bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of plant root exudate and Sym plasmid-localized nodulation genes in the synthesis by Rhizobium leguminosarum of Tsr factor, which causes thick and short roots on common vetch
Authors: Van Brussel A A , Zaat S A , Cremers H C , Wijffelman C A , Pees E , Tak T , Lugtenberg B J
Published: November 2016

In a previous paper it was shown that cocultivation of Rhizobium leguminosarum with the plant Vicia sativa subsp. nigra on solid medium causes a changed mode of growth of the plant roots, resulting in thick and short roots (Tsr). The Sym plasmid present in the bacterium appeared to be essential for causing Tsr (A. A. N. van Brussel, T. Tak, A. Wetselaar, E. Pees, and C. A. Wijffelman, Plant Sci. Lett. 27:317-325, 1982). In the present paper, we show that a role in causing Tsr is general for Sym plasmids of R. leguminosarum and Rhizobium trifolii. Moreover, mutants with transposon insertions in the Sym plasmid-localized nodulation genes nodA, B, C, and D are unable to cause Tsr, in contrast to nodulation mutants localized in other parts of the Sym plasmid. The observation that Tsr could also be brought about in liquid medium enabled us to show that Tsr is caused by a soluble factor. Experiments in which plants and bacteria were grown separately in the sterile supernatant fluids of each other resulted in establishing the following sequence of events. (i) The plant produces a factor, designated as factor A. (ii) Factor A causes the Sym plasmid-harboring bacteria to produce Tsr factor. (iii) Growth of young plants in the presence of Tsr factor results in the Tsr phenotype. Models explaining this example of molecular signalling between bacteria and plants are discussed.
Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance
Authors: Bender C L , Cooksey D A
Published: November 2016

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).
Amplification and deletion of the amyE+-tmrB+ gene region in a Bacillus subtilis recombinant-phage genome by the tmrA7 mutation
Authors: Furusato T , Takano J , Yamane K , Hashiguchi K , Tanimoto A , Mori M , Yoda K , Yamasaki M , Tamura G
Published: November 2016

A 22.4-kilobase DNA fragment containing the tmrA7-amyR2-amyE+-tmrB+-aroI+ region of the Bacillus subtilis N7 chromosomal DNA was cloned into a recombinant B. subtilis bacteriophage, p11-AA248. The amyE+-tmrB+ gene region, approximately 12.6 kilobases, in the phage genome was amplified in a tunicamycin-resistant (Tmr) Amy+ AroI+ transductant of B. subtilis by p11-AA248. On the other hand, the amyE+-tmrB+ region in the genomes of 80 to 90% of the phage particles was deleted when the phages were induced from the Tmr Amy+ AroI+ transductants by treatment with 1.0 micrograms of mitomycin C per ml. From analyses of the physical maps and DNA nucleotide sequences in the junction region of the deleted phage genome and the parental DNA fragments, it is suggested that the deletion occurred within a direct repeat sequence composed of 18 base pairs. The endpoints of the amplified gene region seemed to be closely related to both terminal regions of the deleted DNA.
Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli
Authors: Shanley M S , Neidle E L , Parales R E , Ornston L N
Published: November 2016

This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.
Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli
Authors: Harker A R , Zuber M , Evans H J
Published: November 2016

Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum. The cross-reactive polypeptides in a series of different E. coli strains are of Mrs ca. 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95). The 60-kDa polypeptide from E. coli HB101 comigrated on native gels with detectable Hup activity. The exact role of the 30-kDa polypeptide in E. coli is unclear. E. coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R. japonicum. Anaerobically cultured E. coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct.
Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli
Authors: Finlay B B , Pasloske B L , Paranchych W
Published: November 2016

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.
Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli
Authors: Helmstetter C E , Krajewski C A , Leonard A C , Weinberger M
Published: November 2016

Potential for initiation of chromosome replication present in temperature-sensitive, initiation-defective dnaA5 mutants of Escherichia coli B/r incubated at nonpermissive temperature was expressed by shifting to a more permissive temperature (25 degrees C). Upon expression of initiation potential, the rate of [3H]thymidine incorporation varied in a bimodal fashion, i.e., there was an initial burst of incorporation, which lasted 10 to 20 min, then a sudden decrease in incorporation, and finally a second rapid increase in incorporation. Analyses of this incorporation pattern indicated that a round of replication initiated upon expression of initiation potential, but DNA polymerization stopped after replication of 5 to 10% of the chromosome. This round of replication appeared to resume about 30 min later coincident with initiation of a second round of replication. The second initiation was unusually sensitive to low concentrations of novobiocin (ca. 1 microgram/ml) when this inhibitor was added in the presence of chloramphenicol. In the absence of chloramphenicol, novobiocin at this concentration had no detectable effect on DNA replication. It is suggested that cis-acting inhibition, attributable to an attempted second initiation immediately after the first, caused the first round to stall until both it and the second round could resume simultaneously. This DNA replication inhibition, probably caused by overinitiation, could be a consequence of restraints on replication in the vicinity of oriC, possibly topological in nature, which limit the minimum interinitiation interval in E. coli.
Characterization of the OCT plasmid encoding alkane oxidation and mercury resistance in Pseudomonas putida
Authors: Harder P A , Kunz D A
Published: November 2016

