bluuebunny/crossref_metadata_2025 huggingface dataset:
MotY, a component of the sodium-type flagellar motor
Authors: McCarter L L
Published: November 2016

Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential. Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined. A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain. Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped. Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This size correlated well with the predicted molecular mass of 33,385 Da. Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility. Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E. coli rnt locus. Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins. To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein. This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.
Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12
Authors: Moeck G S , Bazzaz B S , Gras M F , Ravi T S , Ratcliffe M J , Coulton J W
Published: November 2016

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.
Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii
Authors: Orgambide G G , Philip-Hollingsworth S , Hollingsworth R I , Dazzo F B
Published: November 2016

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.
The pacL gene of Synechococcus sp. strain PCC 7942 encodes a Ca(2+)-transporting ATPase
Authors: Berkelman T , Garret-Engele P , Hoffman N E
Published: November 2016

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.
Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response
Authors: Flärdh K , Kjelleberg S
Published: November 2016

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.
Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori
Authors: Kostrzynska M , O'Toole P W , Taylor D E , Trust T J
Published: November 2016

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.
Mutations in the -10 TATAAT sequence of the gyrA promoter affect both promoter strength and sensitivity to DNA supercoiling
Authors: Straney R , Krah R , Menzel R
Published: November 2016

Transcription of the gyrA and gyrB genes, which encode the subunits of DNA gyrase, increases in response to DNA relaxation. Previous studies have shown that a small segment of DNA extending from the -10 consensus hexamer to the start of transcription encodes the sequence determinants for this response. In this study, we examined the role of the -10 region in relaxation-stimulated transcription (RST). A synthetic derivative of the gyrA promoter was designed to allow the modular replacement of the -10 region, and mixed-oligonucleotide mutagenesis was used to obtain a collection of promoter mutants. Most substitutions result in a reduction of the promoter's RST response, and some mutations abolish it altogether. We also note that a variety of promoter changes can increase basal expression twofold above that seen for the gyrA promoter, despite sequences changes (up to three bases) which diverge from the consensus TATAAT of the wild-type gyrA hexamer. The wild-type gyrA promoter, however, is the strongest promoter in this collection on a relaxed DNA template and appears to be repressed on a supercoiled template in vivo. Our results are consistent with a mechanism for RST that involves a step in transcription initiation.
A new gene involved in stationary-phase survival located at 59 minutes on the Escherichia coli chromosome
Authors: Li C , Ichikawa J K , Ravetto J J , Kuo H C , Fu J C , Clarke S
Published: November 2016

We determined the DNA sequence of a 2,232-bp region immediately upstream of the pcm gene at 59 min on the Escherichia coli chromosome that encodes an L-isoaspartyl protein methyltransferase with an important role in stationary-phase survival. Two open reading frames of 477 and 1,524 bp were found oriented in the same direction as that of the pcm gene. The latter open reading frame overlapped the 5' end of the pcm gene by 4 bp. Coupled in vitro transcription-translation analysis of DNA containing the 1,524-bp open reading frame directly demonstrated the production of a 37,000-Da polypeptide corresponding to a RNA species generated from a promoter within the open reading frame. The deduced amino acid sequence showed no similarity to known protein sequences. To test the function of this gene product, we constructed a mutant strain in which a kanamycin resistance element was inserted at a BstEII site in the middle of its coding region in an orientation that does not result in reduction of Pcm methyltransferase activity. These cells were found to survive poorly in stationary phase, at elevated temperatures, and in high-salt media compared with parent cells containing the intact gene, and we thus designate this gene surE (survival). surE appears to be the first gene of a bicistronic operon also containing the pcm gene. The phenotypes of mutations in either gene are very similar and indicate that both gene products are important for the viability of E. coli cells under stressful conditions.
Relationship between ftsZ gene expression and chromosome replication in Escherichia coli
Authors: Zhou P , Helmstetter C E
Published: November 2016

