bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS
Authors: Haak B , Fetzner S , Lingens F
Published: November 2016

The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxylation of 2-halobenzoates with concomitant release of halogenide and carbon dioxide, yielding catechol. The gene cluster encoding this enzyme, cbdABC, was localized on a 70-kbp conjugative plasmid designated pBAH1. The nucleotide sequences of cbdABC and flanking regions were determined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bind the Rieske-type [2Fe-2S] cluster was identified. In the NADH:acceptor reductase component (CbdC), a putative binding site for a chloroplast-type [2Fe-2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show significant homology to benABC, which encode benzoate 1,2-dioxygenase from Acinetobacter calcoaceticus (52% identity at the deduced amino acid level), and to xylXYZ, which encode toluate 1,2-dioxygenase from Pseudomonas putida mt-2 (51% amino acid identity). Recombinant pkT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wild-type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also allowed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Functional NADH:acceptor reductase and oxygenase components of 2-halobenzoate 1,2-dioxygenase were enriched from recombinant Pseudomonas clones.
Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA
Authors: Killmann H , Videnov G , Jung G , Schwarz H , Braun V
Published: November 2016

Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and phi 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and phi 80 and colicin M.
Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia coli mreB morphogene
Authors: Abhayawardhane Y , Stewart G C
Published: November 2016

A gene with substantial sequence similarity to the mreB morphogene of Bacillus subtilis has been identified at 302 degrees on the chromosomal map by A. Decatur, B. Kunkel, and R. Losick (Harvard University; personal communication). Our characterization has revealed that the protein product of this determinant (termed mbl for mreB-like) is 55 and 53% identical in sequence to the MreB proteins of B. subtilis and Escherichia coli, respectively. The protein is 86% identical to a protein identified as MreB from Bacillus cereus, suggesting that the B. cereus protein is actually Mbl. Insertional inactivation of mbl indicated that this gene is not essential for cell viability or sporulation. Cells bearing mutant mbl alleles display a decreased growth rate and an altered cellular morphology. The cells appear bloated and are frequently twisted. Intergenic suppressor mutations which restore the growth rate to an approximately normal level arise within the mutant population. A second site mutation, designated som-1, was mapped to the hisA-mbl region of the chromosome by transduction.
Regulated expression of the histidase structural gene in Streptomyces griseus
Authors: Wu P C , Srinivasan K V , Kendrick K E
Published: November 2016

The histidase structural gene from Streptomyces griseus was expressed from a leaderless, monocistronic transcript. Multiple copies of the DNA located upstream of the hutH transcription initiation site led to a significant level of histidase activity when present in trans in the wild-type strain grown under noninducing conditions.
Methanobacterium formicicum, a mesophilic methanogen, contains three HFo histones
Authors: Darcy T J , Sandman K , Reeve J N
Published: November 2016

The mesophilic methanogen Methanobacterium formicicum JF-1 has been shown to contain three members of the HMf family of archaeal histones, designated HFoA1, HFoA2, and HFoB, and their encodinig genes (hfoA1, hfoA2, and hfoB) have been cloned and sequenced. The HFo histones have primary sequences that are 75 to 82% identical to the HMf sequences and appear to share ancestry with the core histones that form the eukaryal nucleosome. The HFo proteins bind and compact DNA molecules into nucleosome-like structures apparently identical to those formed by the HMf proteins, but, in contrast to the HMf proteins, this activity of the HFo proteins is lost after incubation at 95 degrees C for 5 h.
Proton pumping and the internal pH of yeast cells, measured with pyranine introduced by electroporation
Authors: Peña A , Ramírez J , Rosas G , Calahorra M
Published: November 2016

The internal pH of yeast cells was determined by measuring the fluorescence changes of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), which was introduced into the cells by electroporation. This may be a suitable procedure for the following reasons. (i) Only minor changes in the physiological status of the cells seemed to be produced. (ii) The dye did not seem to leak at a significant rate from the cells. (iii) Different incubation conditions produced large fluorescence changes in the dye, which in general agree with present knowledge of the proton movements of the yeast cell under different conditions. (iv) Pyranine introduced by electroporation seemed to be located in the cytoplasm and to avoid the vacuole, and therefore it probably measured actual cytoplasmic pH. (v) Correction factors to obtain a more precise estimation of the internal pH are not difficult to apply, and the procedure may be useful for other yeasts and microorganisms, as well as for the introduction of other substances into cells. Values for the cytoplasmic pHs of yeast cells that were higher than those reported previously were obtained, probably because this fluorescent indicator did not seem to penetrate into the cell vacuole.
Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers
Authors: Sajjan U S , Sun L , Goldstein R , Forstner J F
Published: November 2016

Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified.
Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454
Authors: Saluja D , Godson G N
Published: November 2016

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.
A test of the directed mutation hypothesis in Escherichia coli MCS2 using replica plating
Authors: Sniegowski P D
Published: November 2016

Excision of phage Mu from Escherichia coli MCS2 was originally put forth as a clear example of directed mutation. This claim was considerably weakened, however, by subsequent evidence that Mu excision occurs during starvation regardless of fitness consequences. Here, I use the classical replica-plating technique to examine Mu excision during starvation of MCS2. The results support the conclusion that Mu excision is not directed but is, instead, nonspecifically induced by starvation.
Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp
Authors: James G A , Korber D R , Caldwell D E , Costerton J W
Published: November 2016

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).
Mutational analysis of flagellum-independent surface spreading of Serratia marcescens 274 on a low-agar medium
Authors: Matsuyama T , Bhasin A , Harshey R M
Published: November 2016

In a previous study (J. O'Rear, L. Alberti, and R. M. Harshey, J. Bacteriol. 174:6125-6137, 1992) we reported the isolation of several transposon mutants of Serratia marcescens 274 that were defective either in swarming alone or in both swimming and swarming motility. All the nonflagellate (Fla-) mutants, while defective in both types of motility, were able to spread rapidly on the surface of low-agar (0.35%) media. We show here that some of the swarming-defective mutants are defective in the production of serrawettin W1, an extracellular cyclic lipopeptide produced by S. marcescens 274. When combined with a Fla defect, the serrawettin (Swt) mutants are deficient in spreading on low-agar media. The spreading deficiency can be overcome by serrawettin supplied extracellularly. Introduction of Fla defects into chemotaxis mutants does not affect this mode of surface translocation. These results suggest that spreading may be a passive form of translocation. We also report that swarming defects in all mutants showing a Dps phenotype (able to swarm within the inoculated area but unable to move outward) in the earlier study can be overcome by changing the commercial source of agar.
Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum
Authors: Kronemeyer W , Peekhaus N , Krämer R , Sahm H , Eggeling L
Published: November 2016

To assess the mechanism and function of the glutamate uptake system of gram-positive Corynebacterium glutamicum, a mutant deficient in glutamate uptake was isolated and was then used to isolate a DNA fragment restoring this deficiency. In a low-copy-number vector, this fragment resulted in an increased glutamate uptake rate of 4.9 nmol/min/mg (wild type, 1.5 nmol/min/mg). In addition, carbon source-dependent regulation of the glutamate uptake system was determined with the fragment, showing that the entire structures required for expression and control reside on the fragment isolated. Sequencing of 3,977 bp revealed the presence of a four-gene cluster (gluABCD) with deduced polypeptide sequences characteristic of a nucleotide-binding protein (GluA), a periplasmic binding protein (GluB), and integral membrane proteins (GluC and GluD), identifying the glutamate transporter as a binding protein-dependent system (ABC transporter). This identification was confirmed by the kinetic characteristics obtained for cells grown in the presence of globomycin, which exhibited an increased Km of 1,400 microM (without globomycin, the Km was 1.5 microM) but a nearly unaltered maximum velocity. By applying gene-directed mutagenesis, a strain with the entire cluster deleted was constructed. With this mutant, the glutamate uptake rate was reduced from 1.4 to less than 0.1 nmol/min/mg, which is proof that this system is the only relevant one for glutamate uptake. With this strain, the glutamate excretion rate was unaffected (18 nmol/min/mg), showing that no component of gluABCD is involved in export but rather that a specific machinery functions for the latter purpose.
Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome
Authors: Rozsa F W , Viollier P , Fussenegger M , Hiestand-Nauer R , Arber W
Published: November 2016

