bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of adenylylated nucleotides in Clostridium acetobutylicum
Authors: Balodimos I A , Kashket E R , Rapaport E
Published: November 2016

In response to the stresses imposed by temperature upshift or addition of butanol, Clostridium acetobutylicum cultures accumulated diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5'-P1,P4-tetraphospho-5'-guanosine (Ap4G) to high levels. The two adenylylated nucleotides were also accumulated in batch culture in the absence of imposed stresses when the clostridia switched from the acidogenic phase of growth to the solventogenic phase. Most of the adenylylated nucleotides were extracellular. The intracellular concentrations of these compounds were low throughout batch growth and in cells stressed by added butanol. In contrast to other procaryotes, these clostridia did not possess enzymes to degrade the dinucleotides, as shown with both intact cells and cell-free preparations. Our findings are consistent with the hypothesis that endogenously produced solvents are stressful to the cells, stimulating the synthesis of adenylylated nucleotides. The nucleotides accumulate extracellularly because they cannot be degraded and because the cell membranes are permeabilized by the solvents produced.
Identification of two new cell division genes that affect a high-molecular-weight penicillin-binding protein in Caulobacter crescentus
Authors: Nathan P , Newton A
Published: November 2016

Penicillin-binding proteins (PBPs) are membrane proteins associated with the synthesis of the bacterial cell wall. We report the characterization of 14 PBPs in Caulobacter crescentus, using in vivo and in vitro penicillin-binding assays and experiments to determine their possible role in cell division. New conditional cell cycle mutants were isolated by selecting cephalosporin-C-resistant mutants of the beta-lactamase strain SC1107 at 30 degrees C that are also defective in cell division at 37 degrees C. They fall into two classes, represented by strains PC8002 and PC8003. Strain PC8002 produced short cells arrested at all stages of cell division at 37 degrees C and was found to contain a high-molecular-weight PBP 1B which was temperature sensitive when assayed in vivo and in vitro. Strain PC8003 was blocked at an early stage of cell division and formed tightly coiled, unpinched filaments. This cephalosporin-C-resistant strain was also defective in PBP 1B, but only when assayed in vivo. PBP 1B behaved like a high-affinity PBP, and in competition assays, beta-lactams that induced filamentation bound preferentially to PBP 1B. These results and the phenotype of mutant PC8002 suggest that PBP 1B is required for cell division, as well as for cell growth, in C. crescentus. The behavior of strain PC8003 suggests that it contains a conditionally defective gene product that interacts in some way with PBP 1B at an early stage of cell division. None of the mutants showed an allele-specific PBP pattern when assayed in vitro at the nonpermissive temperature, but all of them displayed temperature-sensitive PBP 1C (102 kilodaltons) activity. Thus, it appears that PBP 1C is inhibited at 37 degree C as a consequence of filamentous growth.
Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline
Authors: Csonka L N
Published: November 2016

I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that gamma-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to putA mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extracellular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.
Cytochrome c2-independent respiratory growth of Rhodobacter capsulatus
Authors: Daldal F
Published: November 2016

To assess the role of cytochrome c2 as a respiratory electron carrier, we obtained a double mutant of Rhodobacter capsulatus defective in cytochrome c2 and in the quinol oxidase260. This mutant was able to grow chemoheterotrophically, indicating that an electron pathway independent of cytochrome c2 was functional between the ubiquinol:cytochrome c2 oxidoreductase and the cytochrome oxidase410.
Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r
Authors: Knight K L , Hess R M , McEntee K
Published: November 2016

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.
Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation
Authors: Parsons G D , Donly B C , Mackie G A
Published: November 2016

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.
Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions
Authors: Touati D
Published: November 2016