Transformation of Pseudomonas putida and analysis for plasmid DNA revealed that both n-alkane oxidation and mercury resistance are encoded on a single 220-megadalton OCT plasmid molecule. Derivatives of OCT having lost the mercury resistance function could be readily isolated and contained a smaller plasmid estimated to be 170 megadaltons. The results show that segregation of the mercury resistance property occurs not by loss of a separate MER plasmid as previously thought but by a deletion in the OCT plasmid.
Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3'-cyclic phosphodiesterase
Authors: Liu J , Burns D M , Beacham I R
Published: November 2016

The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3'-nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability.
Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair
Authors: Wang T C , Smith K C
Published: November 2016

The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells.
Plasmid-mediated tetracycline resistance in Campylobacter jejuni: expression in Escherichia coli and identification of homology with streptococcal class M determinant
Authors: Taylor D E
Published: November 2016

Plasmid pUA466, a 45-kilobase transmissible tetracycline resistance plasmid from Campylobacter jejuni was mapped with AvaI, AvaII, BclI, HincII, PstI, XhoI, and XbaI. The resistance determinant was cloned and expressed in Escherichia coli and was homologous with a class M determinant from Streptococcus spp.
Processing of Bacillus licheniformis penicillinases lacking a lipoprotein modification site in Escherichia coli
Authors: Hayashi S , Chang S Y , Chang S , Wu H C
Published: November 2016

We have previously shown that the penP Ser-27 prepenicillinase is processed into two forms, Ser-35-penicillinase and Asn-29 penicillinase. Two new penicillinase mutants, penP Ser-27 Pro-28 and penP Ser-27,23' (Pro-Asp)24', were derived from the penP Ser-27 mutant by oligonucleotide-directed site-specific mutagenesis. The penP Ser-27 Pro-28 mutant prepenicillinase was also processed into two forms, Ser-35-penicillinase and Gly-26-penicillinase. On the contrary, the penP Ser-27,23' (Pro-Asp)24' mutant prepenicillinase is unprocessed.
Isolation of a mutation resulting in constitutive synthesis of L-fucose catabolic enzymes
Authors: Bartkus J M , Mortlock R P
Published: November 2016

A ribitol-positive transductant of Escherichia coli K-12, JM2112, was used to facilitate the isolation and identification of mutations affecting the L-fucose catabolic pathway. Analysis of L-fucose-negative mutants of JM2112 enabled us to confirm that L-fucose-1-phosphate is the apparent inducer of the fucose catabolic enzymes. Plating of an L-fuculokinase-negative mutant of JM2112 on D-arabinose yielded an isolate containing a second fucose mutation which resulted in the constitutive synthesis of L-fucose permease, isomerase, and kinase. This constitutive mutation differs from the constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984) in that it is tightly linked to the fucose genes and appears to be located in the gene believed to code for the positive activator of the L-fucose genes.
Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant
Authors: Goldberg I , Mekalanos J J
Published: November 2016

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.
Oxygen-regulated stimulons of Salmonella typhimurium identified by Mu d(Ap lac) operon fusions
Authors: Aliabadi Z , Warren F , Mya S , Foster J W
Published: November 2016

Using the technique of Mu d1(Ap lac)-directed lacZ operon fusions, several oxygen-regulated genetic loci were identified in Salmonella typhimurium. Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified. Based on control by the oxrA and oxrB regulatory loci, the anti-lacZ fusions were grouped into three classes: class I loci were regulated by both oxr loci, class II genes were regulated by oxrA only, and class III loci were not affected by either regulatory locus. Several of the anti-lacZ fusions required growth in complex medium before they exhibited the inducible phenotype. While the expression of some of these loci was repressed when organisms were grown in nitrate, others were stimulated by nitrate. Fusions into the hyd and phs loci were identified among the isolated anti-lacZ fusions. Six oxygen-inducible (oxi) operon fusions were also identified. Two of the oxi loci mapped near oxygen-regulatory loci: oxiC near oxrA and oxiE near oxyR. However, neither fusion appeared to occur within the regulatory locus. The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined.
Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli
Authors: Louie K , Chen Y C , Dowhan W
Published: November 2016

To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate.
Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector
Authors: Wirth R , An F Y , Clewell D B
Published: November 2016