The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1. Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site. An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans. Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used. In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively. In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences. This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion. In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome. The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain. The mechanistic implications and evolutionary relevance of these findings are discussed.
Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin
Authors: Nagahama M , Okagawa Y , Nakayama T , Nishioka E , Sakurai J
Published: November 2016

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.
Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species
Authors: Kim E S , Cramer K D , Shreve A L , Sherman D H
Published: November 2016

Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act beta-ketoacyl synthase (beta-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 beta-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 beta-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.
Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin
Authors: Lewis L A , Dyer D W
Published: November 2016

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).
DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair
Authors: Mendonca V M , Klepin H D , Matson S W
Published: November 2016

DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.
Strain-dependent variation in carbon source regulation of nucleus-encoded mitochondrial proteins of Saccharomyces cerevisiae
Authors: Brown T A , Trumpower B L
Published: November 2016

Nuclear genes encoding mitochondrial proteins are regulated by carbon source with significant heterogeneity among four Saccharomyces cerevisiae strains. This strain-dependent variation is seen both in respiratory capacity of the cells and in the expression of beta-galactosidase reporter fusions to the promoters of CYB2, CYC1, CYC3, MnSOD, and RPO41.
Reshuffling of Rhs components to create a new element
Authors: Zhao S , Hill C W
Published: November 2016

RhsF has been identified as the fourth member of the RhsABCF subfamily of genetic elements. This new element is found in Escherichia coli ECOR-50 and several other strains but not in strain K-12. A novel feature of RhsF is that it represents a new arrangement of components previously uniquely associated with RhsA and RhsC of strain K-12.
Subinhibitory concentrations of antibiotics affect cell surface properties of Streptococcus sobrinus
Authors: Wu Q , Wang Q , Taylor K G , Doyle R J
Published: November 2016

Several antibiotics, at subinhibitory concentrations, caused an increase in the ability of Streptococcus sobrinus to bind alpha-1,6-glucans, whereas other antibiotics decreased glucan binding. In every case, glucan binding was inversely proportional to cell surface hydrophobicity. High levels of glucan-binding activity resulted in low levels of hydrophobicity, whereas low levels of glucan binding caused high levels of cellular hydrophobicity. The results show that low concentrations of antibiotics may modulate lectin and hydrophobin adhesins in streptococci.
The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole
Authors: Chen P , Ailion M , Weyand N , Roth J
Published: November 2016

The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12). Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12. In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon. The cobT gene is the last gene in the operon. Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame. Both the CobT protein of S. typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB. This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide. Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype. J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante Semerena have suggested (J. Bacteriol. 176:3568-3575, 1994) that S. typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity. We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate. Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation. Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide. Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype. Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU). Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.
Identification of a Bacillus subtilis spo0H allele that is necessary for suppression of the sporulation-defective phenotype of a spo0A mutation
Authors: Bramucci M G , Green B D , Ambulos N , Youngman P
Published: November 2016

A mutation in Bacillus subtilis spo0A codon 97 suppressed the sporulation defect caused by the spo0A9V mutation. The suppressor activity of the codon 97 mutation was evident only in the presence of a novel spo0H allele. Our results suggest that the spo0A gene product interacts with the sigma factor subunit of RNA polymerase.
Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein
Authors: Ariza R R , Li Z , Ringstad N , Demple B
Published: November 2016

Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively. These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC. We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes. Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate. The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake. Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology. Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA. Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M). Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS. These data point to a DNA-binding gene activator module used in different protein contexts.
Inhibition and restart of initiation of chromosome replication: effects on exponentially growing Escherichia coli cells
Authors: Bernander R , Akerlund T , Nordström K
Published: November 2016

Escherichia coli strains in which initiation of chromosome replication could be specifically blocked while other cellular processes continued uninhibited were constructed. Inhibition of replication resulted in a reduced growth rate and in inhibition of cell division after a time period roughly corresponding to the sum of the lengths of the C and D periods. The division inhibition was not mediated by the SOS regulon. The cells became elongated, and a majority contained a centrally located nucleoid with a fully replicated chromosome. The replication block was reversible, and restart of chromosome replication allowed cell division and rapid growth to resume after a time delay. After the resumption, the septum positions were nonrandomly distributed along the length axis of the cells, and a majority of the divisions resulted in at least one newborn cell of normal size and DNA content. With a transient temperature shift, a single synchronous round of chromosome replication and cell division could be induced in the population, making the constructed system useful for studies of cell cycle-specific events. The coordination between chromosome replication, nucleoid segregation, and cell division in E. coli is discussed.
Transcription activation at the Escherichia coli uhpT promoter by the catabolite gene activator protein
Authors: Merkel T J , Dahl J L , Ebright R H , Kadner R J
Published: November 2016

Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.
Genetic organization and mRNA expression of enolase genes of Candida albicans
Authors: Postlethwait P , Sundstrom P
Published: November 2016

In previous work, we cloned a Candida albicans cDNA for the glycolytic enzyme enolase and found a single, abundant enolase transcript on Northern (RNA) blots and a single protein on immunoblots, using antiserum raised against a recombinant enolase fusion protein. Because C. albicans enolase is abundantly produced during infection and elicits strong host immune responses, the mechanisms regulating enolase production are important for understanding the growth of C. albicans in vivo. To obtain more information on enolase gene expression by C. albicans, we used the enolase cDNA clone to investigate the genetic organization of enolase genes and the steady-state levels of enolase mRNA under several growth conditions. Gene disruption techniques in combination with Southern blot analyses of genomic DNA showed the presence of two enolase gene loci that could be distinguished by the locations of ClaI and Mn/I sites in their 3' flanking regions. Enolase steady-state mRNA levels were greatest during the middle phase of the logarithmic growth curve and were low during stationary phase. Minimal differences in enolase mRNA levels between yeast cells and hyphae were found. Propagation of C. albicans in glucose did not cause increased enolase mRNA levels compared with growth in a nonfermentable carbon source (pyruvate). It was concluded that two gene loci exist for C. albicans enolase and that enolase mRNA is constitutively produced at high levels during active metabolism.
Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export
Authors: Guilvout I , Carniel E , Pugsley A P
Published: November 2016

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.
Sterol uptake induced by an impairment of pyridoxal phosphate synthesis in Saccharomyces cerevisiae: cloning and sequencing of the PDX3 gene encoding pyridoxine (pyridoxamine) phosphate oxidase
Authors: Loubbardi A , Marcireau C , Karst F , Guilloton M
Published: November 2016

Exogenous sterols do not permeate wild-type Saccharomyces cerevisiae in aerobic conditions. However, mutant strain FKerg7, affected in lanosterol synthase, is a sterol auxotroph which is able to grow aerobically in the presence of ergosterol. Viability of this strain depends on the presence of an additional mutation, aux30, that leads to sterol permeability. Cells bearing the aux30 mutation fail to grow in standard yeast nitrogen base medium containing pyridoxine but grow normally if pyridoxine is replaced by either pyridoxal or pyridoxamine. These mutants are characterized by a lack in pyridoxine (pyridoxamine) phosphate oxidase [P(N/M)P oxidase] (EC 1.4.3.5) activity. The pleiotropic phenotype induced by the aux30 mutation includes a strong perturbation in amino acid biosynthesis. Strains bearing the aux30 mutation also display atypic fatty acid, sterol, and cytochrome patterns. Transformation of an aux30 strain with a replicative vector carrying the wild-type PDX3 gene encoding P(N/M)P oxidase restored wild-type fatty acid, sterol, and cytochrome patterns and suppressed exogenous sterol accumulation. It is proposed that sterol permeation of aux30 strains in mainly the consequence of their leaky Hem- character. The amino acid sequence of S. cerevisiae P(N/M)P oxidase inferred from the nucleotide sequence of PDX3 shows a high percentage of homology with the corresponding enzymes from Escherichia coli and Myxococcus xanthus. Several putative Gcn4p binding sequences are present in the PDX3 promoter region, leading to the assumption that transcription of this gene is under the general control of nitrogen metabolism.
A carbon starvation survival gene of Pseudomonas putida is regulated by sigma 54
Authors: Kim Y , Watrud L S , Matin A
Published: November 2016

