bluuebunny/crossref_metadata_2025 huggingface dataset:
VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid
Authors: Jones A L , Lai E M , Shirasu K , Kado C I
Published: November 2016

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.
The nucleoid protein H-NS facilitates chromosome DNA replication in Escherichia coli dnaA mutants
Authors: Katayama T , Takata M , Sekimizu K
Published: November 2016

Growth inhibition of the dnaA(Cs) mutant, which overinitiates chromosome replication, was shown to be dependent upon the nucleoid protein H-NS. [3H]thymine incorporation experiments indicated that the absence of H-NS inhibited overreplication by the dnaA(Cs) mutant. In addition, the temperature-sensitive phenotype of a dnaA46 mutant was enhanced by disruption of H-NS. These observations suggest that H-NS directly or indirectly facilitates the initiation of chromosome replication.
Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli
Authors: Fralick J A
Published: November 2016

A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype. That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion. These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.
Evidence that the Bacillus subtilis pyrimidine regulatory protein PyrR acts by binding to pyr mRNA at three sites in vivo
Authors: Lu Y , Switzer R L
Published: November 2016

The Bacillus subtilis pyr operon is regulated by a transcriptional attenuation mechanism that requires the PyrR regulatory protein. Multicopy plasmids that could be transcribed to yield segments of RNA from the attenuation regions of the pyr operon induced derepression of chromosomal pyr genes, whereas plasmids that could not yield pyr RNA did not. We conclude that pyr RNA acts by titrating the PyrR protein and preventing it from regulating pyr attenuation.
D-histidine utilization in Salmonella typhimurium is controlled by the leucine-responsive regulatory protein (Lrp)
Authors: Hecht K , Zhang S , Klopotowski T , Ames G F
Published: November 2016

A new class of D-histidine-utilizing mutants which carry mutations in the gene encoding the leucine-responsive regulatory protein (Lrp) has been identified in Salmonella typhimurium. The lrp mutations arise as suppressors of mutations in the genes encoding the histidine permease which drastically decrease the level of histidine transport activity. However, the suppressor effect is not exerted by elevating the level of the permease. Rather, the properties of the suppressor mutants are consistent with the notion that the parent permease mutants transport D-histidine at a low level and that in the suppressor mutants D-histidine is utilized effectively through elevated levels of racemization. The enzymatic activity of D-alanine dehydrogenase (Dad) is shown to be elevated in the suppressor mutants and is a possible pathway of D-histidine utilization. The suppressor mutations are located in the helix-turn-helix region of Lrp.
Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors
Authors: Schripsema J , de Rudder K E , van Vliet T B , Lankhorst P P , de Vroom E , Kijne J W , van Brussel A A
Published: November 2016

Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.
Cell wall sorting of lipoproteins in Staphylococcus aureus
Authors: Navarre W W , Daefler S , Schneewind O
Published: November 2016

Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.
Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis
Authors: Veenstra G J , Cremers F F , van Dijk H , Fleer A
Published: November 2016

Coagulase-negative staphylococci have emerged as important pathogens in infections associated with intravascular devices. Microbial adherence to biomaterial surfaces is a crucial step in the pathogenesis of these infections. Staphylococcal surface proteins (herein referred to as SSP-1 and SSP-2) are involved in the attachment of Staphylococcus epidermidis 354 to polystyrene. In the present study we show that the adhesin protrudes from the cell surface as a fimbria-like polymer. Furthermore, in vitro proteolytic cleavage of SSP-1 produces an SSP-2-like protein which coincides with a loss of adhesive function. SSP-1 expression is down-regulated in a phenotypical variant of S. epidermidis 354 whereas SSP-2 expression is not. These results could suggest that proteolytic cleavage is a key to the regulation of the adhesive state of S. epidermidis in vivo.
A Bacillus subtilis malate dehydrogenase gene
Authors: Jin S , De Jesús-Berríos M , Sonenshein A L
Published: November 2016

