bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in Rhizobium phaseoli that lead to arrested development of infection threads
Authors: Noel K D , Vandenbosch K A , Kulpaca B
Published: November 2016

Two Rhizobium phaseoli mutants, isolated previously by Tn5 mutagenesis, elicited infection threads which ceased development prematurely, usually within root hairs. These infection threads were wide, globular, and otherwise altered in morphology, compared with normal infection threads. Anatomy and division of the root cortical cells during initial stages of nodule morphogenesis appeared normal. However, later nodule differentiation deviated considerably from normal development, and release of bacteria from infection threads was not observed. In tryptone-yeast extract medium the mutants sedimented during growth in shaken cultures and formed rough colonies on agar. Electrophoresis of washed cultures solubilized in dodecyl sulfate revealed that the major carbohydrate band was absent from the mutants. The behavior of this carbohydrate in phenol-water extraction and gel chromatography, its apparent ketodeoxyoctonate content, and its susceptibility to mild acid hydrolysis suggested that it was a lipopolysaccharide. From the results of genetic crosses or reversion analysis, the defect in synthesizing this carbohydrate material and the defect in infection could be attributed to a single mutation in each mutant.
Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus
Authors: Sparling R , Daniels L
Published: November 2016

Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H2O or [13C]formate and analysis by mass spectrometry. When cells (H2-CO2 or formate grown) were transferred to formate medium in 95% 2H water, the proportion of 2H in methane was 95%. When cells (H2-CO2 or formate grown) were transferred to media containing [13C]formate in the presence of H2-CO2 or He-CO2, the ratio of 13CH4 to 12CH4 increased over time parallel to the ratio of 13CO2 to 12CO2. The cells catalyzed a significant exchange of label between [13C]formate and 13CO2.
Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase
Authors: Iuchi S , Kuritzkes D R , Lin E C
Published: November 2016

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.
Transformation of encapsulated Streptococcus pneumoniae
Authors: Yother J , McDaniel L S , Briles D E
Published: November 2016

We describe the high-efficiency transformation of several virulent, encapsulated isolates of Streptococcus pneumoniae. Transformation was effected by the induction of competence with competence factor and was apparently the result both of inducing noncompetent recipients and overcoming the inhibition imposed by the capsule.
Determination of bacterial cell volume with the Coulter Counter
Authors: Kubitschek H E , Friske J A
Published: November 2016

Two methods were used to determine mean volumes of cells of Escherichia coli B/rA in both stationary- and exponential-phase cultures, i.e., electronic measurement with a Coulter Counter-Analyzer system and biophysical measurement of the total volume and number of cells in sedimented cell pellets. Within experimental errors, the methods gave the same mean cell volumes.
Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli
Authors: Kuwajima G , Asaka J , Fujiwara T , Fujiwara T , Node K , Kondo E
Published: November 2016

We determined the DNA sequence of the hag gene of Escherichia coli K-12 and deduced the primary structure of the flagellin consisting of 497 amino acid residues. Comparison of the amino acid sequence with those of other bacterial flagellins revealed a high homology in the NH2- and COOH-terminal regions.
Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12
Authors: Emmett M R , Johnson J R
Published: November 2016

Expression of methionine regulon elements was controlled by the metJ protein gpMetJ. A maxicell system with cloned copies of the metF transcription unit allowed reversible action of gpMetJ. Expression of the metF transcription unit in maxicells was reduced by exogenous vitamin B12 at concentrations of 0.5 nM or greater.
Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria
Authors: Corton J C , Ward J E , Lutkenhaus J
Published: November 2016

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.
Mutations that affect structure and assembly of light-harvesting proteins in the cyanobacterium Synechocystis sp. strain 6701
Authors: Anderson L K , Rayner M C , Eiserling F A
Published: November 2016