By using mini-Tn5 transposon mutagenesis, two mutants of Pseudomonas putida ATCC 12633 were isolated which showed a marked increase in their sensitivity to carbon starvation; these mutants are presumably affected in the Pex type of proteins that P. putida induces upon carbon starvation (M. Givskov, L. Eberl, and S. Molin, J. Bacteriol. 176:4816-4824, 1994). The affected genes in our mutants were induced about threefold upon carbon starvation. The promoter region of the starvation gene in the mutant MK107 possessed a strong sigma 54-type-promoter sequence, and deletion analysis suggested that this was the major promoter regulating expression; this was confirmed by transcript mapping in rpoN+ and rpoN mutant backgrounds. The deletion analysis implicated a sequence upstream of the sigma 54 promoter, as well as a region downstream of the transcription start site, in the functioning of the promoter. Two sigma 70-type Pribnow boxes were also detected in the promoter region, but their transcriptional activity in the wild type was very weak. However, in a sigma 54-deficient background, these promoters became stronger. The mechanism and possible physiological role of this phenomenon and the possibility that the sequence upstream of the sigma 54 promoter may have a role in carbon sensing are discussed.
Enzymatic properties of Escherichia coli peptide deformylase
Authors: Meinnel T , Blanquet S
Published: November 2016

Since its discovery in crude extracts in the late sixties, Escherichia coli peptide deformylase activity could not be further characterized because of an apparent extreme instability. We show that this behavior was caused by an inadequate activity assay, involving substrate concentration inhibition and substrate precipitation in crude extracts. The homogeneous protein, as it was previously purified (T. Meinnel and S. Blanquet J. Bacteriol. 175:7737-7740, 1993), had actually retained its initial activity. The influence on the deformylation reaction of several factors was studied and used to improve the activity assay. Pure peptide deformylase proves to act only on peptide substrates with an N-formylmethionyl moiety. In agreement with the occurrence of zinc in the enzyme, peptide deformylase activity is inhibited by 1,10-phenanthroline.
Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro
Authors: Halberg R , Oke V , Kroos L
Published: November 2016

SpoIIID is a sequence-specific, DNA-binding protein that activates or represses transcription of different genes by sigma K RNA polymerase in vitro. A Bacillus subtilis strain engineered to produce both sigma K and SpoIIID during growth showed effects of SpoIIID on expression of sigma K-dependent genes that were consistent with the effects of a small amount of SpoIIID on transcription of these genes in vitro, indicating that the strain provides a simple, in vivo method to screen for effects of SpoIIID on transcription of sigma K-dependent genes.
Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa
Authors: Koch W H , Henrikson E , Eisenstadt E , Cebula T A
Published: November 2016

The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.
A promoter associated with the neisserial repeat can be used to transcribe the uvrB gene from Neisseria gonorrhoeae
Authors: Black C G , Fyfe J A , Davies J K
Published: November 2016

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrB mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrB proteins of E. coli, Micrococcus luteus, and Streptococcus pneumoniae. A gonococcal uvrB mutant was constructed and found to be extremely sensitive to UV radiation. Transcriptional fusions between portions of the gonococcal uvrB upstream region and a reporter gene were used to localize promoter activity, and the transcriptional start point of the gonococcal uvrB gene was mapped in E. coli by primer extension. A corresponding sigma 70 promoter was identified within a copy of the 26-bp neisserial repeat, and this identification provided the first evidence of a promoter associated with this repetitive element in N. gonorrhoeae.
Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers
Authors: Wiencek K M , Fletcher M
Published: November 2016

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.
Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs
Authors: Garcia M M , Lutze-Wallace C L , Denes A S , Eaglesome M D , Holst E , Blaser M J
Published: November 2016

Campylobacter fetus subsp. venerealis isolated from a case of human vaginosis was inoculated into the uterus of a C. fetus-negative heifer. Isolates obtained weekly from the vaginal mucus exhibited variations in high-molecular-mass-protein profiles from that of the original inoculum, which had a dominant 110-kDa S-layer protein. Immunoblots of the weekly isolates with monoclonal antibody probes against the 110-kDa S-layer protein and other C. fetus S-layer proteins demonstrated antigenic shifts. Genomic digests of the isolates probed with a 75-mer oligonucleotide of the conserved sapA region also indicated that antigenic variation of the S-layer is accompanied by DNA rearrangement.