bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Escherichia coli glnB, prsA, and speA by the purine repressor
Authors: He B , Choi K Y , Zalkin H
Published: November 2016

A strategy was devised to identify Escherichia coli genes subject to coregulation by purR. From a data base search, similarities to the pur regulon cis-acting control site were found in 26 E. coli genes. Of five genes examined in which the putative pur operator is upstream of the coding sequence, glnB, prsA, and speA bound purified purine repressor in vitro. Binding of the repressor to a pur operator in these genes was dependent upon a corepressor. The pur operator in glnB is located between two major transcription start sites that were located by primer extension mapping. The effect of purR on expression of glnB, prsA, and speA was examined by using a lacZ reporter. The results indicated two- to threefold repression of these genes by purR. Coregulation by purR provides evidence that expands the pur regulon to include glnB, prsA, and speA. These genes have functions related to nucleotide metabolism.
Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E
Authors: Smith K , Youngman P
Published: November 2016

We have investigated the temporal and spatial regulation of spoIIM, a gene of Bacillus subtilis whose product is required for complete septum migration and engulfment of the forespore compartment during sporulation. The spoIIM promoter was found to become active about 2 h after the initiation of sporulation. The effects of mutations on the expression of a spoIIM-lacZ fusion were most consistent with its utilization by sigma-E-associated RNA polymerase (E sigma E). A unique 5' end of the in vivo spoIIM transcript was detected by primer extension analysis and was determined to initiate at the appropriate distance from a sequence conforming very closely to the consensus for genes transcribed by E sigma E. A partially purified preparation of E sigma E produced a transcript in vitro that initiated at the same nucleotide as the primer extension product generated from in vivo RNA. Ectopic induction of sigma E synthesis during growth resulted in the immediate and strong expression of a spoIIM-lacZ fusion, but an identical fusion was completely unresponsive to induced synthesis of either sigma F or sigma G under similar conditions. The results of plasmid integration-excision experiments in which the spoIIM gene was reversibly disrupted by a temperature-sensitive integrational vector suggested that spoIIM expression is required in the forespore compartment, but direct examination of subcellular fractions enriched for mother cell or forespore material indicated that spoIIM expression cannot be confined to the forespore. We conclude that spoIIM is a member of the sigma E regulon and that it may be transcribed exclusively by E sigma E. We discuss the implications of this conclusion for models in which activation of sigma E in the mother cell is proposed to be a part of the mechanism responsible for initiating separate programs of gene activity in the two sporangium compartments.
High-efficiency gene inactivation and replacement system for gram-positive bacteria
Authors: Biswas I , Gruss A , Ehrlich S D , Maguin E
Published: November 2016

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.
The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021
Authors: Reuber T L , Walker G C
Published: November 2016

Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules. We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent. The exoZ mutant nodules alfalfa normally.
Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium
Authors: Moukha S M , Wösten H A , Mylius E J , Asther M , Wessels J G
Published: November 2016

Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA.
Oxidation of D-lactate and L-lactate by Neisseria meningitidis: purification and cloning of meningococcal D-lactate dehydrogenase
Authors: Erwin A L , Gotschlich E C
Published: November 2016

Neisseria meningitidis was found to contain at least two lactate-oxidizing enzymes. One of these was purified 460-fold from spheroplast membranes and found to be specific primarily for D-lactate, with low-affinity activity for L-lactate. The gene for this enzyme (dld) was cloned, and a dld mutant was constructed by insertional inactivation of the gene. The mutant was unable to grow on D-lactate but retained the ability to grow on L-lactate, providing evidence for a second lactate-oxidizing enzyme with specificity for L-lactate. High-affinity L-lactate-oxidizing activity was detected in intact bacteria of both the dld+ and dld mutant strains. This L-lactate-oxidizing activity was also seen in sonicated bacteria but was reduced substantially on detergent solubilization or on preparation of spheroplast membranes.
The role of single-mutant intermediates in the generation of trpAB double revertants during prolonged selection
Authors: Hall B G
Published: November 2016

