authors: Yang Y , Chen R F , Shiaris M P
Published: November 2016

A modified cloning procedure was used to obtain large DNA insertions (20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycyclic aromatic hydrocarbon (PAH) transformation activity in Escherichia coli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb in size) were constructed from the initial clones. Naphthalene, fluorene, and phenanthrene transformations were investigated in these eight NCIB 9816 clones by a simple agar plate assay method, which was developed to detect and identify potential PAH metabolites. Results indicated that the necessary genes encoding the initial ring fission of the three PAHs in E. coli cells are located in an 8.5-kb EcoRI-XhoI portion, but the lower-pathway genes are not present in a 38-kb neighborhood region. These NCIB 9816 clones could transform naphthalene and phenanthrene to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. With the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluorenone, and two unidentified compounds. Genetic similarity between the NAH7 upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic similarity, the abilities of the two clusters to transform multiple PAHs were different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detected, whereas the NCIB 9816 clones produced metabolites from all three PAHs.
De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli
Authors: Weiss B , Wang L
Published: November 2016

dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP.
Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12
Authors: Laird M W , Kloser A W , Misra R
Published: November 2016

Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.
Induction of Escherichia coli hydroperoxidase I by acetate and other weak acids
Authors: Mukhopadhyay S , Schellhorn H E
Published: November 2016

Escherichia coli produces two independently regulated hydroperoxidases (catalases) that protect the cell from toxic concentrations of hydrogen peroxide. Hydroperoxidase I (HPI) is induced by hydrogen peroxide in an OxyR-dependent manner, while hydroperoxidase II (HPII) synthesis is regulated by an alternative sigma factor called RpoS (KatF). The activities of both hydroperoxidases increase as exponentially growing cells enter stationary phase. In this study, we examined the growth phase-dependent expression of HPI. Treatment of early-exponential-phase cells with spent culture supernatant resulted in induction of HPI synthesis. Extracellular levels of hydrogen peroxide, accumulating in the culture supernatant during late exponential phase, were found to be lower than the concentrations normally required to induce OxyR-dependent synthesis of HPI. This finding suggested that factors other than hydrogen peroxide may play a role in HPI expression. Weak acids such as acetate, which accumulate in culture supernatant and have been implicated in the regulation of HPII, caused a sixfold increase in HPI expression. Increases in HPI synthesis, mediated by weak acids and spent culture fluid supernatant, could be prevented by chloramphenicol, indicating that de novo protein synthesis is required for induction. Expression studies using a plasmid-borne lacZ transcriptional fusion to katG, the structural gene for HPI, indicated that growth phase-dependent regulation of HPI occurs primarily at the level of transcription and is dependent on RpoS. These results suggest that there may be a common regulatory mechanism of HPI and HPII expression in addition to previously described independent control mechanisms.
Alanine-scanning mutagenesis reveals residues involved in binding of pap-3-encoded pili
Authors: Klann A G , Hull R A , Palzkill T , Hull S I
Published: November 2016

In order to identify functionally important residues in the pap-3-encoded adhesin, oligonucleotide-directed mutagenesis was used to substitute alanine(s) at sixteen positions in the adhesion. These alanine substitutions span nearly every domain and hydrophilic peak of the protein. The effects of these substitutions were measured by evaluating the patterns of hemagglutination exhibited by the mutant strains. It was found that strains harboring alanine substitutions at positions 88 and 89, 128 to 130, and 316 had lost the capacity to hemagglutinate. The presence of the mutated adhesin in the assembled pilus structure was verified by the reactions of purified pili with an adhesin-specific monoclonal antibody in an enzyme-linked immunosorbent assay and with a polyclonal antibody in Western blotting (immunoblotting). Alanine substitutions at positions 68, 110 and 111, and 143 to 146 had no effect upon hemagglutination, whereas substitutions at positions 203 and 204 and position 291 resulted in diminished binding. Thus, the residues necessary for hemagglutination are scattered throughout the adhesin in both the amino and carboxy regions. Delineation of these residues may prove useful in designing a preventive treatment that would cross-react with the essential binding residues from the adhesins of several different pyelonephritis-causing strains.
Role of the TonB amino terminus in energy transduction between membranes
Authors: Jaskula J C , Letain T E , Roof S K , Skare J T , Postle K
Published: November 2016

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.
Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence
Authors: Rajakumar K , Jost B H , Sasakawa C , Okada N , Yoshikawa M , Adler B
Published: November 2016

N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers and was negative in the Serény test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNA was sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of this region revealed four genes, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the biosynthesis of activated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normal levels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membrane fractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in the monolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of the characteristic F-actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing deposition of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.
Molecular characterization of the Erwinia chrysanthemi kdgK gene involved in pectin degradation
Authors: Hugouvieux-Cotte-Pattat N , Nasser W , Robert-Baudouy J
Published: November 2016

