bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene
Authors: Lewis L K , Mount D W
Published: November 2016

The dinG gene was originally isolated during a search for Escherichia coli promoters which are components of the SOS regulon. The regulatory region of this gene contains a potential binding site for LexA repressor which is quite different from other known sites. All previously described chromosomal LexA operators are imperfect palindromes containing the sequence CTG(N10)CAG. The noncanonical dinG sequence breaks the symmetry and takes the form TTG(N10)CAG. In the present study, a search for mutations within dinGop::galK fusion plasmids which render transcription independent of intracellular levels of LexA has yielded mutations only within this 16-bp sequence. Electrophoretic mobility shift assays performed with purified mutant and wild-type operator fragments revealed that the affinity of LexA for each of the mutant sites is greatly reduced compared with that of the wild type. One of the mutants contained an alteration in the putative promoter of dinG which increased the similarity of the -35 region to the consensus sequence (TTGGCT----TTGACT); the apparent promoter activity of this construct was subsequently found to be approximately eight times higher than that of the wild type in vivo. Additional experiments have established the complete nucleotide sequence of the dinG gene. A long open reading frame located immediately downstream of the asymmetric operator segment which could potentially encode a 72.9-kDa DinG protein was identified.
A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens
Authors: Hara O , Hutchinson C R
Published: November 2016

The Streptomyces mycarofaciens mdmB gene encodes a 3-O-acyltransferase that catalyzes the addition of acetyl and propionyl groups to position 3 of the lactone ring in 16-member macrolide antibiotics like midecamycin and spiramycin. A putative O-methyltransferase gene (mdmC) is immediately downstream of mdmB, and both of these genes are closely linked to the mdmA midecamycin resistance gene.
Activation defects caused by mutations in Escherichia coli rpoA are promoter specific
Authors: Gussin G N , Olson C , Igarashi K , Ishihama A
Published: November 2016

Escherichia coli RNA polymerases containing mutated alpha subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two alpha (rpoA) mutants, alpha-256 and alpha-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the lambda pRM promoter by cI protein (repressor) was nearly normal, activation of the lambda pRE promoter by cII protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.
Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids
Authors: Austin S J , Eichorn B G
Published: November 2016

The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.
Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor
Authors: Lottenberg R , Broder C C , Boyle M D , Kain S J , Schroeder B L , Curtiss R
Published: November 2016

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.
The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE
Authors: Wu B , Georgopoulos C , Ang D
Published: November 2016

The grpE gene product is one of three Escherichia coli heat shock proteins (DnaK, DnaJ, and GrpE) that are essential for both bacteriophage lambda DNA replication and bacterial growth at all temperatures. In an effort to determine the role of GrpE and to identify other factors that it may interact with, we isolated multicopy suppressors of the grpE280 point mutation, as judged by their ability to reverse the temperature-sensitive phenotype of grpE280. Here we report the characterization of one of them, designated msgB. The msgB gene maps at approximately 53 min on the E. coli chromosome. The minimal gene possesses an open reading frame that encodes a protein with a predicted size of 41,269 M(r). This open reading frame was confirmed the correct one by direct amino-terminal sequence analysis of the overproduced msgB gene product. Genetic experiments demonstrated that msgB is essential for E. coli growth in the temperature range of 22 to 37 degrees C. Through a sequence homology search, MsgB was shown to be identical to N-succinyl-L-diaminopimelic acid desuccinylase (the dapE gene product), which participates in the diaminopimelic acid-lysine pathway involved in cell wall biosynthesis. Consistent with this finding, the msgB null allele mutant is viable only when the growth medium is supplemented with diaminopimelic acid. These results suggest that GrpE may have a previously unsuspected function(s) in cell wall biosynthesis in E. coli.
Characterization of the lipopolysaccharide O antigens of Actinobacillus pleuropneumoniae serotypes 9 and 11: antigenic relationships among serotypes 9, 11, and 1
Authors: Beynon L M , Griffith D W , Richards J C , Perry M B
Published: November 2016