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.
Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway
Authors: Styrvold O B , Falkenberg P , Landfald B , Eshoo M W , Bjørnsen T , Strøm A R
Published: November 2016

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Strøm, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.
Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp
Authors: Hartmann A , Fu H , Burris R H
Published: November 2016

Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Direct role of the Escherichia coli Dam DNA methyltransferase in methylation-directed mismatch repair
Authors: Schlagman S L , Hattman S , Marinus M G
Published: November 2016

The T4 dam+ gene has been cloned (S. L. Schlagman and S. Hattman, Gene 22:139-156, 1983) and transferred into an Escherichia coli dam-host. In this host, the T4 Dam DNA methyltransferase methylates mainly, if not exclusively, the sequence 5'-GATC-3'; this sequence specificity is the same as that of the E. coli Dam enzyme. Expression of the cloned T4 dam+ gene suppresses almost all the phenotypic traits associated with E. coli dam mutants, with the exception of hypermutability. In wild-type hosts, 20- to 500-fold overproduction of the E. coli Dam methylase by plasmids containing the cloned E. coli dam+ gene results in a hypermutability phenotype (G.E. Herman and P. Modrich, J. Bacteriol. 145:644-646, 1981; M.G. Marinus, A. Poteete, and J.A. Arraj, Gene 28:123-125, 1984). In contrast, the same high level of T4 Dam methylase activity, produced by plasmids containing the cloned T4 dam+ gene, does not result in hypermutability. To account for these results we propose that the E. coli Dam methylase may be directly involved in the process of methylation-instructed mismatch repair and that the T4 Dam methylase is unable to substitute for the E. coli enzyme.
Transport of p-nitrophenyl-alpha-maltoside by the maltose transport system of Escherichia coli and its subsequent hydrolysis by a cytoplasmic alpha-maltosidase
Authors: Reyes M , Treptow N A , Shuman H A
Published: November 2016

In wild-type Escherichia coli the activity of the maltose transport system is dependent on a periplasmic maltose-binding protein. It has been possible, however, to isolate mutants in which transport activity is mediated by the membrane components of the system and is no longer dependent on the periplasmic binding protein. In this manuscript we show that in these binding protein-independent strains, p-nitrophenyl-alpha-maltoside is a potent inhibitor of maltose transport. In contrast, p-nitrophenyl-alpha-maltoside is only a weak inhibitor of maltose transport in wild-type bacteria. In addition, we show that p-nitrophenyl-alpha-maltoside is transported by the binding protein-independent strains but not by wild-type bacteria. We were able to detect transport of this compound because there is a cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside. This enzyme has not previously been described. We show that although the synthesis of this enzyme is subject to the same regulation as the components of the maltose regulon, and is MalT dependent, it is not coded for by a known mal gene. We refer to this enzyme as alpha-maltosidase. These results strengthen our proposal that the membrane components of the maltose transport system comprise a recognition site for maltose and related substrates.
Isolation and properties of Streptomyces spore membranes
Authors: Quirós L M , Hardisson C , Salas J A
Published: November 2016

A simple procedure for the isolation of membranes from Streptomyces spores is described which produces about 12 mg of membrane protein per g of dry weight. The membrane fractions were contaminated by low levels of DNA, RNA, and hexosamines. The functional integrity of the membrane is conserved through the isolation procedure, as evaluated by the presence of several activities of the membrane-bound electron transport chain. This isolation procedure allowed the determination of the biosynthesis of proteins and phospholipids of the membrane. Both biosynthetic processes started in the first 5 min of germination and increased progressively during spore germination. A stable mRNA fraction of the dormant spore encoded 44% of the membrane proteins synthesized early in germination, but most of the phospholipid biosynthesis was not dependent on this fraction.
Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation
Authors: Condemine G , Hugouvieux-Cotte-Pattat N , Robert-Baudouy J
Published: November 2016

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.
In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides
Authors: Hoger J H , Chory J , Kaplan S
Published: November 2016

The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo- with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.
Apparently unidirectional polyamine transport by proton motive force in polyamine-deficient Escherichia coli
Authors: Kashiwagi K , Kobayashi H , Igarashi K
Published: November 2016

A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake.
Pore formation by LamB of Escherichia coli in lipid bilayer membranes
Authors: Benz R , Schmid A , Nakae T , Vos-Scheperkeuter G H
Published: November 2016

Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.
Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa
Authors: Poole K , Hancock R E
Published: November 2016