The pathways of pectin and galacturonate catabolism in Erwinia chrysanthemi converge to form a common intermediate, 2-keto-3-deoxygluconate (KDG), which is phosphorylated by KDG kinase encoded by the kdgK gene. We cloned the kdgK gene of E. chrysanthemi 3937 by complementing an Escherichia coli kdgK mutation, using an RP4-derivative plasmid. One of the kdgK R-prime plasmids harbored a DNA insert of about 80 kb and carried the uxuA and uxuB genes involved in glucuronate catabolism and the celY gene coding for an E. chrysanthemi cellulase. The kdgK and celY genes were precisely located on this plasmid, and their respective transcriptional directions were determined. The nucleotide sequence of the kdgK region indicated that the kdgK reading frame is 981 bases long, corresponding to a protein of 329 amino acids with a molecular mass of 36,377 Da. Analysis of the deduced primary amino acid sequence showed that this enzyme is a new member of the PfkB family of carbohydrate kinases. Expression of kdgK is controlled by a negative regulatory gene, kdgR, which represses all the steps of pectin degradation. Near the putative promoter of the kdgK gene, we identified a putative KdgR-binding site and demonstrated that the KdgR protein specifically binds in vitro to this DNA region. The KdgR-KDG couple directly mediates the phenomenon of repression or induction. The KDG kinase, by limiting the intracellular inducer concentration, appears to be a key enzyme in induction of the whole catabolic pathway.
Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants
Authors: Zhao N , Oh W , Trybul D , Thrasher K S , Kingsbury T J , Larson T J
Published: November 2016

The glp operons of Escherichia coli are negatively controlled by the glp repressor. Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding. Substitutions at other positions had a severe to no effect on repressor binding, depending on the base substitution. The results obtained indicate that the repressor binds with highest affinity to operators with the half-site WATKYTCGWW, where W is A or T, K is G or T, and Y is C or T. Strong cooperative binding of the repressor to tandem operators was demonstrated in vivo. Cooperativity was maximal when two 20-bp operators were directly repeated or when 2 bp separated the two operators. Cooperativity decreased with the deletion of 2 bp or the addition of 4 bp between the individual operators. Cooperativity was eliminated with a 6-bp insertion between the operators.
Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82
Authors: Takizawa N , Kaida N , Torigoe S , Moritani T , Sawada T , Satoh S , Kiyohara H
Published: November 2016

Naphthalene and phenanthrene are transformed by enzymes encoded by the pah gene cluster of Pseudomonas putida OUS82. The pahA and pahB genes, which encode the first and second enzymes, dioxygenase and cis-dihydrodiol dehydrogenase, respectively, were identified and sequenced. The DNA sequences showed that pahA and pahB were clustered and that pahA consisted of four cistrons, pahAa, pahAb, pahAc, and pahAd, which encode ferredoxin reductase, ferredoxin, and two subunits of the iron-sulfur protein, respectively.
Analysis of the lipid moiety of lipopolysaccharide from Rhizobium tropici CIAT899: identification of 29-hydroxytriacontanoic acid
Authors: Gil-Serrano A M , González-Jiménez I , Tejero-Mateo P , Megías M , Romero-Vazquez M J
Published: November 2016

The lipid moieties of two lipid A's isolated from the phenolic and aqueous fractions of lipopolysaccharide from Rhizobium tropici CIAT899 have been studied. Several 3-hydroxy fatty acids and two long-chain hydroxy fatty acids, 27-hydroxyoctacosanoic acid, and 29-hydroxytriacontanoic acid were identified; the ratios of these acids are the same in both lipid A's. These results can be used for chemotaxonomic purposes.
Processing and surface presentation of the Mycoplasma hyorhinis variant lipoprotein VlpC
Authors: Cleavinger C M , Kim M F , Wise K S
Published: November 2016

The variant surface lipoprotein VlpC of Mycoplasma hyorhinis was shown to be processed by cleavage of a characteristic prokaryotic prolipoprotein signal peptide. In addition, a vlpC::phoA fusion protein expressed and translocated in Escherichia coli was recognized by surface-binding monoclonal antibodies, which identified the characteristic region II of Vlps, containing divergent external sequences proximal to the membrane, as an exposed portion of these surface proteins subject to immune recognition and selection.
Cloning and high-level expression of the glutathione-independent formaldehyde dehydrogenase gene from Pseudomonas putida
Authors: Ito K , Takahashi M , Yoshimoto T , Tsuru D
Published: November 2016

A DNA fragment of 485 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified formaldehyde dehydrogenase (EC 1.2.1.46) from Pseudomonas putida and on that of a cyanogen bromide-derived peptide. With this product as a probe, a gene coding for formaldehyde dehydrogenase (fdhA) in P. putida chromosomal DNA was cloned in Escherichia coli DH5 alpha. Sequencing analysis revealed that the fdhA gene contained 1,197-bp open reading frame, encoding a protein composed of 399 amino acid residues whose calculated molecular weight was 42,082. The transformant of E. coli DH5 alpha harboring the hybrid plasmid, pFDHK3DN71, showed about 50-fold-higher formaldehyde dehydrogenase activity than P. putida. The predicted amino acid sequence contained several features characteristic of the zinc-containing medium-chain alcohol dehydrogenase (ADH) family. Most of the glycine residues strictly conserved within the family, including a Gly-Xaa-Gly-Xaa-Xaa-Gly pattern in the coenzyme binding domain, were well conserved in this enzyme. Regions around both the catalytic and the structural zinc atoms were also conserved. Analyses of structural and enzymatic characteristics indicated that P. putida FDH belongs to the medium-chain ADH family, with mixed properties of mammalian class I and III ADHs.
Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1
Authors: Becher B , Müller V
Published: November 2016