Protein and operon fusions between the manganese superoxide dismutase (MnSOD) gene, sodA, and genes of the lactose operon were constructed in an attempt to explore the effects of various factors on MnSOD expression and the level at which they operate. In sodA-lacZ protein fusions, induction of beta-galactosidase perfectly mimicked MnSOD induction (i.e., beta-galactosidase was not expressed in anaerobiosis and was induced by oxygen, redox-cycling compounds in aerobiosis, and iron chelators in anaerobiosis). In tac-sodA operon fusions, MnSOD induction was monitored only by the lactose operon inducer isopropyl-beta-D-thiogalactopyranoside. Various plasmids carrying part or all of the sodA regulatory and structural region inhibited aerobic beta-galactosidase induction in sodA-lacZ fusions. This included plasmids carrying only the transcription start and upstream region and also plasmids which did not contain this region and in which MnSOD was under foreign transcriptional control. The role of metal ions was also investigated. Addition of Mn(II) enhanced MnSOD activity but did not affect induction. The anaerobic expression of MnSOD from the oxygen-insensitive tac promoter was enhanced threefold by iron-chelating agents, implying a posttranscriptional or most likely a posttranslational modulation of enzyme activity via metal ions. To accommodate all these data, multiregulation of MnSOD is proposed.
Cloning and complete nucleotide sequences of the type II restriction-modification genes of Salmonella infantis
Authors: Karreman C , de Waard A
Published: November 2016

The complete type II restriction-modification system of Salmonella infantis was cloned in Escherichia coli as an R . Sau3AI fragment of 3,430 base pairs. The clone was shown to express the restriction endonuclease as well as the modification methylase. The nucleotide sequence of the above fragment showed two open reading frames of 461 and 230 codons in tail-to-tail orientation. These were shown to represent the modification methylase M . SinI and the restriction endonuclease R . SinI, respectively. The methylase M . SinI amino acid sequence revealed a considerable similarity to those of other deoxycytidylate methylases. In contrast, endonuclease R . SinI did not exhibit such a similarity to other restriction enzymes.
Isolation and characterization of the cell-associated region of group A streptococcal M6 protein
Authors: Pancholi V , Fischetti V A
Published: November 2016

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.
Sequence of the dnaB gene of Salmonella typhimurium
Authors: Wong A , Kean L , Maurer R
Published: November 2016

The dnaB gene of Escherichia coli encodes a helicase that operates at replication forks of the bacterium and certain of its bacteriophages to produce separated strands suitable for subsequent use by primase and DNA polymerase III. Here, we present the sequence of the dnaB gene of Salmonella typhimurium, a functionally interchangeable analog of the E. coli dnaB gene. The DnaB proteins of these two organisms, inferred from the DNA sequences, are identical in length and in 93% of amino acid residues. Extended portions of the DnaB proteins are also similar to two phage-encoded DNA replication proteins: the gene 4 helicase-primase of coliphage T7 and, as reported previously (H. Backhaus and J. B. Petri, Gene 32: 289-303, 1984), the gene 12 protein of Salmonella phage P22. In contrast, little similarity was found between DnaB and either the UvrD repair helicase or transcription termination factor Rho (an RNA-DNA helicase). These results identify S. typhimurium DnaB as a member of the DnaB family of proteins by structural, as well as functional, criteria and provide the basis for the eventual identification, by mutational studies, of residues in DnaB critical for its function.
Characteristics of Ureaplasma urealyticum urease
Authors: Blanchard A , Razin S , Kenny G E , Barile M F
Published: November 2016

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.
Genetics of the iron dicitrate transport system of Escherichia coli
Authors: Pressler U , Staudenmaier H , Zimmermann L , Braun V
Published: November 2016

Escherichia coli B and K-12 express a citrate-dependent iron(III) transport system for which three structural genes and their arrangement and products have been determined. The fecA gene of E. coli B consists of 2,322 nucleotides and encodes a polypeptide containing a signal sequence of 33 amino acids. The cleavage site was determined by amino acid sequence analysis of the unprocessed protein and the mature protein. For the processed form a length of 741 amino acids was calculated. The mature FecA protein in the outer membrane contains at the N terminus the "TonB box," a pentapeptide, which has hitherto been found in all receptors and colicins which functionally require the TonB protein. In addition, the dyad repeat sequence GAAAATAATTCTTATTTCG is proposed to serve as the binding site of the Fur iron repressor protein. The fecB gene was mapped downstream of fecA and encodes a protein with an apparent molecular weight of 30,000. It was synthesized as a precursor, and the mature form was found in the periplasm. The fecD gene follows fecB and was related to a membrane-bound protein with an apparent molecular weight of 28,000. In Mu d1 insertion mutants upstream of fecA, the fec genes were not inducible by iron limitation and citrate, indicating a regulatory region, termed fecI, which controls fec gene expression.
Transcriptional regulation of a promoter in the men gene cluster of Bacillus subtilis
Authors: Miller P , Mueller J , Hill K , Taber H
Published: November 2016