The unicellular cyanobacterium Synechocystis sp. strain 6701 was mutagenized with UV irradiation and screened for pigment changes that indicated genetic lesions involving the light-harvesting proteins of the phycobilisome. A previous examination of the pigment mutant UV16 showed an assembly defect in the phycocyanin component of the phycobilisome. Mutagenesis of UV16 produced an additional double mutant, UV16-40, with decreased phycoerythrin content. Phycocyanin and phycoerythrin were isolated from UV16-40 and compared with normal biliproteins. The results suggested that the UV16 mutation affected the alpha subunit of phycocyanin, while the phycoerythrin beta subunit from UV16-40 had lost one of its three chromophores. Characterization of the unassembled phycobilisome components in these mutants suggests that these strains will be useful for probing in vivo the regulated expression and assembly of phycobilisomes.
Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere
Authors: Sebastian J , Chandra A K , Kolattukudy P E
Published: November 2016

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Organization of developing Escherichia coli colonies viewed by scanning electron microscopy
Authors: Shapiro J A
Published: November 2016

Colony growth was initiated by inoculating minimal glucose agar with 1-microliter. spots of a plasmid-free Escherichia coli culture and incubating at 32 degrees C. Inoculations took place over a 3-day period, at the end of which the plates were fixed and dried for scanning electron microscopy. In this way, it was possible to examine the surfaces of colonies ranging in age from 0 to 68 h. Macroscopically, the colonies were organized into different concentric zones, and several morphological features could be seen to develop over this period. These included a shallow depression ring marking the site of inoculation, a deeper indentation ring whose position moved outward as the colony grew, an expanding plateau region between the two rings, a mound outside the indentation ring, and a flat brim extending onto the substrate which was either present or absent at different times. Microscopically, a variety of cell morphologies and cell arrangements were detected. Upon inoculation, the bacteria accumulated at the periphery of the inoculation spot but showed no other kind of order. For the first 7.5 h, all bacteria were rod shaped; at the end of this initial phase, a high degree of alignment was seen in the cells at the colony edge. By 24.5 h, both shorter more ovoid cells and longer filaments had begun to appear, and large multicellular arrays had formed. At later stages of colony development, morphologically distinguishable zones involving cells of different shapes and sizes had formed, and these zones often marked the boundaries of macroscopic features. The edges were particularly interesting and at 68 h displayed very sharp saw-toothed boundaries between concentrically organized groups of bacteria. There were some transient irregularities in the concentric organizations of growing colonies, and one colony had entered upon a distinct developmental pathway.
Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein
Authors: Morris E J , Ganeshkumar N , Song M , McBride B C
Published: November 2016

Cell surface fibrils could be released from Streptococcus sanguis 12 but not from strains 12na or N by freeze-thawing followed by brief homogenization. Fibrils were isolated from the homogenate by ultracentrifugation or ammonium sulfate precipitation. Electron microscopy demonstrated the presence of dense masses of aggregated fibrils in these preparations. Under nondenaturing conditions, no proteins were seen in polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS)-PAGE analysis revealed a single band stained with Coomassie blue and periodic acid Schiff stain with a molecular weight in excess of 300,000. The protein has been given the name long-fibril protein (LFP). The molecule was susceptible to digestion with subtilisin, pronase, papain, and trypsin, but was unaffected by chymotrypsin or muramidases. Attempts to dissociate the protein into smaller subunits with urea, guanidine, sodium thiocyanate, and HCl were unsuccessful. Gel filtration on a column of Sephacryl S-400 in the presence of 2% SDS resulted in elution of the protein at the void volume. Antibody raised against the LFP excised from an SDS-PAGE gel reacted with long fibrils on the surface of strain 12 and with isolated fibrils by an immunogold labeling technique. Monoclonal antibody reactive with LFP in SDS-PAGE also reacted with fibrils present on the cell. Antisera raised against the fibrils inhibited adherence to saliva-coated hydroxyapatite.
Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness
Authors: Li J D , Hu C Z , Yoch D C
Published: November 2016

Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+- or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool remained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4+ azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4+ substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivation. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4+ switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4+ switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4+ (glutamine) switch-off.
Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA
Authors: Miyamoto C , Byers D , Graham A F , Meighen E A
Published: November 2016

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.
Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules
Authors: Scott D B , Wilson R , Shaw G J , Petit A , Tempe J
Published: November 2016

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.
DNA element of Corynebacterium diphtheriae with properties of an insertion sequence and usefulness for epidemiological studies
Authors: Rappuoli R , Perugini M , Ratti G
Published: November 2016