Selection-induced mutations are nonrandom mutations that occur as specific, direct responses to environmental challenges and that occur more often when they are selectively advantageous than when they are selectively neutral. One of the most puzzling examples of selection-induced mutations involved the simultaneous reversions of two mutations, one in trpA and the other in trpB, at rates that were several orders of magnitude greater than would have been predicted if the two mutations had occurred as independent events (B. G. Hall, Proc. Natl. Acad. Sci. USA 88:5882-5886, 1991). Here I examine the possibility that the double mutations might be accounted for by sequential mutations with intervening growth.
Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli
Authors: Lai X , Ingram L O
Published: November 2016

Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.
recA mutations that reduce the constitutive coprotease activity of the RecA1202(Prtc) protein: possible involvement of interfilament association in proteolytic and recombination activities
Authors: Liu S K , Eisen J A , Hanawalt P C , Tessman I
Published: November 2016

Twenty-eight recA mutants, isolated after spontaneous mutagenesis generated by the combined action of RecA1202(Prtc) and UmuDC proteins, were characterized and sequenced. The mutations are intragenic suppressors of the recA1202 allele and were detected by the reduced coprotease activity of the gene product. Twenty distinct mutation sites were found, among which two mutations, recA1620 (V-275-->D) and recA1631 (I-284-->N), were mapped in the C-terminal portion of the interfilament contact region (IFCR) in the RecA crystal. An interaction of this region with the part of the IFCR in which the recA1202 mutation (Q-184-->K) is mapped could occur only intermolecularly. Thus, altered IFCR and the likely resulting change in interfilament association appear to be important aspects of the formation of a constitutively active RecA coprotease. This observation is consistent with the filament-bundle theory (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 335:318-325, 1992). Furthermore, we found that among the 20 suppressor mutations, 3 missense mutations that lead to recombination-defective (Rec-) phenotypes also mapped in the IFCR, suggesting that the IFCR, with its putative function in interfilament association, is required for the recombinase activity of RecA. We propose that RecA-DNA complexes may form bundles analogous to the RecA bundles (lacking DNA) described by Story et al. and that these RecA-DNA bundles play a role in homologous recombination.
Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation
Authors: Andrup L , Damgaard J , Wassermann K
Published: November 2016

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.
Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli
Authors: Atlung T , Hansen F G
Published: November 2016

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.
Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti
Authors: Margolin W , Long S R
Published: November 2016

A 4-kb fragment active as an autonomously replicating sequence (ARS) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R. meliloti. The resulting Escherichia coli-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required. The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection. An 0.8-kb DNA subfragment was sufficient for replication in both R. meliloti and A. tumefaciens. The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats. Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication. These results suggest that the R. meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication origins. Since the mini-pSym-b shuttle vector can coexist with IncP-1 broad-host-range plasmids, it is also now possible to use two compatible plasmids for cloning and genetic manipulation in R. meliloti.
A cdc-like autolytic Saccharomyces cerevisiae mutant altered in budding site selection is complemented by SPO12, a sporulation gene
Authors: Molero G , Yuste-Rojas M , Montesi A , Vázquez A , Nombela C , Sanchez M
Published: November 2016

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lyt1-1 allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37 degrees C, lyt1 haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyt1-1/lyt1-1 diploids failed to sporulate at both 24 and 37 degrees C. Taking into account these characteristics, the lyt1 mutant could be considered a cdc-like mutant. By genetic transformation of an appropriate lyt1 strain with a genomic library, ligated to the multicopy vector YEp13, we isolated a gene capable of complementing mitotic alterations but not the meiotic defect. This was the sporulation-specific gene SPO12, which is expressed under the control of the locus MAT in meiosis and is also expressed in the mitotic cycle (V. Parkes and L. H. Johnston, Nucleic Acids Res. 20:5617-5623, 1992). A significant level of SPO12 mRNA can be detected when this gene is inserted in a multicopy plasmid.
The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence
Authors: Mantis N J , Winans S C
Published: November 2016