The control of men gene expression during growth and sporulation of Bacillus subtilis was examined at the transcriptional level. Two different approaches were used. (i) Steady-state levels of men-specific mRNA were measured directly. (ii) A men'-lacZ gene fusion was constructed. In both cases, it was observed that men promoter activity was maximal at the onset of sporulation and declined soon thereafter. These kinetics were similar to the pattern of menaquinone accumulation previously observed. Expression from the men promoter was independent of the presence of the products of the spo0A and spo0H genes and was enhanced by addition of glucose and glutamine to the culture medium. DNA sequence analysis of the promoter region revealed a potential recognition site for the principal vegetative form of RNA polymerase but not for any of the known minor polymerase forms. The functionality in vivo of the promoter sequence was confirmed by high-resolution S1 nuclease mapping of the transcript start site. An additional sequence element was identified that is shared by the sdhA, citG, and ctaA promoters and may indicate a common regulatory mechanism in the expression of these genes.
A rapid population method for action spectra applied to Halobacterium halobium
Authors: Stoeckenius W , Wolff E K , Hess B
Published: November 2016

We have developed a simple and rapid technique for measuring the action spectra for phototaxis of populations of microorganisms and applied it to halobacteria. A microscope with a dark-field condenser was used to illuminate the cell suspension in a sealed chamber with light of wavelength greater than 750 nm; in this region of the spectrum, the halobacteria show no phototactic response. A 150-micron spot of light from a xenon arc lamp, whose wavelength and intensity can be varied, was projected through the objective lens into the center of the dark field. The objective lens imaged this measuring spot through a 780-nm cut-off filter on an aperture in front of a photomultiplier. The intensity of the scattered 750-nm light, and therefore the photomultiplier current, is proportional to the number of cells in the measuring spot. A third lamp provided background light of variable wavelength and intensity through the dark-field condenser. To minimize secondary effects due to large changes in cell density, we recorded the initial changes in the photomultiplier current over 1 min after the actinic light had been switched on. By plotting the rate of change against wavelength, we obtained action spectra after the proper corrections for changes in light intensity with wavelength were applied and saturation effects were avoided.
The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2
Authors: Black P N
Published: November 2016

The fadL+ gene of Escherichia coli encodes an outer membrane protein (FadL) essential for the uptake of long-chain fatty acids (C12 to C18). The present study shows that in addition to being required for uptake of and growth on the long-chain fatty acid oleate (C18:1), FadL acts as a receptor of bacteriophage T2. Bacteriophage T2-resistant (T2r) strains lacked FadL and were unable to take up and grow on long-chain fatty acids. Upon transformation with the fadL+ clone pN103, T2r strains became sensitive to bacteriophage T2 (T2s), became able to take up long-chain fatty acids at wild-type levels, and contained FadL in the outer membrane.
Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis
Authors: Sá-Nogueira I , Paveia H , de Lencastre H
Published: November 2016

Constitutive mutants for L-arabinose utilization were isolated from Bacillus subtilis 168T+ and showed resistance to D-fucose, a nonmetabolizable analog of L-arabinose. The mutations that conferred the constitutive phenotype (Arac) were mapped between cysB and hisA. All the mutants showed an isomerase activity which was reduced to 50 to 70% in the presence of L-arabinose and to 10% in the presence of glucose.
Saccharomyces cerevisiae acquires resistance to 2-deoxyglucose at a very high frequency
Authors: Heredia M F , Heredia C F
Published: November 2016

We have found that Saccharomyces cerevisiae acquires spontaneously increasing resistance to 2-deoxyglucose at a very high frequency. This finding allows the easy isolation of different types of resistant strains of interest for metabolic studies with 2-deoxyhexoses. On the other hand, it sounds a note of caution in the widespread use of 2-deoxyglucose as a selective agent for the isolation of yeast mutants with impaired hexose transport or phosphorylation systems.
Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato
Authors: Mellano M A , Cooksey D A
Published: November 2016

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.
Illegitimate recombination in an Escherichia coli plasmid: modulation by DNA damage and a new bacterial gene
Authors: Yi T M , Stearns D , Demple B
Published: November 2016