The segment of DNA which is inserted within the tox gene of bacteriophage gamma and is responsible for its Tox- phenotype was found to be present and repeated approximately 30 times in the chromosome of Corynebacterium diphtheriae Belfanti 1030. Other C. diphtheriae strains contained a variable number of copies (1 to 25) of the same element. Sequence analysis showed that this repeated and interspersed DNA element was flanked by 9-base-pair direct repeats and that the 5' and 3' ends of the insertion contained sequences forming an imperfect inverted repeat. Therefore, the DNA segment here described has most of the typical structural features of a bacterial insertion sequence element. We show that different C. diphtheriae isolates derived from the same outbreak of diphtheria have an identical genomic distribution of this DNA element and that such DNA can be useful for epidemiological studies.
Characterization of nonattaching mutants of Agrobacterium tumefaciens
Authors: Matthysse A G
Published: November 2016

The first step in tumor formation by Agrobacterium tumefaciens is the site-specific binding of the bacteria to plant host cells. Transposon mutants of the bacteria which fail to attach to carrot suspension culture cells were isolated. These mutants showed no significant attachment to carrot cells with either microscopic or viable cell count assays of bacterial binding. The nonattaching mutants were all avirulent. When revertants of the mutants were obtained by enriching for bacteria which do bind to carrot cells, the bacteria were found to have regained the ability to bind to carrot cells and virulence simultaneously. These results suggest that the ability of the bacteria to bind to plant cells is required for virulence. Like the parent strain, all of the nonattaching mutants synthesized cellulose, but unlike the parent strain, they failed to aggregate carrot suspension culture cells. The transposon Tn5, which was used to obtain the mutants, was located on a 12-kilobase EcoRI fragment of the bacterial chromosomal DNA in all of the nonattaching mutants from strain C58. That the mutant phenotype was due to the Tn5 insertion was shown by cloning the Tn5-containing DNA fragment from the mutant bacteria and using it to replace the wild-type fragment in the parent strain by marker exchange. The resulting bacteria had the same mutant phenotype as the original Tn5 mutants; they did not attach to carrot cells, they did not cause the aggregation of carrot cells, and they were avirulent. No difference was seen between the parent strain and the nonattaching mutants in hydrophobicity, motility, flagella, fimbriae, beta-2-glucan content, size of lipopolysaccharide, or ability of the lipopolysaccharide to inhibit bacterial attachment to tissue culture cells. Differences were seen between the parent strain and the nonattaching mutants in the polypeptides removed from the bacteria during the preparation of spheroplasts. Three of the mutants were lacking a polypeptide of about 34 kilodaltons (kDa). One mutant was lacking the 34-kDa polypeptide and another polypeptide of about 38 kDa. The fifth mutant was lacking a polypeptide slightly smaller than the 34-kDa polypeptide missing in the other four mutants. These missing polypeptides all reappeared in the revertants of the mutants. Thus, bacterial binding to plant cells appears to require the presence of these polypeptides.
Rec dependence of mu transposition from P22-transduced fragments
Authors: Hughes K T , Olivera B M , Roth J R
Published: November 2016

Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics. Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22. When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome. Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion. First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome. Second, transposition requires the host recA, B, and C functions. Transposition of Mu following injection by a Mu virion is rec independent. While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22. We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.
Benzoate and muconate, structurally dissimilar metabolites, induce expression of catA in Acinetobacter calcoaceticus
Authors: Neidle E L , Ornston L N
Published: November 2016

Biosynthetic regulation of catA, the gene encoding catechol 1,2-dioxygenase (EC 1.13.1.1), was studied in an Acinetobacter calcoaceticus mutant strain unable to metabolize benzoate. Benzoate and muconate independently induced the enzyme. In glucose-grown cells, benzoate yielded higher enzyme levels than did muconate, whereas muconate was the more effective inducer in succinate-grown cells.
Intracellular 5',5'-dinucleoside polyphosphate levels remain constant during the Escherichia coli cell cycle
Authors: Plateau P , Fromant M , Kepes F , Blanquet S
Published: November 2016

All AppppN and ApppN nucleotides (N = A, C, G, or U) occur in Escherichia coli. Measured cellular concentrations were 2.42 microM AppppA, 0.61 microM AppppC, 0.95 microM AppppG, 1.17 microM AppppU, 0.47 microM ApppA, 0.14 microM ApppC, 0.20 microM ApppG, and 0.12 microM ApppU. These concentrations remained constant during the cell cycle in synchronized exponentially growing cells.
Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system
Authors: Jenkins L S , Nunn W D
Published: November 2016

The structural organization and regulation of the genes involved in short-chain fatty acid degradation in Escherichia coli, referred to as the ato system, have been studied by a combination of classic genetic and recombinant DNA techniques. A plasmid containing a 6.2-kilobase region of the E. coli chromosome was able to complement mutations in the ato structural genes, atoA (acetyl-coenzyme A [CoA]:acetoacetyl [AA]-CoA transferase) and atoB (thiolase II), as well as mutations in the ato regulatory locus, atoC. Complementation studies performed with mutants defective in acetyl-CoA:AA-CoA transferase suggest that two loci, atoD and atoA, are required for the expression of functional AA-CoA transferase. The ato gene products were identified by in vitro transcription and translation and maxicell analysis as proteins of 48, 26.5, 26, and 42 kilodaltons for atoC, atoD, atoA, and atoB, respectively. In vitro and insertional mutagenesis of the ato hybrid plasmid indicated that the ato structural genes were arranged as an operon, with the order of transcription atoD-atoA-atoB. Although transcribed in the same direction as the atoDAB operon, the atoC gene appeared to use a promoter which was distinct from that used by the atoDAB operon. A delta atoC plasmid expressed the atoD, atoA, and atoB gene products only in strains containing a functional atoC gene. Although the exact mechanism of control was not evident from these studies, the data suggest that the atoC gene product is an activator which is required for the synthesis or activation of the atoDAB-encoded enzymes.
Transposition of IS91 does not generate a target duplication
Authors: Diaz-Aroca E , Mendiola M V , Zabala J C , de la Cruz F
Published: November 2016

We determined the DNA sequences surrounding the junctions of IS91 in two insertion derivatives: pSU234 (pACYC184::IS91) and pSU240 (pBR322::IS91). The termini of IS91 consist of two imperfect inverted repeats eight base pairs long. Their sequence is 5'-TCGAGTAGG. . . CCTATCGA-3'. Insertion of IS91 did not generate direct repetitions in the target DNAs.
Haemophilus influenzae immunoglobulin A1 protease genes: cloning by plasmid integration-excision, comparative analyses, and localization of secretion determinants
Authors: Grundy F J , Plaut A , Wright A
Published: November 2016

Many bacteria which establish infections after invasion at human mucosal surfaces produce enzymes which cleave immunoglobulin A (IgA), the primary immunoglobulin involved with protection at these sites. Bacterial species such as Haemophilus influenzae which produce IgA1 proteases secrete this enzyme into their environment. However, when the gene encoding this protein was isolated from H. influenzae serotype d and introduced into Escherichia coli, the activity was not secreted into the medium but was localized in the periplasmic space. In this study, the IgA1 protease gene (iga) from an H. influenzae serotype c strain was isolated and the gene from the serotype d strain was reisolated. The IgA1 proteases produced in E. coli from these genes were secreted into the growth medium. A sequence linked to the carboxyl terminus of the iga gene but not present in the original clone was shown to be necessary to achieve normal secretion. Tn5 mutagenesis of the additional carboxyl-terminal region was used to define a 75- to 100-kilodalton coding region required for complete secretion of IgA1 protease but nonessential for protease activity. The iga genes were isolated by a plasmid integration-excision procedure. In this method a derivative of plasmid pBR322 containing a portion of the protease gene and the kanamycin resistance determinant of Tn5 was introduced into H. influenzae by transformation. The kanamycin resistance gene was expressed in H. influenzae, but since pBR322 derivatives are unable to replicate in this organism, kanamycin-resistant transformants arose by integration of the plasmid into the Haemophilus chromosome by homologous recombination. The plasmid, together with the adjoining DNA encoding IgA1 protease, was then excised from the chromosome with DNA restriction enzymes, religated, and reintroduced into E. coli. Comparisons between the H. influenzae protease genes were initiated which are useful in locating functional domains of these enzymes.
Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis
Authors: Guffanti A A , Clejan S , Falk L H , Hicks D B , Krulwich T A
Published: November 2016