In an effort to identify the Agrobacterium tumefaciens phosphate regulatory gene(s), we isolated a clone from an A. tumefaciens cosmid library that restored regulated alkaline phosphatase activity to an Escherichia coli phoB mutant. The gene that complemented phoB was localized by subcloning and deletion analysis, and the DNA sequence was determined. An open reading frame, denoted chvI, was identified that encoded a predicted protein with amino acid similarity to the family of bacterial response regulators and 35% identify to PhoB. Surprisingly, an A. tumefaciens chvI mutant showed normal induction of phosphatase activity and normal virG expression when grown in phosphate-limiting media. However, this mutant was unable to grow in media containing tryptone, peptone, or Casamino Acids and was also more sensitive than the wild type to acidic extracellular pH. This mutant was avirulent on Kalanchoeë diagremontiana and was severely attenuated in vir gene expression. The pH-inducible expression of virG was also abolished. Growth of the chvI mutant was inhibited by K. diagremontiana wound sap, suggesting that avirulence may be due, in part, to the inability of this mutant to survive the plant wound environment.
Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture
Authors: Salman M , Shirazi I , Tarshis M , Rottem S
Published: November 2016

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.
Stringency and relaxation among the halobacteria
Authors: Cimmino C , Scoarughi G L , Donini P
Published: November 2016

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.
Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa
Authors: Chapalmadugu S , Chaudhry G R
Published: November 2016

A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was necessary for deaggregation and purification of the hydrolase. This is the first membrane-bound hydrolase involved in the hydrolysis of any methylcarbamate pesticide purified from a bacterial source to date. The enzyme exhibited a unique specificity of hydrolyzing only carbaryl (1-naphthyl N-methylcarbamate) but not any other methylcarbamates. The purified enzyme was a monomer with a molecular mass of 65,000 Da. The pH and temperature optima for the enzyme activity were 8.5 and 45 degrees C, respectively. No cofactor requirement for the hydrolase activity could be demonstrated, and none of the divalent cations studied affected the activity of the enzyme. Also, the enzyme activity was not affected by the thiols: dithioerythritol, dithiothreitol, and 2-mercaptoethanol. The Km and Vmax values for carbaryl were 9 microM and 7.9 mumol/min/mg of protein, respectively.
Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems
Authors: Girbés T , Barbieri L , Ferreras M , Arias F J , Rojo M A , Iglesias R , Alegre C , Escarmis C , Stirpe F
Published: November 2016

The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations.
Absence of a direct role for RNase HI in initiation of DNA replication at the oriC site on the Escherichia coli chromosome
Authors: Hong X , Kogoma T
Published: November 2016

On the basis of the experiments carried out with rnhA224 mutants, we previously concluded that RNase HI is not essential for initiation of Escherichia coli chromosome replication at oriC (T. Kogoma, N.L. Subia, and K. von Meyenburg, Mol. Gen. Genet. 200:103-109, 1985). In light of the recent finding that rnhA224 is a UGA nonsense mutation which can be leaky in certain genetic backgrounds, we reexamined this conclusion with the use of rnhA339 (Null)::cat mutants. The possibility that recB+ is required for initiation at the alternative origins (oriKs) of replication in rnhA mutants was also tested. The results clearly indicated that RNase HI is not essential for oriC initiation and that recB+ is not required for initiation at oriK sites.
Hypersensitive sites in the 5' promoter region of nit-3, a highly regulated structural gene of Neurospora crassa
Authors: Brito N , Gonzalez C , Marzluf G A
Published: November 2016

The nit-3 gene of the filamentous fungus Neurospora crassa encodes nitrate reductase, the enzyme which catalyzes the first step in nitrate assimilation. The nit-3 gene is subject to a high degree of regulation by metabolic inducers and repressors, and its expression requires two distinct trans-acting regulatory proteins. Hypersensitive sites in the 5' DNA sequence upstream of the nit-3 gene were mapped with the use of three different nucleases as molecular probes. Six hypersensitive sites, three of which are very strong, were detected at essentially identical positions by all three nucleases. The hypersensitive sites appear to develop in a constitutive fashion and are present under conditions in which the nit-3 structural gene is expressed but also when this gene is inactive, although these sites are considerably less prominent in cells subjected to nitrogen catabolite repression. The presence of the hypersensitive sites appears to depend upon both the positively acting NIT2 and the positively acting NIT4 regulatory proteins, which might play a role in positioning of chromatin protein.
Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus
Authors: Heinrichs J H , Beecher D J , MacMillan J D , Zilinskas B A
Published: November 2016