We studied DNA rearrangements in Escherichia coli by using a plasmid-based system with a transcriptionally silent tet gene and selecting for Tetr isolates. The predominant activating event was a 1.3-kilobase-pair deletion in the plasmid between two sites, with 14 of 19 base pairs being identical. These deletions occurred equally frequently in a recA+ strain and a recA13 mutant. However, the frequency of Tetr occurrence was stimulated 50-fold by treatment of the cells with UV light in a process that was at least partly independent of the SOS response. Bacterial mutants deleted for the xth-pnc region of the chromosome exhibited a strongly elevated spontaneous frequency of Tetr isolates, all with the same 1.3-kilobase-pair deletion. This phenotype of high-frequency deletion could be complemented by an episome covering this region, but not by the cloned xth gene. These studies helped to define the role of different DNA damages in illegitimate recombination and identify a region of the E. coli chromosome that contains a gene whose product normally suppresses illegitimate deletions.
Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene
Authors: Kim U J , Ambulos N P , Duvall E J , Lorton M A , Lovett P S
Published: November 2016

Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.
Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation
Authors: Dessaux Y , Guyon P , Petit A , Tempé J , Demarez M , Legrain C , Tate M E , Farrand S K
Published: November 2016

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.
Cloning of the genes of the chitin utilization regulon of Serratia liquefaciens
Authors: Joshi S , Kozlowski M , Selvaraj G , Iyer V N , Davies R W
Published: November 2016

The set of genes that determine the expression of the enzymes involved in chitin degradation by Serratia liquefaciens was cloned. The role of each gene was investigated, and for the first time regulatory genes were identified in this system. The chiA and chiB genes coded for separate chitinase activities. The chiC region coded for a chitobiase activity, but it was not formally separated from chiB. Transposon mutagenesis and deletion analysis identified a region, chiD, whose absence led to higher expression of chiA, chiB, and chiC. chiD may therefore be a gene that codes for a repressor. Loss of function of another adjacent region, chiE, prevented induction unless a chiE+ strain was a near neighbor, suggesting that this gene may code for a protein that is involved in the synthesis of the inducer. chiB, chiC, chiD, and chiE are closely linked, while chiA is in a separate location on the chromosome.
Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae
Authors: Pines O , Yoon H J , Inouye M
Published: November 2016

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.
Sequence analysis of termini of conjugative transposon Tn916
Authors: Clewell D B , Flannagan S E , Ike Y , Jones J M , Gawron-Burke C
Published: November 2016

Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.
cis-acting sites in the transcript of the Bacillus subtilis trp operon regulate expression of the operon
Authors: Kuroda M I , Henner D , Yanofsky C
Published: November 2016

Transcription of the trp operon of Bacillus subtilis is regulated by attenuation. A trpE'-'lacZ gene fusion preceded by the wild-type trp promoter-leader region was used to analyze regulation. Overproduction of the trp leader transcript in trans from a multicopy plasmid caused constitutive expression of the chromosomal trpE'-'lacZ fusion, presumably by titrating a negative regulatory factor encoded by the mtr locus. Subsegments of the trp leader region cloned onto the multicopy plasmid were examined for their abilities to elevate beta-galactosidase activity. An RNA segment spanning the portion of the leader transcript that forms the promoter-proximal strand of the proposed antiterminator structure was most active in this trans test. The data suggest that the mtr gene product, when activated by tryptophan, binds to this RNA segment and prevents formation of the antiterminator. In this manner, the trans-acting factor promotes formation of the RNA structure that causes transcription termination. Secondary-structure predictions for the leader segment of the trp operon transcript suggest that if the mtr factor bound this RNA segment in a nonterminated transcript, the ribosome-binding site for the first structural gene, trpE, could be sequestered in a stable RNA structure. We tested this possibility by comparing transcriptional and translational fusions containing the initial segments of the trp operon. Our findings suggest that the mtr product causes both transcription attenuation and inhibition of translation of trpE mRNA. Inhibition of translation initiation would reduce ribosome density on trpE mRNA, perhaps making it more labile. Consistent with this interpretation, the addition of tryptophan to mtr+ cultures increased the rate of trpE'-'lacZ mRNA decay.
Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli
Authors: Polayes D A , Rice P W , Garner M M , Dahlberg J E
Published: November 2016