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.
Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli
Authors: Balke V L , Gralla J D
Published: November 2016

The supercoiling levels of plasmid DNA were determined from Escherichia coli which was grown in ways that are known to alter global patterns of gene expression and metabolism. Changes in DNA supercoiling were shown to occur during several types of these nutrient upshifts and downshifts. The most dramatic change in supercoiling was seen in starved cells, in which two populations of differentially relaxed plasmids were shown to coexist. Thus, some changes in the external nutritional environment that cause the cells to reorganize their global metabolism also cause accompanying changes in DNA supercoiling. Results of experiments with dinitrophenol suggested that the observed relaxations were probably not due to reduced pools of ATP. When rifampin was used to release supercoils restrained by RNA polymerase, the cellular topoisomerases responded by removing these new, unrestrained supercoils. We interpret these results as implying that the cellular topological machinery maintains a constant superhelical energy in the DNA except during certain growth transitions, when changes in metabolism and gene expression are accompanied by changes in DNA supercoiling.
Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function
Authors: Torres-Cabassa A , Gottesman S , Frederick R D , Dolph P J , Coplin D L
Published: November 2016

A primary determinant of pathogenicity in Erwinia stewartii is the production of extracellular polysaccharide (EPS). A single mutation can abolish both EPS synthesis and pathogenicity; both properties are restored by a single cosmid clone. Subcloning and insertion analysis have defined a single positive regulatory function which shares a number of similarities with the rcsA function of Escherichia coli K-12, a positive regulator for capsular polysaccharide synthesis. In E. stewartii, the gene promotes the transcription of at least two operons (cps) involved in EPS synthesis; we have previously demonstrated a similar function for rcsA in E. coli. Both genes code for proteins of 25 to 27 kilodaltons; both proteins are unstable in E. coli. The E. stewartii RcsA protein was stabilized in E. coli lon mutants, as the RcsA product from E. coli is. The E. stewartii function complemented E. coli rcsA mutants, and the E. coli RcsA function increased cps expression and restored virulence in E. stewartii mutants. Therefore, these two gram-negative organisms share a similar component of their regulatory circuitry for the control of capsular polysaccharide synthesis.
Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity
Authors: Van Dyk T K , Smulski D R , Chang Y Y
Published: November 2016

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.
Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA
Authors: Sharma R C , Smith K C
Published: November 2016

Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation. The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair. The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene. When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain. Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation. The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain. These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.
Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin
Authors: Sandkvist M , Hirst T R , Bagdasarian M
Published: November 2016

The gene encoding the B subunit of heat-labile enterotoxin (etxB) was mutated at its 3' end by targeted addition of random nucleotide sequences. Gene products from five mutated etxB genes, all of which were shown to encode B subunits with short carboxy-terminal amino acid extensions, were analyzed with respect to a range of functional and structural properties. One class of altered B subunits, exemplified by EtxB124 and EtxB138, which both have seven extra amino acid residues, were found to be specifically defective in their ability to stably associate with A subunits and form holotoxin. Other altered B subunits were less subtlely affected by extensions at their C termini and were, in addition to their failure to associate with A subunits, unable to translocate into the periplasm of Escherichia coli, to pentamerize, or to bind to GM1 ganglioside. This suggests that the carboxy-terminal domain of EtxB mediates A subunit-B subunit interaction.
Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes
Authors: Shen B F , Tai P C , Pritchard A E , Vasil M L
Published: November 2016

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.
dnaA, an essential host gene, and Tn5 transposition
Authors: Yin J C , Reznikoff W S
Published: November 2016

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.
Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance
Authors: Boe L , Gerdes K , Molin S
Published: November 2016

Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.
Allantoin transport in Saccharomyces cerevisiae is regulated by two induction systems
Authors: Cooper T G , Chisholm V T , Cho H J , Yoo H S
Published: November 2016