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.
Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi
Authors: Rosa P , Samuels D S , Hogan D , Stevenson B , Casjens S , Tilly K
Published: November 2016

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.
The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase
Authors: Siegele D A , Imlay K R , Imlay J A
Published: November 2016

The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions. surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase. Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E. coli. Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants. In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1. Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase. Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro. Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport.
Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation
Authors: Atrih A , Zöllner P , Allmaier G , Foster S J
Published: November 2016

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.
Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis
Authors: Perkins J B , Bower S , Howitt C L , Yocum R R , Pero J
Published: November 2016

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.
Role of the two-component signal transduction and the phosphoenolpyruvate: carbohydrate phosphotransferase systems in competence development of Haemophilus influenzae Rd
Authors: Gwinn M L , Yi D , Smith H O , Tomb J F
Published: November 2016

Haemophilus influenzae Rd becomes competent for transformation by nutritional downshift or transient anaerobic growth through a process that requires cyclic AMP receptor protein and adenylate cyclase. Insertion mutations in crr or ptsI of the phosphoenolpyruvate:carbohydrate phosphotransferase system lowered transformation frequencies, and the effect was reversed by the addition of cyclic AMP. However, insertions into H. influenzae homologs of two-component signal transduction genes had no effect on competence.
Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction
Authors: Carlson K , Kosturko L D , Nyström A C
Published: November 2016

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.
Determination of the structure of a novel glycolipid from Thermus aquaticus 15004 and demonstration that hydroxy fatty acids are amide linked to glycolipids in Thermus spp
Authors: Carreto L , Wait R , Nobre M F , da Costa M S
Published: November 2016

The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.
Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis
Authors: Pavelka M S , Jacobs W R
Published: November 2016

Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and the construction of the peptidoglycan of many species of bacteria, including mycobacteria. DAP is synthesized by bacteria as part of the aspartate amino acid family, which includes methionine, threonine, isoleucine, and lysine. Aspartokinase, the first enzyme in this pathway, is encoded by the ask gene in mycobacteria. Previous attempts to disrupt this gene in Mycobacterium smegmatis were unsuccessful, even when the cells were supplied with all the members of the aspartate family, suggesting that unlike other bacteria, mycobacteria may have an absolute requirement for this pathway even when growing in rich medium containing DAP. The purpose of this study was to determine if the ask gene and the aspartate pathway are essential to M. smegmatis. This study describes a test for gene essentiality in mycobacteria, utilizing a counterselectable marker (streptomycin resistance) in conjunction with a specially constructed merodiploid strain. We have used this system to show that the ask gene could not be disrupted in wild-type M. smegmatis, using selective rich medium supplemented with DAP unless there was an extra copy of ask provided elsewhere in the chromosome. Disruption of ask was also possible in a lysine auxotroph incapable of converting DAP to lysine. The ask mutant, mc21278 (ask1::aph), exhibits multiple auxotrophy (Met-, Thr-, DAP-, and Lys-) and is complemented by the ask gene. This is the first description of DAP auxotrophy in mycobacteria. The ask mutant lyses when deprived of DAP in culture, a characteristic which can be exploited for the reproducible preparation of protoplasts and mycobacterial extracts. The evidence presented here indicates that the aspartate pathway is essential to M. smegmatis and that DAP is the essential product of this pathway.
Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling
Authors: Givskov M , de Nys R , Manefield M , Gram L , Maximilien R , Eberl L , Molin S , Steinberg P D , Kjelleberg S
Published: November 2016

Acylated homoserine lactones (AHLs) play a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems.
Role of mismatch repair in the Escherichia coli UVM response
Authors: Murphy H S , Palejwala V A , Rahman M S , Dunman P M , Wang G , Humayun M Z
Published: November 2016

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.
Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication
Authors: Hobbs L J , Nossal N G
Published: November 2016

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.
Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter
Authors: Tang Y , Murakami K , Ishihama A , deHaseth P L
Published: November 2016