The spf gene of Escherichia coli encodes an unstable 109-nucleotide RNA, spot 42 RNA; the level of this RNA was reduced three- to fivefold when cells were grown in the presence of 3',5'-cyclic AMP (cAMP). We show that this regulation occurs through reduction in transcription and depends on both cAMP and the cAMP receptor protein (CRP) but is independent of the de novo protein synthesis. Through deletion analysis of the spf gene promoter, we have identified sequences that are important in the synthesis of spot 42 RNA. Deletion of sequences upstream of -77 completely eliminated the negative control of cAMP-CRP and resulted in high constitutive levels of transcription. This region contained a sequence that both conformed to the consensus binding site for cAMP-CRP in positively regulated promoters and acted as a cAMP-CRP binding site in a gel retardation assay. Deletion of sequences between positions -77 and -60 greatly reduced the level of transcription in the presence or absence of cAMP-CRP, indicating that at least part of this region is a binding site for a positive-acting transcription factor (or RNA polymerase itself). We propose that the proximity of the two sites defined here allows for the negative control of spf gene transcription by cAMP-CRP. In particular, if only one site at a time can be occupied, the binding of cAMP-CRP would interfere with the binding of a transcription factor.
Molecular characterization of the hemolysin determinant of Serratia marcescens
Authors: Poole K , Schiebel E , Braun V
Published: November 2016

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.
Lipopolysaccharides of Thiocystis violacea, Thiocapsa pfennigii, and Chromatium tepidum, species of the family Chromatiaceae
Authors: Meissner J , Pfennig N , Krauss J H , Mayer H , Weckesser J
Published: November 2016

The lipopolysaccharides (LPS) of three species of purple sulfur bacteria (Chromatiaceae), Thiocystis violacea, Thiocapsa pfennigii, and the moderately thermophilic bacterium Chromatium tepidum, were isolated. The LPS of Thiocystis violacea and Chromatium tepidum contained typical O-specific sugars, indicating O-chains. Long O-chains were confirmed for these species by sodium deoxycholate gel electrophoresis of their LPS. Thiocapsa pfennigii, however, had short or no O-chains. The core region of the LPS of all three species comprised D-glycero-D-mannoheptose as the only heptose and 2-keto-3-deoxyoctonate. The lipid A, obtained from the LPS by mild acid hydrolysis, contained glucosamine as the main amino sugar. Amide-bound 3-hydroxymyristic acid was the only hydroxy fatty acid. The main ester-bound fatty acid in all lipid A fractions was 12:0. Mannose and small amounts of 2,3-diamino-2,3-dideoxy-D-glucose were common constituents of the lipid A of the three Chromatiaceae species investigated. All lipid A fractions were essentially free of phosphate.
Physiology of behavioral mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway
Authors: Bergman K , Gulash-Hoffee M , Hovestadt R E , Larosiliere R C , Ronco P G , Su L
Published: November 2016

Wild-type and nonchemotactic mutant strains of Rhizobium meliloti were tested for attraction to localized sites on alfalfa roots and for attraction to numerous small molecules, including sugars, amino acids, and two fractions derived from alfalfa root extracts. Four strains (carrying mutations che-6, che-11, che-12, and che-26) lost all responses and were classified as generally nonchemotactic mutants. One strain (carrying mutation che-7) lost responses to a group of structurally unrelated amino acids but retained all other responses and was classified as a putative sensory transducer mutant. Two strains (carrying mutations che-1 and che-3) lost responses to all the amino acids and sugars tested but retained normal responses to localized sites on roots and to the root fractions. These two mutant strains could not be classified according to the generally accepted model for a sensory pathway, derived from studies of enteric bacteria, and provided evidence for a dual chemotaxis pathway in R. meliloti.
Cloning of nifHD from Nostoc commune UTEX 584 and of a flanking region homologous to part of the Azotobacter vinelandii nifU gene
Authors: Defrancesco N , Potts M
Published: November 2016

The heterocystous cyanobacterium Nostoc commune UTEX 584 contains two nifH-like sequences (nifH1 and nifH2) in addition to nifHD. A region of DNA 1 kilobase upstream from the 5' end of nifH showed considerable sequence similarity to part of the published nifU sequences of Azotobacter vinelandii and Klebsiella pneumoniae.
Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene
Authors: Phoenix P , Drapeau G R
Published: November 2016