We show that the allantoin transport system of Saccharomyces cerevisiae responds to two induction systems, one mediated by allophanate or its analog oxalurate and the other mediated by allantoin or its analog hydantoin acetate. The effects of the two inducers were additive in strain M85. Like other allantoin pathway genes, oxalurate-mediated induction of allantoin transport required a functional DAL81 gene product. Hydantoin acetate-mediated induction of the system, on the other hand, occurred normally in dal81 mutants. This suggests that induction was not only mediated by two separate inducers, but also involved different regulatory proteins. Induction is probably a transcriptionally regulated process, because addition of hydantoin acetate or oxalurate to the culture medium increased the steady-state levels of mRNA encoded by a gene required for allantoin transport (DAL4).
Regulation and initiation of cenB transcripts of Cellulomonas fimi
Authors: Greenberg N M , Warren R A , Kilburn D G , Miller R C
Published: November 2016

We characterized the in vivo transcription of the Cellulomonas fimi cenB gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4). By Northern blot (RNA blot) analysis, cenB mRNA was detected in C. fimi RNA preparations from glycerol-, glucose-, and carboxymethyl cellulose (CMC)-grown cells. The relative abundance of the specific mRNAs in these preparations appeared to depend on the carbon source provided, with the preparations from CMC-grown cells having the greatest amount of cenB transcripts, followed by glycerol- and glucose-grown cells. Therefore, the transcription of this gene could be regulated by the carbon source provided to C. fimi. High-resolution nuclease S1 protection studies were used to map cenB mRNA 5' termini with a unique 5'-labeled DNA probe and C. fimi RNA isolated in vivo. With this procedure, three 5' termini were found in abundance upstream of the translational initiation ATG codon in RNA preparations from C. fimi grown on CMC, while less-abundant 5' termini were found 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results are indicative of a tandem promoter arrangement, with the ATG-proximal promoter directing constitutive low-level cenB transcription and the more distal promoter directing higher levels of transcription under the inducing effects of the cellulosic substrate. The corresponding transcripts were not detected in S1 mapping experiments with RNA isolated in vivo from Escherichia coli clones harboring recombinant plasmids carrying C. fimi genomic inserts. Comparative analysis of the 5' -flanking DNA sequences of the cenB gene and the cenA and cex genes of C. fimi (N. M. Greenberg, R. A. J. Warren, D. G. Kilburn, and R. C. Miller, Jr., J. Bacteriol. 169:646-653, 1987) revealed a region of 50 bases in which these sequences displayed at least 64% homology.
Characterization of Pi-repressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants
Authors: Furukawa K , Hasunuma K , Shinohara Y
Published: November 2016

In wild-type mycelial cultures of Neurospora crassa under Pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases I, II, III, and IV, 5'-nucleotidase, acid and alkaline nucleases, RNase N1, and a newly detected endonuclease were secreted into the culture media. These enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4. The proteins were examined by polyacrylamide gel electrophoresis in a manner which allowed the identification of each of them.
Replication control genes of plasmid pE194
Authors: Villafane R , Bechhofer D H , Narayanan C S , Dubnau D
Published: November 2016

pE194, a 3.7-kilobase plasmid, confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. The previously identified cop and repF genes of pE194 have been further localized by molecular cloning and mutational analysis together with DNA sequencing. The CfoIB fragment of pE194 is capable of autonomous replication and contains both genes. Most of this region has been resequenced, and two errors reported in a previous study have been corrected. The corrected sequence indicates that the replication region contains a single large open reading frame, which we propose encodes the repF product. Northern blot (RNA blot) analysis of this region detected six transcripts, all transcribed in the same direction as one another and opposite to repF. A 350-base transcript is synthesized from the region containing cop. No in vivo transcript for the repF gene was detected, but a protein was observed in an in vitro transcription-translation system which appears to be its product. An ochre mutation was inserted in the putative repF open reading frame, and a nonsense fragment was detected in the in vitro system. When carried passively on a pUB110 replicon, this mutant product appears capable of inhibiting pE194 replicons in trans. The pE194 origin of replication has been localized to within 200 bases.