The antigenic lipopolysaccharide O polysaccharides of capsular serotypes 9 and 11 were examined by chemical, immunological, and nuclear magnetic resonance methods. Immunodiffusion tests carried out on these O antigens indicated that both contained common epitopes which were also shared by Actinobacillus pleuropneumoniae serotype 1. Chemical analysis and high-field nuclear magnetic resonance spectroscopy showed that the O antigens of serotypes 9 and 11 were high-molecular-weight polymers consisting of a backbone of repeating trisaccharide units composed of alpha-L-rhamnopyranosyl and alpha-D-glucopyranosyl residues (2:1). One of the alpha-L-rhamnose units forms a branch point and is stoichiometrically substituted with terminal 2-acetamido-2-deoxy-beta-D-glucose residues in the serotype 11 O polysaccharide, but only to the extent of 25% in the serotype 9 O polysaccharide. Thus, the serotype 9 O polysaccharide contains two different repeating units: a tetrasaccharide unit with the same structure as that of the serotype 11 O polysaccharide and a trisaccharide unit: [formula: see text] where R = beta-D-GlcpNAc for serotype 1 and 11 O polysaccharides, and R = H (75%) and R = beta-D-GlcpNAc (25%) for serotype 9. The structure of the previously determined serotype 1 O polysaccharide (E. Altman, J.-R. Brisson, and M. B. Perry, Biochem. Cell. Biol. 64:17-25, 1986) is identical to that of the serotype 11 O polysaccharide. We propose a more complete serotyping scheme for A. pleuropneumoniae which includes designation of both the capsular (K) and O antigens.
NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III)
Authors: Suzuki T , Miyata N , Horitsu H , Kawai K , Takamizawa K , Tai Y , Okazaki M
Published: November 2016

An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme. Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.
Decay of ompA mRNA and processing of 9S RNA are immediately affected by shifts in growth rate, but in opposite manners
Authors: Georgellis D , Arvidson S , von Gabain A
Published: November 2016

By growing Escherichia coli in continuous cultures at various growth rates, we provide definitive evidence that the stability of the ompA mRNA is growth rate dependent. Shifting fast-growing cells into physiological salt buffer led to an immediately increased rate of ompA mRNA decay and to an instantly decreased rate of 9S RNA conversion into 5S rRNA. Shifting slowly growing cells into fresh medium had the opposite effect for each of the two RNA species. The observed regulatory patterns underline the need of cells to adjust the output of ompA and 9S RNAs in response to growth rate changes. At all growth rates and throughout all shift experiments, the half-life of bla mRNA was constant. A stabilization of the ompA transcript was even observed when slowly growing cells were shifted into fresh medium already containing the transcriptional inhibitor rifampicin. A hybrid bla transcript with the 5' untranslated region from the ompA gene behaved similarly to the wild-type ompA messenger in response to a shift in growth rate. In agreement with this result, we found that the same type of 5' cleavages as have been previously shown to initiate the decay of the ompA transcript seem to be involved in stability regulation. In E. coli the degradation of mRNA has been shown to depend on the ams/rne gene. This gene controls the stability-related cleavages in the ompA transcript, catabolic processes, and the cleavages which process the 9S rRNA into 5S RNA, an anabolic process. We discuss these results with respect to the ams/rne gene and the related nuclease activities that control the ompA and 9S RNA cleavages.
Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli
Authors: Podkovyrov S M , Zeikus J G
Published: November 2016

Clostridium thermohydrosulfuricum 39E, a gram-positive thermophilic anaerobic bacterium, produced a cyclodextrin (CD)-degrading enzyme, cyclodextrinase (CDase) (EC 3.2.1.54). The enzyme was purified to homogeneity from Escherichia coli cells carrying a recombinant multicopy plasmid that contained the gene encoding for thermophilic CDase. The purified enzyme was a monomer with an M(r) of 66,000 +/- 2,000. It showed the highest activity at pH 5.9 and 65 degrees C. The enzyme hydrolyzed alpha-, beta-, and gamma-CD and linear maltooligosaccharides to yield maltose and glucose. The Km values for alpha-, beta-, and gamma-CD were 2.5, 2.1, and 1.3 mM, respectively. The rates of hydrolysis for polysaccharides (starch, amylose, amylopectin, and pullulan) were less than 5% of the rate of hydrolysis for alpha-CD. The entire nucleotide sequence of the CDase gene was determined. The deduced amino acid sequence of CDase, consisting of 574 amino acids, showed some similarities with those of various amylolytic enzymes.
Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis
Authors: Buchanan C E , Gustafson A
Published: November 2016

Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore. The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized. The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores. This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure. Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane. Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B. subtilis chromosome. This is a region where there are no other known spo, ger, or PBP genes. In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis. In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5*. The two PBPs could be distinguished by immunoassays.
Amino acid substitution in the lactose carrier protein with the use of amber suppressors
Authors: Huang A M , Lee J I , King S C , Wilson T H
Published: November 2016

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.
Use of electroporation to construct isogenic mutants of Haemophilus ducreyi
Authors: Hansen E J , Latimer J L , Thomas S E , Helminen M , Albritton W L , Radolf J D
Published: November 2016

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.
Physical distance between the site of type II DNA binding to the membrane and oriC on the Bacillus subtilis 168 chromosome
Authors: Itaya M , Laffan J J , Sueoka N
Published: November 2016

The precise physical locations of the oriC region and the region for type II DNA binding to the membrane on the Bacillus subtilis 168 chromosome were determined. The DNA regions were physically mapped by creating new restriction sites (NotI and SfiI) within these regions. The physical distance between oriC and the type II DNA-binding region was verified with the creation of a novel sequence cleaved by endonuclease I-SceI in each of the above regions. Complete removal of the defined type II membrane-binding region produced no noticeable phenotype.
Genetic evidence for an activator required for induction of pectin lyase in Erwinia carotovora subsp. carotovora by DNA-damaging agents
Authors: McEvoy J L , Murata H , Chatterjee A K
Published: November 2016

In Erwinia carotovora subsp. carotovora 71, the induction of pectin lyase (Pnl), the bacteriocin carotovoricin (Ctv), and cellular lysis (Lss) requires a RecA function. We obtained mutants wherein a pleiotropic defect, i.e., the lack of induction with mitomycin C, is not restored by the recA+ DNA. From a genomic library of strain 71, a cosmid (pAKC280) that restored induction of Pnl, Ctv, and Lss by mitomycin C was isolated. The activator function, designated Rdg for regulator of damage-inducible genes, was localized by subcloning and insertional mutagenesis to a 2.6-kb region within a 6.7-kb EcoRI fragment. An rdg-lacZ operon fusion was inducible by mitomycin C in RecA+ but not RecA- derivatives of E. carotovora subsp. carotovora 71 and Escherichia coli. A RecA+ E. coli strain carrying only a PnlA+ plasmid was not inducible for Pnl production; however, when both a PnlA+ plasmid and a Rdg+ plasmid were present, the transcription of pnlA and the production of the enzyme were activated by mitomycin C. The size of the pnlA transcript produced in E. coli was identical to that of the transcript produced by E. carotovora subsp. carotovora 71, suggesting that the same promoter and termination sequences were being utilized in these bacteria.
Mutations causing aminotriazole resistance and temperature sensitivity reside in gyrB, which encodes the B subunit of DNA gyrase
Authors: Toone W M , Rudd K E , Friesen J D
Published: November 2016

Certain mutations in gyrA and gyrB, the genes encoding the two subunits of DNA gyrase, are known to influence expression of the his operon (K. E. Rudd and R. Menzel, Proc. Natl. Acad. Sci. USA 84:517-521, 1987). Such mutations lead to a decrease in tRNA(His) levels and consequently to an attenuator-dependent increase in his operon expression. This effect presumably is due to the dependence of the hisR promoter (hisR encodes tRNA(His) on supercoiling for maximal activity. We used a relaxed (Rel-) strain of Escherichia coli to isolate gyrB mutants by selecting for resistance to the histidine antimetabolite 3-amino-1,2,4-triazole and then screening for temperature-sensitive growth on rich medium. Rel- mutants, which generally have lower basal levels of ppGpp (a positive regulator of his operon transcription), are more sensitive than wild-type E. coli to aminotriazole. The chance of isolating spoT mutants, which can be selected with a similar procedure, was decreased by selecting in the presence of a multicopy plasmid that carries the wild-type spoT gene. Under these conditions, gyrB mutants were isolated preferentially. This scheme selects for loss of function of DNA gyrase, rather than for its alteration due to resistance to specific gyrase inhibitors, and thus a greater variety of gyrase mutations might be obtainable.
Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus
Authors: Becker-Rudzik M , Young D A , Marrs B L
Published: November 2016

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.
A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome
Authors: Kieser H M , Kieser T , Hopwood D A
Published: November 2016

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.
GCR3 encodes an acidic protein that is required for expression of glycolytic genes in Saccharomyces cerevisiae
Authors: Uemura H , Jigami Y
Published: November 2016