The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.
Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use
Authors: Chakravorty Soumitesh , Roh Sandy S. , Glass Jennifer , Smith Laura E. , Simmons Ann Marie , Lund Kevin , Lokhov Sergey , Liu Xin , Xu Peng , Zhang Guolong , Via Laura E. , Shen Qingyu , Ruan Xianglin , Yuan Xing , Zhu Hong Zhu , Viazovkina Ekaterina , Shenai Shubhada , Rowneki Mazhgan , Lee Jong Seok , Barry Clifton E. , Gao Qian , Persing David , Kwiatkawoski Robert , Jones Martin , Gall Alexander , Alland David
Published: November 2016

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in
gyrA
,
gyrB
,
katG
, and
rrs
genes and the promoters of
inhA
and
eis
genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of
Mycobacterium tuberculosis
in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.
Comparison of Clostridium difficile Ribotypes Circulating in Australian Hospitals and Communities
Authors: Furuya-Kanamori Luis , Riley Thomas V. , Paterson David L. , Foster Niki F. , Huber Charlotte A. , Hong Stacey , Harris-Brown Tiffany , Robson Jenny , Clements Archie C. A.
Published: November 2016

Clostridium difficile
infection (CDI) is becoming less exclusively a health care-associated CDI (HA-CDI). The incidence of community-associated CDI (CA-CDI) has increased over the past few decades. It has been postulated that asymptomatic toxigenic
C. difficile
(TCD)-colonized patients may play a role in the transfer of
C. difficile
between the hospital setting and the community. Thus, to investigate the relatedness of
C. difficile
across the hospital and community settings, we compared the characteristics of symptomatic and asymptomatic host patients and the pathogens from these patients in these two settings over a 3-year period. Two studies were simultaneously conducted; the first study enrolled symptomatic CDI patients from two tertiary care hospitals and the community in two Australian states, while the second study enrolled asymptomatic TCD-colonized patients from the same tertiary care hospitals. A total of 324 patients (96 with HA-CDI, 152 with CA-CDI, and 76 colonized with TCD) were enrolled. The predominant
C. difficile
ribotypes isolated in the hospital setting corresponded with those isolated in the community, as it was found that for 79% of the
C. difficile
isolates from hospitals, an isolate with a matching ribotype was isolated in the community, suggesting that transmission between these two settings is occurring. The toxigenic
C. difficile
strains causing symptomatic infection were similar to those causing asymptomatic infection, and patients exposed to antimicrobials prior to admission were more likely to develop a symptomatic infection (odds ratio, 2.94; 95% confidence interval, 1.20 to 7.14). Our findings suggest that the development of CDI symptoms in a setting without establishment of hospital epidemics with binary toxin-producing
C. difficile
strains may be driven mainly by host susceptibility and exposure to antimicrobials, rather than by
C. difficile
strain characteristics.
Impact of High Diversity of Achromobacter Populations within Cystic Fibrosis Sputum Samples on Antimicrobial Susceptibility Testing
Authors: Dupont Chloé , Jumas-Bilak Estelle , Michon Anne-Laure , Chiron Raphaël , Marchandin Hélène
Published: November 2016

Chronic colonization by opportunistic environmental bacteria is frequent in the airways of cystic fibrosis (CF) patients. Studies of
Pseudomonas aeruginosa
evolution during persistence have highlighted the emergence of pathoadaptive genotypes and phenotypes, leading to complex and diversified inpatient colonizing populations also observed at the intraspecimen level. Such diversity, including heterogeneity in resistance profiles, has been considered an adaptive strategy devoted to host persistence. Longitudinal genomic diversity has been shown for the emergent opportunistic pathogen
Achromobacter
, but phenotypic and genomic diversity has not yet been studied within a simple CF sputum sample. Here, we studied the genomic diversity and antimicrobial resistance heterogeneity of 132
Achromobacter
species strains (8 to 27 strains of identical or distinct colonial morphotypes per specimen) recovered from the sputum samples of 9 chronically colonized CF patients. We highlighted the high within-sample and within-morphotype diversity of antimicrobial resistance (disk diffusion) and genomic (pulsed-field gel electrophoresis) profiles. No sputum sample included strains with identical pulsotypes or antibiotic susceptibility patterns. Differences in clinical categorization were observed for the 9 patients and concerned 3 to 11 antibiotics, including antibiotics recommended for use against
Achromobacter
. Within-sample antimicrobial resistance heterogeneity, not predictable from colonial morphology, suggested that it may represent a selective advantage against antibiotics in an
Achromobacter
persisting population and potentially compromise the antibiotic management of CF airway infections.