The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of pRM's ATR. We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM. However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions. The possible involvement of the alpha subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.
Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain
Authors: Odenbreit S , Wieland B , Haas R
Published: November 2016

The N-terminal sequence of a protein, originally described as an adhesin of Helicobacter pylori, was used in an oligonucleotide-based screening procedure of an H. pylori plasmid library in Escherichia coli. Five independent plasmid clones were isolated, all mapping to the same chromosomal region and encoding the H. pylori catalase. The gene, designated katA, comprises 1,518 nucleotides and encodes a putative protein of 505 amino acids with a predicted Mr of 58,599. A second open reading frame, orf2, encoding a putative 32,715-Da protein of unknown function, follows katA. The transcriptional start site of katA mRNA was determined, but no typical consensus promoter sequence was present. A potential binding site for the Fur protein is located upstream of katA. When introduced into the catalase-deficient E. coli double-mutant UM255, the cloned gene readily complemented E. coli for catalase activity. H. pylori KatA is highly homologous to catalases in both prokaryotes and eukaryotes, with the highest homology being shown to Bordetella pertussis (64.9%), Bacteroides fragilis (59.8%), and Haemophilus influenzae (57.9%) catalases. Transposon insertion mutants were generated in three independent H. pylori strains by TnMax5-mediated transposon shuttle mutagenesis. In contrast to the wild-type strains, no significant catalase-specific enzymatic activity could be detected in the mutant strains, consistent with the fact that no additional katA-homologous gene copies were found in the H. pylori chromosome. No significant difference between wild-type and mutant strains for binding to epithelial cells was apparent, suggesting that KatA is not involved in H. pylori adhesion. The cloning and genetic characterization of katA are essential steps for further investigation of the role of catalase in the defense of H. pylori against oxygen-dependent killing mechanisms by polymorphonuclear granulocytes, a process not well understood for this chronically persisting pathogen.
Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA
Authors: Baker K E , Ditullio K P , Neuhard J , Kelln R A
Published: November 2016

Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli. The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization. Further, succinate was found to compete with orotate for entry into the cell. The S. typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA+ plasmid. The DNA immediately upstream of the deduced -35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source. The E. coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.
dacD, an Escherichia coli gene encoding a novel penicillin-binding protein (PBP6b) with DD-carboxypeptidase activity
Authors: Baquero M R , Bouzon M , Quintela J C , Ayala J A , Moreno F
Published: November 2016

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [3H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.
Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated
Authors: Luka S , Patriarca E J , Riccio A , Iaccarino M , Defez R
Published: November 2016

Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragment was cloned and used as a hybridization probe for cloning of the R. etli sigA gene. Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors. The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides. Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase. On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors.
The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA
Authors: Baggio L , Morrison M
Published: November 2016

Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful.
Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange
Authors: Zientz E , Six S , Unden G
Published: November 2016

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.
Analysis of the region between amino acids 30 and 42 of intact UmuD by a monocysteine approach
Authors: Guzzo A , Lee M H , Oda K , Walker G C
Published: November 2016

On the basis of characterizations of a set of UmuD monocysteine derivatives, we had suggested that positions 24, 34, and 44 are closer to the intact UmuD homodimer interface than other positions tested (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994). Because this region of UmuD also appeared to be important for interactions with RecA, we followed up on our previous study by constructing a second set of monocysteine UmuD derivatives with single cysteine substitutions at positions 30 to 42. We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 showed deficiencies in in vivo and in vitro RecA-mediated cleavage as well as in UV mutagenesis, suggesting that the position 32 to 35 region may be important for RecA-mediated cleavage of UmuD. Interestingly, UmuD with monocysteine substitutions at residues 33 and 40 showed a reduction in UV mutagenesis while retaining the ability to be cleaved by RecA in vivo, suggesting a deficiency in the subsequent role of the UmuD' derivatives in mutagenesis. All of the UmuD monocysteine derivatives in the position 30 to 42 series purified indistinguishably from the wild-type protein. The observations that purified proteins of the UmuD derivatives RC37 and IC38 could be disulfide cross-linked quantitatively upon addition of iodine and yet were poorly modified with iodoacetate led us to suggest that the pairs of residues at positions 37 and 38 are extremely close to the UmuD2 homodimer interface. These observations indicate that the structure of the UmuD2 homodimer in solution is very different from the crystal structure of the UmuD'2 homodimer reported by Peat et al. (T. S. Peat, E. G. Frank, J. P. McDonald, A. S. Levine, R. Woodgate, and W. A. Hendrickson, Nature [London] 380:727-730, 1996).
Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae
Authors: Clairmont C A , Sweasy J B
Published: November 2016

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.
Mutations affecting mRNA processing and fimbrial biogenesis in the Escherichia coli pap operon
Authors: Nilsson P , Naureckiene S , Uhlin B E
Published: November 2016

The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in "crew-cut" bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.
Soft metal thiol chemistry is not involved in the transport of arsenite by the Ars pump
Authors: Chen Y , Dey S , Rosen B P
Published: November 2016

The single cysteine in the ArsB protein subunit of the arsenite resistance pump was changed to serine and alanine residues. Resistance in cells expressing the two mutant arsB genes was the same as in the wild type, and the serine substitution had no effect on the arsenite transport properties. These results eliminate possible thiol chemistry in translocation. Thus, the pump uses soft metal chemistry for metalloactivation and nonmetal chemistry for oxyanion transport.
dTMP biosynthesis in Archaea
Authors: Nyce G W , White R H
Published: November 2016

The biosynthesis of dTMP has been studied in cell extracts of two different members of the domain Archaea, Methanosarcina thermophila and Sulfolobus solfataricus. In M. thermophila, the dTMP was formed from dUMP and [methylene-2H2]-5,10-methylenetetrahydrosarcinapterin generated in situ from added [methylene-2H2] formaldehyde and the tetrahydrosarcinapterin present in the cell extract. In S. solfataricus, the 5,10-methyl-enetetrahydro derivative of a synthetic fragment of sulfopterin, the modified folate present in these cells, served as the C1 donor. These data indicate that the Archaea thymidylate synthases carry out the same basic reaction which occurs in other organisms but use the 5,10-methylenetetrahydro derivatives of modified folates as C1 donors.
The repetitive element Rep MP 1 of Mycoplasma pneumoniae exists as a core element within a larger, variable repetitive mosaic
Authors: Forsyth M H , Geary S J
Published: November 2016

The repetitive element Rep MP 1 has been previously described as a 300-bp element present within the chromosome of Mycoplasma pneumoniae in at least 10 copies. Sequence found flanking Rep MP 1 in lambda clone 5B52 has been demonstrated to exist in multiple copies within the genome of M. pneumoniae. A HindIII M. pneumoniae fragment containing the lambda MP 5B52 insert and its flanking sequences was cloned into pBluescript. Sequence comparisons of this clone, designated pMPH 624-20, with nucleotide database entries showed that in addition to the 300-bp Rep MP 1 element, flanking sequence blocks were conserved within several other cloned sequences. These short repeated sequences, approximately 80, 56, and 71 nucleotides long, are termed sRep A, sRep B, and sRep C, respectively. These sRep regions were used as probes in the selection of cloned sequences containing these repetitive flanking regions. Comparison of these sequences demonstrated that the 300-bp Rep MP 1 elements is actually the core element within a larger, variable repetitive mosaic. The repetitive regions surrounding the core element are found in various combinations, arrangements, and distances from the core in a mosaic pattern. These newly identified portions of the mosaic do not exist independently of the core element. The core appears to be the only invariant portion of this repetitive mosaic.
Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin
Authors: Renauld-Mongénie G , Cornette J , Mielcarek N , Menozzi F D , Locht C
Published: November 2016

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.