Bacteria from members of the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 to 0.2 mM Pi) conditions and examined for the production of novel membrane proteins. Of the 17 strains examined, 12 expressed a phosphate-starvation-induced outer membrane protein which was heat modifiable in that after solubilization in sodium dodecyl sulfate at low temperature the protein ran on gels as a diffuse band of higher apparent molecular weight, presumably an oligomer form, which shifted to an apparent monomer form after solubilization at high temperature. These proteins fell into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The first class, expressed by species of the Pseudomonas fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible, channel-forming protein P of Pseudomonas aeruginosa. The second class resembled the major enterobacterial porin proteins and the phosphate-regulated PhoE protein of Escherichia coli. Using a protein P-trimer-specific polyclonal antiserum, we were able to demonstrate cross-reactivity of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. With a protein P-monomer-specific antiserum, no reactivity was seen with any of the phosphate-starvation-inducible membrane proteins (in either oligomeric or monomeric form), with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins.
Two patterns in the Aeromonas salmonicida A-layer may reflect a structural transformation that alters permeability
Authors: Stewart M , Beveridge T J , Trust T J
Published: November 2016

Electron micrographs of negatively stained regular surface layers (A-layers) of Aeromonas salmonicida showed two square patterns having p4 symmetry. Computer image processing demonstrated that, at a resolution of 2.3 nm, both square arrays were composed of two different morphological units arranged alternatively to give a face-centered lattice in which the four nearest neighbors of each unit were the other type of unit. The lattice constant was slightly but significantly different in the two patterns, and the orientation of one of the two morphological units changed by about 20 degrees between patterns. These patterns were probably not derived from different strains present in the preparation, since both were seen in material that appeared to come from a single layer. This and the difference in lattice constant made it unlikely that they represented different sides of the A-layer. However, it is possible that the two patterns may reflect a structural transformation of the layer. In this respect, it is interesting that the rotation of one morphological subunit changed the size of the gaps between units in the layer. This raised the possibility that the transformation could be related to a change in permeability of the A-layer, possibly analogous to that proposed for gap junctions in eucaryotic cells.
L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product
Authors: Spring K J , Jerlström P G , Burns D M , Beacham I R
Published: November 2016

Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments. Therefore, the genes encoding asparaginases I and II are highly sequence related.
Physical and genetic analysis of the ColD plasmid
Authors: Frey J , Ghersa P , Palacios P G , Belet M
Published: November 2016

The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.
Naphthalene association and uptake in Pseudomonas putida
Authors: Bateman J N , Speer B , Feduik L , Hartline R A
Published: November 2016

Two methods for bacterial membrane transport, filtration and flow dialysis, were used to study cellular association of Pseudomonas putida with naphthalene. It is not technically possible to determine the exact cellular or vesicular location of the naphthalene, and because it is hydrophobic, it could be at the membrane(s) rather than inside the cells. As an index of naphthalene having crossed the inner membrane we used the intracellular formation of its first catabolite. An energized membrane or ATP was not essential for association or movement into the cell. Evidence for a nonspecific association and a movement into cells by simple diffusion are the lack of saturation of association, an absence of inhibition of association by protein inhibitors and structural analogs, and the passage of naphthalene through cell membranes in the presence of iodoacetamide. Specific naphthalene metabolism gene expression was not required for association.
Effect of stage 0 sporulation mutations on subtilisin expression
Authors: Ferrari E , Howard S M , Hoch J A
Published: November 2016

Subtilisin expression as a function of growth and sporulation was determined using a presubtilisin-beta-galactosidase gene fusion. An approximately 500-base-pair region upstream of the subtilisin gene and including the first eight codons of the presubtilisin protein was fused at the eighth codon of beta-galactosidase in the integrative vector pJF751. This gene fusion does not carry a signal sequence, and therefore its synthesis is uncoupled from maturation of presubtilisin. The fusion protein gene was integrated into a variety of recipient strains to test for the effect of various mutations on the initial rate of presubtilisin-beta-galactosidase synthesis. Among the spo0 mutations tested, the spo0A mutations showed a strong, 10-fold decrease in the rate of beta-galactosidase synthesis. This effect of the spo0A mutations was not evident when the presubtilisin-beta-galactosidase fusion was present on a multicopy plasmid. The sacU mutation, which was known to increase the extracellular level of levansucrase and proteases, was found to increase the synthesis of the presubtilisin-beta-galactosidase gene fusions 7-fold, and the hpr mutations were shown to increase the rate of presubtilisin-beta-galactosidase gene fusions 17-fold, indicating that these mutations influence either transcription or translation of the presubtilisin gene. However, the effect of these mutations was only observed in the stationary phase of growth, indicating they did not render synthesis constitutive. By using multicopy plasmids and an integrated gene fusion, it was shown that there is likely to be a titratable repressor controlling subtilisin synthesis.
Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching
Authors: Yamaguchi S , Fujita H , Ishihara A , Aizawa S , Macnab R M
Published: November 2016

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.
Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis
Authors: Shivakumar A G , Gundling G J , Benson T A , Casuto D , Miller M F , Spear B B
Published: November 2016

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.