Screening of a mutagenized strain carrying a multicopy ENO1-'lacZ fusion plasmid revealed a new mutation affecting several glycolytic enzyme activities. The recessive single nuclear gene mutation, named gcr3, caused an extremely defective growth phenotype on fermentable carbon sources such as glucose, while growth on respiratory media was almost normal. The GCR3 gene was obtained by growth complementation from a genomic DNA library, and the complemented strains had normal enzyme levels. GCR3 gene was sequenced, and a 99,537-Da protein was predicted. The predicted GCR3 protein was fairly acidic (net charge, -34). The C-terminal region was highly charged, and an acidic stretch was found in it.
Energy transduction in the methanogen Methanococcus voltae is based on a sodium current
Authors: Dybas M , Konisky J
Published: November 2016

We provide experimental support for the proposal that ATP production in Methanococcus voltae, a methanogenic member of the archaea, is based on an energetic system in which sodium ions, not protons, are the coupling ions. We show that when grown at a pH of 6.0, 7.1, or 8.2, M. voltae cells maintain a membrane potential of approximately -150 mV. The cells maintain a transmembrane pH gradient (pH(in) - pH(out)) of -0.1, -0.2, and -0.2, respectively, values not favorable to the inward movement of protons. The cells maintain a transmembrane sodium concentration gradient (sodium(out)/sodium(in)) of 1.2, 3.4, and 11.6, respectively. While the protonophore 3,3',4',5-tetrachlorosalicylanilide inhibits ATP formation in cells grown at pH 6.5, neither ATP formation nor growth is inhibited in cells grown in medium at pH 8.2. We show that when grown at pH 8.2, cells synthesize ATP in the absence of a favorably oriented proton motive force. Whether grown at pH 6.5 or pH 8.2, M. voltae extrudes Na+ via a primary pump whose activity does not depend on a proton motive force. The addition of protons to the cells leads to a harmaline-sensitive efflux of Na+ and vice versa, indicating the presence of Na+/H+ antiporter activity and, thus, a second mechanism for the translocation of Na+ across the cell membrane. M. voltae contains a membrane component that is immunologically related to the H(+)-translocating ATP synthase of the archaeabacterium Sulfolobus acidocaldarius. Since we demonstrated that ATP production can be driven by an artificially imposed membrane potential only in the presence of sodium ions, we propose that ATP production in M. voltae is mediated by an Na+-translocating ATP synthase whose function is coupled to a sodium motive force that is generated through a primary Na+ pump.
Efficiency of homologous intermolecular recombination at different locations on the Bacillus subtilis chromosome
Authors: Biswas I , Vagner V , Ehrlich S D
Published: November 2016

The efficiencies of intermolecular recombination at 12 different locations on the Bacillus subtilis chromosome were determined by transforming competent cells with a nonreplicative plasmid. The efficiencies varied by only about threefold but were significantly different (P less than 0.05 by a chi-square test) for approximately 20% of the locations. The recA gene product is required for recombination, and the addA gene product appears to affect the variation in a site-specific way.
Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strA1 of Haemophilus influenzae Rd
Authors: Stuy J H , Walter R B
Published: November 2016

The high-level streptomycin resistance strA1 gene of Haemophilus influenzae Rd was cloned in plasmid pAT4 as a 2.1-kbp EcoRI insert. It was later replaced in pAT4 by the wild-type strA+ gene. Plasmid pAT4 carrying the strA+ gene is highly unstable and renders chromosomally resistant recipients sensitive to streptomycin. The strA+ gene and the instability factor both reside on a 500-base HindIII-EcoRI subfragment. The two biological activities are also expressed in Escherichia coli. Both wild-type (strA+) and mutant (strA1) genes were sequenced. They show considerable nucleotide homology with the E. coli strA+ gene and its product.
Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron
Authors: Tancula E , Feldhaus M J , Bedzyk L A , Salyers A A
Published: November 2016

Previous studies of starch utilization by the gram-negative anaerobe Bacteroides thetaiotaomicron have demonstrated that the starch-degrading enzymes are cell associated rather than extracellular, indicating that the first step in starch utilization is binding of the polysaccharide to the bacterial surface. Five transposon-generated mutants of B. thetaiotaomicron which were defective in starch binding (Ms-1 through Ms-5) had been isolated, but initial attempts to identify membrane proteins missing in these mutants were not successful. We report here the use of an immunological approach to identify four maltose-inducible membrane proteins, which were missing in one or more of the starch-binding mutants of B. thetaiotaomicron. Three of the maltose-inducible proteins were outer membrane proteins (115, 65, and 43 kDa), and one was a cytoplasmic membrane protein (80 kDa). The genes encoding these proteins were shown to be clustered in an 8.5-kbp segment of the B. thetaiotaomicron chromosome. Two other loci defined by transposon insertions, which appeared to contain regulatory genes, were located within 7 kbp of the cluster of membrane protein genes. The 115-kDa outer membrane protein was essential for utilization of maltoheptaose (G7), whereas loss of the other proteins affected growth on starch but not on G7. Not all of the proteins missing in the mutants were maltose regulated. We also detected two constitutively produced proteins (32 and 50 kDa) that were less prominent in all of the mutants than in the wild type. Both of these were outer membrane proteins.
Purification and phosphorylation of the Arc regulatory components of Escherichia coli
Authors: Iuchi S , Lin E C
Published: November 2016

In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration. ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family. 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells. Autophosphorylation of 'ArcB was revealed when the protein was incubated with [gamma-32P]ATP but not with [alpha-32P]ATP or [gamma-32P]GTP. When ArcA was incubated in the presence of 'ArcB and [gamma-32P]ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P. When a limited amount of 'ArcB was incubated with excess ArcA and [gamma-32P]ATP, ArcA-32P increased linearly with time. Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB. Thus, 'ArcB acted as a kinase for ArcA. Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s).
A temperature-regulated, retrotransposon-like element from Candida albicans
Authors: Chen J Y , Fonzi W A
Published: November 2016

A repetitive element was isolated from the genome of Candida albicans. This repetitive element, which we designated alpha, was localized to a 500-bp fragment of genomic DNA. The alpha element was dispersed in the genome and varied in copy number and genomic location in the strains examined. Analyses of various loci containing the alpha element identified a locus containing a composite element. This composite element consisted of two direct repeats of the alpha element separated by approximately 5.5 kb of DNA, a structural arrangement similar to that of retrovirus-like transposable elements. The flanking alpha elements of the composite structure were 388 bp in length and were identical in sequence. They were bounded by the nucleotides 5'-TG. ... CA-3', which were part of a delimiting inverted repeat, a feature conserved in the long terminal repeats of retroviruses and retrovirus-like elements. As in retrovirus-like elements, the entire composite element, including the alpha elements, was transcribed into an approximately unit-length mRNA. The expression of this transcript was greatly increased when cells were grown at 25 versus 37 degrees C. As has been found in many retrotransposons, the composite element was flanked by a 5-bp duplication and varied in both copy number and genomic location in various strains. We conclude that the composite element is a retrotransposon-like element, and we have designated this element Tca1. We suggest that Tca1 may be relevant to the genomic evolution of C. albicans and the pathogenic potential of the organism.
A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides
Authors: Nissen-Meyer J , Holo H , Håvarstein L S , Sletten K , Nes I F
Published: November 2016

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL-4B, and reverse-phase chromatography. The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed alpha 1, alpha 2, and beta, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, alpha 1 and alpha 2 were the same gene product. Differences in their configurations presumably resulted in alpha 2 having a slightly lower affinity for the reverse-phase column than alpha 1 and a reduced bacteriocin activity when combined with beta. Bacteriocin activity required the complementary action of both the alpha and the beta peptides. When neither alpha 1 nor beta was in excess, about 0.3 nM alpha 1 and 0.04 nM beta induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, alpha 1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for alpha 1 and beta, respectively, were obtained by mass spectrometry. The N-terminal halves of both the alpha and beta peptides may form amphiphilic alpha-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.
Behavioral analysis of Vibrio parahaemolyticus variants in high- and low-viscosity microenvironments by use of digital image processing
Authors: Lawrence J R , Korber D R , Caldwell D E
Published: November 2016

Digital image analysis and light microscopy were used to study and quantify the growth and behavior of two variants and selected flagellar mutants of Vibrio parahaemolyticus in glass flow cells under high- and low-viscosity conditions. The observations showed a series of surface-associated behaviors, including attachment, microcolony formation, migration, chemotactic movements, and aggregation, indicating a substantial degree of adaptive flexibility and multicellular behavior during growth of V. parahaemolyticus at interfaces.
Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations
Authors: Lam H M , Winkler M E
Published: November 2016