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.
UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme
Authors: Weissborn A C , Liu Q , Rumley M K , Kennedy E P
Published: November 2016

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.
The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium
Authors: Bergman T , Erickson K , Galyov E , Persson C , Wolf-Watz H
Published: November 2016

Virulent bacteria of the genus Yersinia secrete a number of virulence determinants called Yops. These proteins lack typical signal sequences and are not posttranslationally processed. Two gene loci have been identified as being involved in the specific Yop secretion system (G. Cornelis, p. 231-265, In C. E. Hormache, C. W. Penn, and C. J. Smythe, ed., Molecular Biology of Bacterial Infection, 1992; S. C. Straley, G. V. Plano, E. Skrzypek, P. L. Haddix, and K. A. Fields, Mol. Microbiol. 8:1005-1010, 1993). Here, we have shown that the lcrB/virB locus (yscN to yscU) encodes gene products essential for Yop secretion. As in previously described secretion apparatus mutants, expression of the Yop proteins was decreased in the yscN/U mutants. An lcrH yscR double mutant expressed the Yops at an increased level but did not secrete Yops into the culture supernatant. The block in Yop expression of the ysc mutants was also circumvented by overexpression of the activator LcrF in trans. Although the Yops were expressed in elevated amounts, the Yops were still not exported. This analysis showed that the ysc mutants were unable to secrete Yops and that they were also affected in the negative Ca(2+)-regulated loop. The yscN/U genes showed remarkably high homology to the spa genes of Shigella flexneri and Salmonella typhimurium with respect to both individual genes and gene organization. These findings indicate that the genes originated from a common ancestor.
Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis
Authors: Gocht M , Marahiel M A
Published: November 2016

The D-phenylalanine-activating enzyme tyrocidine synthetase I (TycA) from Bacillus brevis ATCC 8185 was overexpressed in Escherichia coli, purified to homogeneity, and assayed for ATP-PPi exchange and covalent binding of phenylalanine by the thiotemplate mechanism. Amino acid exchanges in four different cores of TycA created by site-directed mutagenesis revealed the amino acid residues involved in aminoacyladenylate formation and in covalent thioester formation. Mutations in the putative ATP-binding site SGTTGKPKG caused a decreased phenylalanine-dependent ATP-PPi exchange activity to 10% of the wild-type level for a Lys-186-to-Arg substitution and an almost complete loss of activity (< 1%) for a Lys-186-to-Thr exchange. Alteration of Asp-401 to Asn in the ATPase motif TGDL of TycA decreased the phenylalanine-dependent ATP-PPi exchange activity to 75% of wild type, while an Asp-401-to-Ser mutation decreased the activity to 10% of the wild-type level. Replacement of Ser-562 in the putative thioester-binding motif LGGDSI to Ala or Gly caused a reduction in trichloroacetic acid-precipitable TycA-[14C]phenylalanine complex to one-third of the wild-type level. However, no cleavable [14C]phenylalanine could be detected after treatment with performic acid, indicating that the resulting mutant was unable to form thioester with phenylalanine. In E. coli, TycA was labeled with beta-[3H]alanine, a precursor of 4'-phosphopantetheine, indicating that TycA is modified with a beta-alanine-containing cofactor.
Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor
Authors: Kwakman J H , Postma P W
Published: November 2016

A glucose kinase (glkA) mutant of Streptomyces coelicolor A3(2) M145 was selected by the ability to grow in the presence of the nonmetabolizable glucose analog 2-deoxyglucose. In this glkA mutant, carbon catabolite repression of glycerol kinase and agarase was relieved on several carbon sources tested, even though most of these carbon sources are not metabolized via glucose kinase. This suggests that catabolite repression is not regulated by the flux through glucose kinase and that the protein itself has a regulatory role in carbon catabolite repression. A 10-fold overproduction of glucose kinase also results in relief of catabolite repression, suggesting that excess glucose kinase can titrate the repressing signal away. This could be achieved directly by competition of excess glucose kinase with its repressing form for binding sites on DNA promoter regions or indirectly by competition for binding of another regulatory protein.
5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA
Authors: Chen W , Russell C S , Murooka Y , Cosloy S D
Published: November 2016

hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned. We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs. Overexpression of hemM alone did not produce ALA. More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone. We conclude that hemA alone is required for ALA synthesis but hemA and hemM are required for maximal ALA synthesis.