Transcriptional levels within the ftsQAZ region of the Escherichia coli chromosome were correlated with chromosome replication and the division cycle. The transcripts were measured either in synchronous cultures generated by the baby machine technique or in dnaC2(Ts) mutants that had been aligned for initiation of chromosome replication by temperature shifts. Transcription within the ftsZ reading frame was found to fluctuate during the cell cycle, with maximal levels about midcycle and a minimum level at division, in cells growing with a doubling time of 24 min at 37 degrees C. Examination of transcription in dnaC(Ts) mutants aligned for chromosome replication indicated that the periodicity was due to a reduction in transcripts coincident with replication of the ftsQAZ region. Transcription originating upstream of the ftsA gene exhibited the periodicity and accounted for a significant proportion of the transcripts entering ftsZ. The most obvious interpretation of the data is that replication of the region transiently inhibits transcription, but alternative explanations have not been ruled out. However, no other relationship between transcription and either replication or division was detected.
The min locus, which confers topological specificity to cell division, is not involved in its coupling with nucleoid separation
Authors: Dassain M , Bouché J P
Published: November 2016

In Escherichia coli, nucleoid separation and cell constriction remain tightly linked when division is retarded by altering the level of synthesis of the protein FtsZ. In this study, we have examined the role of the min locus, which is responsible for the inactivation of polar division sites, in the partition-septation coupling mechanism. We conclude that the coupling persists in a delta min strain and that its timing relative to replication remains dependent on the level of FtsZ synthesis. We suggest that the retarded nucleoid segregation observed in min mutants is the result of this coupling in cells with a perturbed pattern of nonpolar divisions.
Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses
Authors: Hurek T , Reinhold-Hurek B , Van Montagu M , Kellenberger E
Published: November 2016

The invasive properties of Azoarcus sp. strain BH72, an endorhizospheric isolate of Kallar grass, on gnotobiotically grown seedlings of Oryza sativa IR36 and Leptochloa fusca (L.) Kunth were studied. Additionally, Azoarcus spp. were localized in roots of field-grown Kallar grass. To facilitate localization and to assure identity of bacteria, genetically engineered microorganisms expressing beta-glucuronidase were also used as inocula. beta-Glucuronidase staining indicated that the apical region of the root behind the meristem was the most intensively colonized. Light and electron microscopy showed that strain BH72 penetrated the rhizoplane preferentially in the zones of elongation and differentiation and colonized the root interior inter- and intracellularly. In addition to the root cortex, stelar tissue was also colonized; bacteria were found in the xylem. No evidence was obtained that Azoarcus spp. could reside in living plant cells; rather, plant cells were apparently destroyed after bacteria had penetrated the cell wall. A common pathogenicity test on tobacco leaves provided no evidence that representative strains of Azoarcus spp. are phytopathogenic. Compared with the control, inoculation with strain BH72 significantly promoted growth of rice seedlings. This effect was reversed when the plant medium was supplemented with malate (0.2 g/liter). N2 fixation was apparently not involved, because the same response was obtained with a nifK mutant of strain BH72, which has a Nif- phenotype. Also, Western blot (immunoblot) analysis of protein extracts from rice seedlings gave no indication that nitrogenase was present. PCR and Western immunoblotting, using primers specific for eubacteria and antibodies recognizing type-specific antigens, respectively, indicated that strain BH72 could colonize rice plants systemically, probably mediated by longitudinal spreading through vessels.
The nature of the traK4 mutation in the F sex factor of Escherichia coli
Authors: Penfold S S , Usher K , Frost L S
Published: November 2016