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.
Role of the imp operon of the Streptomyces coelicolor genetic element SLP1: two imp-encoded proteins interact to autoregulate imp expression and control plasmid maintenance
Authors: Shiffman D , Cohen S N
Published: November 2016

The Streptomyces coelicolor genetic element SLP1 can exist either integrated into the host chromosome or as an autonomously replicating plasmid. The integrated form of SLP1 includes a locus (imp, for inhibition of plasmid maintenance) that can act both in cis and in trans to prevent propagation of SLP1 as an extrachromosomal replicon (S. R. Grant, S. C. Lee, K. Kendall, and S. N. Cohen, Mol. Gen. Genet. 217:324-331, 1989). We report here that a 1.8-kb Eco47III DNA fragment previously shown to encode the Imp+ phenotype contains two genes (impA and impC) that must be expressed in cis to each other and whose products interact functionally and probably physically to interfere with SLP1 plasmid maintenance and repress expression of the imp operon. Partial repression of the imp promoter (P(imp)), which is located immediately 5' of impA, by the 29.7-kDa ImpA protein is enhanced by the impC gene product. Gel shift analysis indicates that ImpA binds to a 16-bp sequence located within the DNA segment containing P(imp) and that ImpC interferes with this binding. Our data suggest that binding of ImpA to the P(imp) region mediates DNA looping in this region.
Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus
Authors: Yakunin A F , Gennaro G , Hallenbeck P C
Published: November 2016

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.
chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100
Authors: Masuda Y , Miyakawa K , Nishimura Y , Ohtsubo E
Published: November 2016

The pem locus is responsible for stable maintenance of plasmid R100 and consists of two genes, pemI and pemK. The pemK gene product is a growth inhibitor, while the pemI gene product is a suppressor of this inhibitory function. We found that the PemI amino acid sequence is homologous to two open reading frames from Escherichia coli called mazE and orf-83, which are located at 60 and 100 min on the chromosome, respectively. We cloned and sequenced these loci and found additional open reading frames, one downstream of each pemI homolog, both of which encode proteins homologous to PemK. The pem locus homolog at 60 min was named chpA and consists of two genes, chpAI and chpAK; the other, at 100 min, was named chpB and consists of two genes, chpBI and chpBK. The distal portion of chpBK was found to be adjacent to the ppa gene that encodes pyrophosphatase, whose map position had not been previously determined. We then demonstrated that the chpAK and chpBK genes encode growth inhibitors, while the chpAI and chpBI genes encode suppressors for the inhibitory function of the ChpAK and ChpBK proteins, respectively. These E. coli pem locus homologs may be involved in regulation of cell growth.
Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes
Authors: Reyrat J M , David M , Blonski C , Boistard P , Batut J
Published: November 2016

Oxygen concentration regulates the expression of nitrogen fixation genes in the symbiotic bacterium Rhizobium meliloti. We demonstrate that two proteins, FixL and FixJ, that belong to the two-component family of regulatory proteins are necessary and sufficient for oxygen-regulated in vitro transcription of the two key regulatory genes, nifA and fixK. We show directly that FixJ is a transcriptional activator, working in conjunction with the RNA polymerase sigma 70 holoenzyme. Addition of FixL122, a soluble form of the sensor FixL protein, to the transcription assay enhanced FixJ transcriptional activity in response to low oxygen concentration. This enhancement of FixJ activity was correlated with FixJ phosphorylation.
Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway
Authors: Denome S A , Stanley D C , Olson E S , Young K D
Published: November 2016

From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.
Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters
Authors: de Lorenzo V , Cases I , Herrero M , Timmis K N
Published: November 2016