We report the first molecular genetic analysis of a pyridoxine 5'-phosphate oxidase, the PdxH gene product of Escherichia coli K-12. Chromosomal insertions in and around pdxH were generated with various transposons, and the resulting phenotypes were characterized. The DNA sequence of pdxH was determined, and the promoters of pdxH and the downstream gene tyrS, which encodes tyrosyl-tRNA synthetase, were mapped by RNase T2 protection assays of chromosomal transcripts. These combined approaches led to the following conclusions: (i) pdxH is transcribed from a sigma 70-type promoter and shares its transcript with tyrS; (ii) tyrS is additionally transcribed from a relatively strong, nonconventional internal promoter that may contain an upstream activating sequence but whose expression is unaffected by a fis mutation; (iii) PdxH oxidase is basic, has a molecular mass of 25,545 Da, and shares striking homology (greater than 40% identity) with the developmentally regulated FprA protein of Myxococcus xanthus; (iv) mild pyridoxal 5'-phosphate limitation of pdxH mutants inhibits cell division and leads to formation of unsegregated nucleoids; (v) E. coli PdxH oxidase is required aerobically and anaerobically, but second-site suppressors that replace pdxH function entirely can be isolated; and (vi) pdxH mutants excrete significant amounts of L-glutamate and a compound, probably alpha-ketoisovalerate, that triggers L-valine inhibition of E. coli K-12 strains. These findings extend earlier observations that pyridoxal 5'-phosphate biosynthetic and aminoacyl-tRNA synthetase genes are often members of complex, multifunctional operons. Our results also show that loss of pdxH function seriously disrupts cellular metabolism in unanticipated ways.
Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis
Authors: Zhu Y , Englebert S , Joris B , Ghuysen J M , Kobayashi T , Lampen J O
Published: November 2016

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.
Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway
Authors: Sherman D H , Kim E S , Bibb M J , Hopwood D A
Published: November 2016

Streptomyces coelicolor A3(2) and Streptomyces violaceoruber Tü22 produce the antibiotics actinorhodin and granaticin, respectively. Both the aglycone of granaticin and the half-molecule of actinorhodin are derived from one acetyl coenzyme A starter unit and seven malonyl coenzyme A extender units via the polyketide pathway to produce benzoisochromane quinone moieties with identical structures (except for the stereochemistry at two chiral centers). In S. coelicolor and S. violaceoruber, the type II polyketide synthase (PKS) is encoded by clusters of five and six genes, respectively. We complemented a series of S. coelicolor mutants (act) defective in different components of the PKS (actI for carbon chain assembly, actIII for ketoreduction, and actVII for cyclization-dehydration) by the corresponding genes (gra) from S. violaceoruber introduced in trans on low-copy-number plasmids. This procedure showed that four of the act PKS components could be replaced by a heterologous gra protein to give a functional PKS. The analysis also served to identify which of three candidate open reading frames (ORFs) in the actI region had been altered in each of a set of 13 actI mutants. It also proved that actI-ORF2 (whose putative protein product shows overall similarity to the beta-ketoacyl synthase encoded by actI-ORF1 but whose function is unclear) is essential for PKS function. Mutations in each of the four complemented act genes (actI-ORF1, actI-ORF2, actIII, and actVII) were cloned and sequenced, revealing a nonsense or frameshift mutation in each mutant.
Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB
Authors: Vimr E R
Published: November 2016

The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded by region 2 neu genes of the multigenic kps cluster. To facilitate analysis of capsule synthesis and translocation, an E. coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction. The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid. By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium. Complementation was detected by bacteriophage K1F adsorption or infectivity assays. Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface. Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37 degrees C. Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide. The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase. The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms.
Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development
Authors: Soliveri J , Brown K L , Buttner M J , Chater K F
Published: November 2016

Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping. Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M. J. Buttner, K. F. Chater, and M. J. Bibb, J. Bacteriol. 172:3367-3378, 1990) around -40, -10, and +1. In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB. Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture. In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S. coelicolor. Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH). Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.
A 14-kilodalton inner membrane protein of Vibrio cholerae biotype e1 tor confers resistance to group IV choleraphage infection to classical vibrios
Authors: Biswas S K , Chowdhury R , Das J
Published: November 2016

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.