The sequence of traK gene of the F sex factor of Escherichia coli is presented; the traK gene product is predicted to be a protein of 25,627 Da with a signal sequence of 21 amino acids to give a mature protein of 23,307 Da. The traK4 mutation is an extremely polar mutation in the F plasmid that affects F pilus synthesis and plasmid transfer. traK genes carrying the traK4 mutation and a nonpolar mutation traK105 were cloned, sequenced, and identified as an amber nonsense and a frameshift mutation, respectively. The traK4 mutation occurred within one predicted rho-dependent transcription termination element (TTE) and immediately upstream of another, while the traK105 mutation occurred after the two potential TTEs within the traK gene. S1 nuclease protection analysis and Northern (RNA) blot analysis were used to confirm that the traK4 mutation, but not the traK105 mutation, caused premature termination of transcription. Computer analysis of the F transfer region suggested the presence of TTE motifs at regular intervals throughout the 33.4-kb sequence.
Expression of the Prevotella loescheii adhesin gene (plaA) is mediated by a programmed frameshifting hop
Authors: Manch-Citron J N , London J
Published: November 2016

The 2.4-kb plaA gene, which encodes a Prevotella loescheii galactoside-specific adhesin, contains a programmed frameshifting hop. The frameshift region consists of two UAA termination codons, two repeats of four identical bases between the terminators, and a stem-loop structure that has the potential to form a pseudoknot located downstream from the second UAA. The stem-loop and pseudoknot are features found in a number of retroviruses where frameshifting is a more common occurrence. The terminators, sequence repeats, and secondary structures were identified in both the P. loescheii plaA gene and the mRNA transcript. An in-frame fusion of the entire plaA frameshift region between codons 9 and 10 of the lacZ gene permitted relatively efficient expression (4 to 25% of that of the control) of beta-galactosidase in Escherichia coli.
Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA
Authors: Chistoserdova L V , Lidstrom M E
Published: November 2016

In a previous paper, we reported identification of the 5' part of hprA of Methylobacterium extorquens AM1, which encodes the serine cycle enzyme hydroxypyruvate reductase (L. V. Chistoserdova and M. E. Lidstrom, J. Bacteriol. 174:71-77, 1992). Here we present the complete sequence of hprA and partial sequence of genes adjacent to hprA. Upstream of hprA, the 3' part of an open reading frame was discovered, separated from hprA by 263 bp. This open reading frame was identified as the gene encoding another serine cycle enzyme, serine glyoxylate aminotransferase (sgaA). Cells containing an insertion mutation into sgaA were unable to grow on C1 compounds, demonstrating that the gene is required for C1 metabolism. Sequencing downstream of hprA has revealed the presence of another open reading frame (mtdA), which is probably cotranscribed with hprA. This open reading frame was identified as the gene required for the synthesis of 5,10-methylenetetrahydrofolate dehydrogenase. Our data suggest that this enzyme plays an integral role in methylotrophic metabolism in M. extorquens AM1, either in formaldehyde oxidation or as part of the serine cycle.
Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa
Authors: Ochsner U A , Koch A K , Fiechter A , Reiser J
Published: November 2016

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.
Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum
Authors: Lee C Y , O'Kane D J , Meighen E A
Published: November 2016

Four genes immediately downstream of luxG in the Photobacterium phosphoreum lux operon (ribEBHA) have been sequenced and shown to be involved in riboflavin synthesis. Sequence analyses and complementation of Escherichia coli riboflavin auxotrophs showed that the gene products of ribB and ribA are 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthetase and GTP cyclohydrolase II, respectively. By expression of P. phosphoreum ribE in E. coli using the bacteriophage T7 promoter-RNA polymerase system, ribE was shown to code for riboflavin synthetase, which catalyzes the conversion of lumazine to riboflavin. Increased thermal stability of RibE on expression with RibH indicated that ribH coded for lumazine synthetase. The organization of the rib genes in P. phosphoreum is quite distinct, with ribB and ribA being linked but separated by ribH, whereas in E. coli, they are unlinked and in Bacillus subtilis, RibB and RibA functions are coded by a single gene.
A modified two-component regulatory system is involved in temperature-dependent biosynthesis of the Pseudomonas syringae phytotoxin coronatine
Authors: Ullrich M , Peñaloza-Vázquez A , Bailey A M , Bender C L
Published: November 2016

Biosynthesis of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is regulated by temperature at the transcriptional level. A 3.4-kb DNA fragment from the COR biosynthetic gene cluster restored temperature-regulated phytotoxin production to Tn5 mutants defective in COR production. Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR. Although only one response regulator, CorR, had a DNA-binding domain, the phosphate-receiving domains of both response regulator proteins were highly conserved. Transcriptional fusions of the corP and corR promoters to a promoterless glucuronidase gene (uidA) indicated that these two genes are expressed constitutively at 18 and 28 degrees C. In contrast, a corS::uidA fusion exhibited the temperature dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18 degrees C and low activity at 28 degrees C. Furthermore, glucuronidase activity for corS::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA). This difference was not observed for corP::uidA and corR::uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background. The three regulatory genes functioned in a P. syringae strain lacking the COR gene cluster to achieve temperature-dependent activation of an introduced COR biosynthetic promoter, indicating that this triad of genes is the primary control for COR biosynthesis and responsible for thermoregulation. Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.
Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2
Authors: Small F J , Ensign S A
Published: November 2016

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.
A periplasm in Bacillus subtilis
Authors: Merchante R , Pooley H M , Karamata D
Published: November 2016

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.
Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133
Authors: Summers M L , Wallis J G , Campbell E L , Meeks J C
Published: November 2016

Heterocysts, sites of nitrogen fixation in certain filamentous cyanobacteria, are limited to a heterotrophic metabolism, rather than the photoautotrophic metabolism characteristic of cyanobacterial vegetative cells. The metabolic route of carbon catabolism in the supply of reductant to nitrogenase and for respiratory electron transport in heterocysts is unresolved. The gene (zwf) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the oxidative pentose phosphate pathway, was inactivated in the heterocyst-forming, facultatively heterotrophic cyanobacterium, Nostoc sp. strain ATCC 29133. The zwf mutant strain had less than 5% of the wild-type apparent G6PD activity, while retaining wild-type rates of photoautotrophic growth with NH4+ and of dark O2 uptake, but it failed to grow either under N2-fixing conditions or in the dark with organic carbon sources. A wild-type copy of zwf in trans in the zwf mutant strain restored only 25% of the G6PD specific activity, but the defective N2 fixation and dark growth phenotypes were nearly completely complemented. Transcript analysis established that zwf is in an operon also containing genes encoding two other enzymes of the oxidative pentose phosphate cycle, fructose-1,6-bisphosphatase and transaldolase, as well as a previously undescribed gene (designated opcA) that is cotranscribed with zwf. Inactivation of opcA yielded a growth phenotype identical to that of the zwf mutant, including a 98% decrease, relative to the wild type, in apparent G6PD specific activity. The growth phenotype and lesion of G6PD activity in the opcA mutant were complemented in trans with a wild-type copy of opcA. In addition, placement in trans of a multicopy plasmid containing the wild-type copies of both zwf and opcA in the zwf mutant resulted in an approximately 20-fold stimulation of G6PD activity, relative to the wild type, complete restoration of nitrogenase activity, and a slight stimulation of N2-dependent photoautotrophic growth and fructose-supported dark growth. These results unequivocally establish that G6PD, and most likely the oxidative pentose phosphate pathway, represents the essential catabolic route for providing reductant for nitrogen fixation and respiration in differentiated heterocysts and for dark growth of vegetative cells. Moreover, the opcA gene product is involved by an as yet unknown mechanism in G6PD synthesis or catalytic activity.
hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors
Authors: Wei Z M , Beer S V
Published: November 2016

hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed.
Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus
Authors: Hecht G B , Newton A
Published: November 2016

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.
Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus
Authors: Feng G H , Leonard T J
Published: November 2016

Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCR-derived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A)+ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, beta-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.
Oxygen, iron, carbon, and superoxide control of the fumarase fumA and fumC genes of Escherichia coli: role of the arcA, fnr, and soxR gene products
Authors: Park S J , Gunsalus R P
Published: November 2016

The tricarboxylic acid cycle enzyme fumarase catalyzes the interconversion of fumarate to L-malate. Escherichia coli contains three biochemically distinct fumarases. While the fumA and fumB genes encode heat-labile, iron-containing fumarases, the fumC gene product is a heat-stable fumarase which does not require iron for activity. To study how the fumA and fumC genes are regulated, we constructed lacZ operon fusions to the fumA and/or fumC upstream regions. Expression of the fumA and fumC genes was lowest during anaerobic cell growth, in support of the proposed roles of FumA and FumC as aerobic fumarases. Transcription of the fumC gene was shown to be complex: it was dependent on both the fumA and fumC promoters. Anaerobic expression from the fumA promoter was derepressed in both an arcA and a fnr mutant, while expression from the fumC promoter was derepressed in only the arcA strain. The fumA promoter was also shown to be catabolite controlled, whereas the fumC promoter was relatively unaffected by the type of carbon used for cell growth. Cellular iron limitation stimulated fumC but not fumA expression. Superoxide radicals also caused increased fumC gene expression; fumA expression was unaffected. Both the superoxide control and the iron control of fumC expression required the SoxR regulatory protein. These studies suggest different physiological roles for the FumA and FumC fumarases. The iron-containing FumA fumarase is the more abundant enzyme under most conditions of aerobic cell growth except when iron is limiting; FumC, which lacks iron, appears to be a backup enzyme that is synthesized optimally only when iron is low or when superoxide radicals accumulate.
Rhizobium NodI and NodJ proteins play a role in the efficiency of secretion of lipochitin oligosaccharides
Authors: Spaink H P , Wijfjes A H , Lugtenberg B J
Published: November 2016

Thin-layer chromatographic analysis of extracts of D-[1-14C]glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules. Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions. A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-[1-14C]glucosamine-labelled cells grown in medium supplemented with chitinase. At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs. In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis. In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction. At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains. By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion. Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.
Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis
Authors: Douglas A L , Hatch T P
Published: November 2016

We determined that the putative upstream promoter of the long transcript (P1) of ompA, the gene encoding the major outer membrane protein of Chlamydia trachomatis L2, is recognized in vitro by the major sigma factor of chlamydiae, sigma 66. We found no evidence that the putative downstream promoter of the short ompA transcript (P1) is functional in vitro. RNase protection of guanylyltransferase-capped transcripts made in vivo confirmed that P2 is a primary transcript while P1 is a processed product of a longer transcript, possibly P2.
Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes
Authors: Essenberg R C
Published: November 2016

A clone from Brucella abortus 19 complemented an Escherichia coli strain deficient in phosphorylation of glucose. Open reading frames similar to E. coli mepA, glk, and genes encoding ATP-coupled exporters were found in the sequence. A fourth affected growth on minimal media of the ptsI glk strain with various carbon sources.
Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm
Authors: Eismann K , Mlejnek K , Zipprich D , Hoppert M , Gerberding H , Mayer F
Published: November 2016

Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.
Attention deficit hyperactivity disorder: overdiagnosed or diagnoses missed?
Authors: Taylor Eric
Published: November 2016

The prevalence of attention deficit hyperactivity disorder is about 5% of the child population, and it carries risks for mental health, educational and personal dysfunction, antisociality and death. The diagnosis in practice is made with remarkable differences in frequency between nations. Low rates in the UK, by comparison with research estimates, suggest that the condition may often be missed in clinical practice; the reasons are considered.
Hyperhomocysteinaemia in children receiving phenytoin and carbamazepine monotherapy: a cross-sectional observational study
Authors: Chandrasekaran Saravanan , Patil Sooraj , Suthar Renu , Attri Savita Verma , Sahu Jitendra Kumar , Sankhyan Naveen , Tageja Mini , Singhi Pratibha
Published: November 2016

Objective

Long-term therapy with phenytoin and carbamazepine is known to cause hyperhomocysteinaemia. We evaluated the prevalence of hyperhomocysteinaemia in North Indian children receiving phenytoin or carbamazepine monotherapy for >6 months duration and the effect of folic acid supplementation on plasma homocysteine.

Methods

In this cross-sectional observational study we enrolled consecutive children aged 2–12 years with epilepsy who had received phenytoin or carbamazepine monotherapy for >6 months. Plasma total homocysteine, folic acid, vitamin B12 and antiepileptic drug concentrations were measured. Healthy age- and sex-matched controls were recruited. Children with homocysteine >10.4 µmol/L received folic acid supplementation for 1 month and homocysteine and folic acid concentrations were measured after 1 month follow-up.

Results

A total of 112 children receiving antiepileptic monotherapy for >6 months were enrolled. Hyperhomocysteinaemia was present in 54 children (90%) receiving phenytoin, 45 children (90%) receiving carbamazepine therapy and 17 (34%) controls (p<0.05). Mean plasma homocysteine concentrations were significantly higher (18.9±10.2 vs 9.1±3 µmol/L) and serum folic acid concentrations (10.04±8.5 ng/ml vs 12.6±4.8 p<0.001) and vitamin B12 concentrations (365±155 pg/mL vs 474±332 pg/mL, p=0.02) were significantly lower in the study group compared with the control group. Duration of antiepileptic drug therapy correlated significantly with elevated homocysteine and reduced folic acid concentrations (p<0.05). Supplementation with folic acid for 1 month led to a reduction in plasma homocysteine concentrations in the study group (from 20.9±10.3 µmol/L to 14.2±8.2 µmol/L, p<0.05).

Conclusions

Phenytoin or carbamazepine monotherapy for >6 months duration is associated with hyperhomocysteinaemia in 90% of North Indian children. Elevated homocysteine concentrations were normalised in these children with folic acid supplementation.
Protein and glycomic plasma markers for early detection of adenoma and colon cancer
Authors: Rho Jung-hyun , Ladd Jon J , Li Christopher I , Potter John D , Zhang Yuzheng , Shelley David , Shibata David , Coppola Domenico , Yamada Hiroyuki , Toyoda Hidenori , Tada Toshifumi , Kumada Takashi , Brenner Dean E , Hanash Samir M , Lampe Paul D
Published: November 2016

Objective

To discover and confirm blood-based colon cancer early-detection markers.

Design

We created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years after blood was drawn (ie, prediagnostic) and cancer-free, matched controls. Potential markers were tested on plasma samples from people diagnosed with adenoma or cancer, compared with controls. Components of an optimal 5-marker panel were tested via immunoblotting using a third sample set, Luminex assay in a large fourth sample set and immunohistochemistry (IHC) on tissue microarrays.

Results

In the prediagnostic samples, we found 78 significantly (t-test) increased proteins, 32 of which were confirmed in the diagnostic samples. From these 32, optimal 4-marker panels of BAG family molecular chaperone regulator 4 (BAG4), interleukin-6 receptor subunit beta (IL6ST), von Willebrand factor (VWF) and CD44 or epidermal growth factor receptor (EGFR) were established. Each panel member and the panels also showed increases in the diagnostic adenoma and cancer samples in independent third and fourth sample sets via immunoblot and Luminex, respectively. IHC results showed increased levels of BAG4, IL6ST and CD44 in adenoma and cancer tissues. Inclusion of EGFR and CD44 sialyl Lewis-A and Lewis-X content increased the panel performance. The protein/glycoprotein panel was statistically significantly higher in colon cancer samples, characterised by a range of area under the curves from 0.90 (95% CI 0.82 to 0.98) to 0.86 (95% CI 0.83 to 0.88), for the larger second and fourth sets, respectively.

Conclusions

A panel including BAG4, IL6ST, VWF, EGFR and CD44 protein/glycomics performed well for detection of early stages of colon cancer and should be further examined in larger studies.