Transcriptional lacZ fusions to the Pu and Pm promoters of the TOL (toluene degradation) plasmid inserted in monocopy in the chromosome of Pseudomonas putida showed a very different responsiveness to their respective aromatic effectors regarding growth phase. While a substantial XylS-dependent activation of Pm-lacZ was detected nearly instantly after m-toluate addition, XylR- and xylene-mediated induction of the sigma 54 promoter Pu became significant only after cells slowed down exponential growth and entered stationary phase. When Pu and Pm were fused to lacZ-tet reporters (i.e., promoterless lacZ genes coupled to a tet gene which confers resistance to tetracycline when cotranscribed with the leading gene) instead of lacZ alone, the resulting colonies displayed a distinct phenotype consisting of hyperfluorescence on agar plates after being sprayed with 4-methylumbelliferyl-beta-D-galactoside, simultaneously with being either sensitive (Pu) or resistant (Pm) to tetracycline. To examine whether the same phenotype could be scored in strains carrying transcriptional fusions of the lacZ-tet cassettes to other genes or promoters whose expression is silenced during growth and activated in stationary phase, we constructed mini-Tn5 lacZ-tet transposons for random genetic probing of promoters preferentially active at advanced stages of growth. Chromosomal insertions of this mobile element were selected by means of the constitutive resistance to kanamycin which is also specified by the transposon. A number of kanamycin-resistant colonies which are hyperfluorescent with 4-methylumbelliferyl-beta-D-galactosidase but sensitive to tetracycline and which reached full induction only at postexponential growth stages were obtained.
Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti
Authors: Glucksmann M A , Reuber T L , Walker G C
Published: November 2016

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.
Antioxidant function of fungal melanin
Authors: Jacobson E S , Tinnell S B
Published: November 2016

Polyphenols have been implicated in the virulence and oxidant resistance of Cryptococcus neoformans. Although monomeric polyphenols did not protect against the prooxidant, plumbagin, polymeric dopamine-melanin conferred resistance both to hypochlorite and to permanganate. The physiologic antioxidant capacity conferred by melanin was found to be 21.3 x 10(-15) mole-equivalents per cell, a value which approximates oxidant production by stimulated macrophages.
Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii
Authors: Coleman J P , Hudson L L , Adams M J
Published: November 2016

A bile acid-inducible NADP-linked 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Clostridium sordellii ATCC 9714 was purified 310-fold by ion-exchange, gel filtration, and dye-ligand affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed one predominant peptide band (30,000 Da). The N-terminal sequence was determined, and the corresponding oligonucleotides were synthesized and used to screen EcoRI and HindIII genomic digests of C. sordellii. Two separate fragments (4,500 bp, EcoRI; 3,200 bp, HindIII) were subsequently cloned by ligation to pUC19 and transformation into Escherichia coli DH5 alpha-MCR. The EcoRI fragment was shown to contain a truncated 7 alpha-HSDH gene, while the HindIII fragment contained the entire coding region. E. coli clones containing the HindIII insert expressed high levels of an NADP-linked 7 alpha-HSDH. Nucleotide sequence analyses suggest that the 7 alpha-HSDH is encoded by a monocistronic transcriptional unit, with DNA sequence elements resembling rho-independent terminators located in both the upstream and downstream flanking regions. The transcriptional start site was located by primer extension analysis. Northern (RNA) blot analysis indicated that induction is mediated at the transcriptional level in response to the presence of bile acid in the growth medium. In addition, growth-phase-dependent expression is observed in uninduced cultures. Analysis of the predicted protein sequence indicates that the enzyme can be classified in the short-chain dehydrogenase group.
Characterization of pilin genes from seven serologically defined prototype strains of Moraxella bovis
Authors: Atwell J L , Tennent J M , Lepper A W , Elleman T C
Published: November 2016

Numerous field isolates of Moraxella bovis have previously been classified by serological techniques into seven serogroups, each defined by homologous cross-reaction with antisera prepared against purified pili of a single prototype strain. The gene encoding pilin from each of the prototype strains has been characterized by nucleotide sequence determination. The coding sequences show extensive homology (70 to 80%) while the proximal downstream sequences show a dichotomy into nonhomologous sets. The pilin genes of three more strains were also characterized. The presence of an additional, partial pilin gene in each prototype strain was confirmed by Southern blot analysis, and the partial pilin genes from two strains of one serogroup were characterized by sequence determination. Features of the pilin gene sequences are considered in relation to pilin gene inversion and the serological variants of strains which may arise from gene inversion events.
Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus
Authors: Lee J C , Xu S , Albus A , Livolsi P J
Published: November 2016

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.
Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis
Authors: Savarino S J , Fox P , Deng Y , Nataro J P
Published: November 2016

The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit.