bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted mutagenesis of the b subunit of F1F0 ATP synthase in Escherichia coli: Glu-77 through Gln-85
Authors: McCormick K A , Cain B D
Published: November 2016

Subunit b of Escherichia coli F1F0 ATP synthase contains a large hydrophilic region thought to be involved in the interaction between F1 and F0. Oligonucleotide-directed mutagenesis was used to evaluate the functional importance of a segment of this region from Glu-77 through Gln-85. The mutagenesis procedure employed a phagemid DNA template and a doped oligonucleotide primer designed to generate a predetermined collection of missense mutations in the target segment. Sixty-one mutant phagemids were identified and shown to contain nucleotide substitutions encoding 37 novel missense mutations. Mutations were isolated singly or in combinations of up to four mutations per recombinant phagemid. F1F0 ATP synthase function was studied by mutant phagemid complementation of a novel E. coli strain in which the uncF (b) gene was deleted. Complementation was assessed by observing growth on solid succinate minimal medium. Many phagemid-encoded uncF (b) gene mutations in the targeted segment resulted in growth phenotypes indistinguishable from those of strains expressing the native b subunit, suggesting abundant F1F0 ATP synthase activity. In contrast, several specific mutations were associated with a loss of enzyme function. Phagemids specifying the Ala-79----Pro, Arg-82----Pro, Arg-83----Pro, or Gln-85----Pro mutation failed to complement uncF (b) gene-deficient E. coli. F1F0 ATP synthase displayed the greatest sensitivity to mutations altering a single site in the target segment, Ala-79. The evidence suggests that Ala-79 occupies a restricted position in the enzyme complex.
Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria
Authors: Sherman M Y , Goldberg A L
Published: November 2016

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.
Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus
Authors: Steinberg N A , Meeks J C
Published: November 2016

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.
Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations
Authors: Zhan H J , Lee C C , Leigh J A
Published: November 2016

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.
Characterization of the major promoter for the plasmid-encoded sucrose genes scrY, scrA, and scrB
Authors: Cowan P J , Nagesha H , Leonard L , Howard J L , Pittard A J
Published: November 2016

Sucrose genes from a Salmonella thompson plasmid were cloned in Escherichia coli K-12. A physical map and a genetic map of the genes were constructed, revealing strong homology with the scr regulon from the Salmonella typhimurium plasmid pUR400. Two promoters were examined after being subcloned into transcriptional fusion vectors. Primer extension analysis and site-directed mutagenesis were used to identify the precise location of the promoter of scrY, scrA, and scrB. Transcription from this promoter was regulated over a 1,000-fold range by the combined effects of ScrR-mediated repression and catabolite repression. A putative cyclic AMP receptor protein binding site centered 72.5 bp upstream of the start point of transcription of scrY appeared to be essential for full activity of the scrY promoter. Transcription from the putative scrK promoter was far less sensitive to repression by ScrR. In ScrR+ cells, readthrough transcription from the putative scrK promoter into scrY accounted for less than 10% of scrY expression.
Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis
Authors: Frisby D , Zuber P
Published: November 2016

The transcription from the spoVG promoter of Bacillus subtilis is induced at the start of the stationary phase of growth and is dependent on the expression of the spoOA, spoOB, and spoOH genes. It is repressed in cells grown in the presence of excess glucose and glutamine and is under the negative control of the abrB gene. The spoOA and spoOB gene products function to suppress the negative control exerted by abrB. Transcription initiation requires the form of RNA polymerase holoenzyme that contains the spoOH gene product, sigma H. Optimal transcription also requires an upstream A-T-rich region termed the upstream activating sequence (UAS). The mechanism of UAS function was examined through mutational analysis of the spoVG promoter region. Deletion of the UAS or positioning the UAS one half turn or one full turn of the DNA helix upstream of its location in wild-type spoVG resulted in a severe reduction in promoter activity. Deletion of most of the UAS abolished the abrB-dependent repression of spoVG transcription. Higher activity was observed when the UAS was inserted 10 bp (one turn of the helix) upstream than when the sequence was repositioned either 5 or 13 bp upstream. Sequences upstream of the UAS were found not to be involved with the position-dependent function of the UAS. Positioning the UAS 42 or 116 bp upstream eliminated the stimulatory effect of the sequence on spoVG transcription. These data indicate that the UAS functions effectively when it is in close proximity to the -35 region. In vitro transcription analysis indicated that the deletion and insertion mutation affecting the UAS impair RNA polymerase-spoVG promoter interaction. Deletion of the UAS showed that the negative effect of exogenous glucose and glutamine is not dependent on the UAS but is exerted at a site within or near the -35 and -10 regions.
Defects in gliding motility in mutants of Cytophaga johnsonae lacking a high-molecular-weight cell surface polysaccharide
Authors: Godchaux W , Lynes M A , Leadbetter E R
Published: November 2016

We previously observed (W. Godchaux, L. Gorski, and E.R. Leadbetter, J. Bacteriol. 172:1250-1255, 1990) that two mutants (strains 21 and NS-1) of the gliding bacterium Cytophaga johnsonae that were totally deficient in motility-dependent colony spreading, movement of rafts (groups) of cells as observed with a microscope, and movement of polystyrene-latex spheres that attached to the cell surface (observed in wet mounts) were also deficient in a high-molecular-weight cell surface polysaccharide (HMPS) and suggested a role for that substance in gliding motility. Antisera have been prepared against the purified HMPS, and these were used to select mutants specifically and highly deficient in the polysaccharide. All five such mutants had rates of colony spreading and raft movement that were much lower than those of the parent strain, but the rate of increase in colony diameter was higher than that found for strains NS-1 and 21 (which do not undergo raft movement at all). Unlike these latter two strains, the HMPS mutants retained the ability to move polystyrene-latex spheres over their surfaces. Hence, HMPS deficiency results in defective motility but not nonmotility, and the HMPS deficiency cannot fully explain the phenotype of mutants 21 and NS-1; in these strains, gliding must be affected by additional biochemical lesions. The HMPS may, nonetheless, be advantageous in that it supports greater gliding speeds.
Sequence analysis of the clpG gene, which codes for surface antigen CS31A subunit: evidence of an evolutionary relationship between CS31A, K88, and F41 subunit genes
Authors: Girardeau J P , Bertin Y , Martin C , Der Vartanian M , Boeuf C
Published: November 2016

The clpG gene coding for the CS31A subunit was localized on a 0.9-kb SphI fragment from the recombinant plasmid pAG315. This was established by testing the ability of subclones to hybridize with a 17-meric oligonucleotide probe obtained from N-terminal analysis of the CS31A subunit. The nucleotide sequence of the region coding for CS31A was determined. From primer extension analysis, two initiation translation start sites were detected. Two possible promoterlike sequences were identified; the ribosome binding site and the translation terminator are proposed. Inverted repeat sequences leading to the formation of possible hairpin structures of the transcripts were found on the 5' untranslated region of clpG. The deduced amino acid composition was in close agreement with the chemical amino acid composition and sequence match with the first 25 N-terminal amino acids from the published N-terminal sequence of the purified CS31A subunit. The clpG gene codes for a mature protein of 257 amino acids with a molecular size of 26,777 Da. An obvious homology was observed when the amino acid sequence of CS31A was compared with those of K88 and F41. This homology includes five different conserved sequences of up to 19 identical amino acids, which is associated with conserved proline. An extensive change in the CS31A region homologous to that identified to contain the K88 receptor binding site might be responsible for the functional divergence between CS31A and K88.
Presence of 5-methylcytosine in CC(A/T)GG sequences (Dcm methylation) in DNAs from different bacteria
Authors: Gomez-Eichelmann M C , Levy-Mustri A , Ramirez-Santos J
Published: November 2016

The presence of CC(A/T)GG sequences with methylated internal cytosine (Dcm methylation) was determined in DNA from different genera of eubacteria. This methylation was studied by using restriction enzymes EcoRII and BstNI, which cleave unmethylated or methylated CC(A/T)GG sequences. Dcm methylation was only detected in genera of the family Enterobacteriaceae closely related to Escherichia: Shigella, Citrobacter, Salmonella, and Klebsiella.
Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12
Authors: Sarsero J P , Pittard A J
Published: November 2016

Expression of the mtr gene, which encodes a tryptophan-specific transport system in Escherichia coli K-12, is activated by the TyrR protein. Two TyrR protein binding sites (TYR R boxes) are positioned upstream of the -35 promoter region. Mutational and DNase protection studies indicate that TyrR protein binds preferentially to the TYR R box closest to the promoter, and this is essential for activation of gene expression. In the presence of tyrosine and ATP, a second TyrR molecule is able to cooperatively bind to the second box and cause a further increase in the level of activation.
Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein
Authors: Sharples G J , Lloyd R G
Published: November 2016

The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.
Examination of the phosphate in conjugative F-like pili by use of electron spectroscopic imaging
Authors: Frost L S , Bazett-Jones D P
Published: November 2016

F-like pili specified by conjugative plasmids have been reported to contain phosphate which may be noncovalently incorporated into the pilus. Electron spectroscopic imaging was able to detect phosphate in the filamentous, single-stranded DNA phage f1, used as positive control, but could not detect phosphate in F-like pili. Thus, the phosphate in purified pili which has been reported is probably derived from contaminating cell envelope material.
Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein
Authors: Kawasaki Y , Wada C , Yura T
Published: November 2016

Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32. We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.
Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN)
Authors: Kullik I , Fritsche S , Knobel H , Sanjuan J , Hennecke H , Fischer H M
Published: November 2016

Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida. Both B. japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant. B. japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes. In symbiosis both rpoN genes could replace each other functionally. The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely. Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally. By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ. Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant. The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.
DNA sequence divergence and functional conservation at the STB locus of yeast 2 microns circle variants
Authors: Wei X A , Pelcher L E , Rank G H
Published: November 2016

2 microns DNA isolated from industrial Saccharomyces cerevisiae yeasts exhibited extensive restriction fragment length polymorphisms. At least five 2 microns species were identified from eleven [cir+] strains. Southern hybridization mapped restriction fragment length polymorphisms at STB, a cis-acting locus essential for plasmid partitioning. Some 2 microns variants (e.g., 4110-2 microns and 4108-2 microns) had an altered number of 125-bp consensus repeats at STB. However, the corresponding region of 7754-2 microns has only approximately 70% nucleotide sequence homology with the 125-bp STB consensus repeat. YRp plasmids containing 7754-2 microns STB behave as YEp plasmids in laboratory yeasts, thereby indicating STB sequence divergence coupled to conservation of function.
Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase
Authors: Rossi S C , Topal M D
Published: November 2016

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.
Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12
Authors: Inoue N , Uchida H
Published: November 2016

By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently.
Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227
Authors: Eaton R W , Karns J S
Published: November 2016

Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. Tn5-containing-EcoRI fragments from these mutants were cloned and identified by selection for Tn5-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindIII fragments, as well as on a PstI fragment and a BglII fragment. Restriction mapping and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.
Isolation and characterization of ilvA, ilvBN, and ilvD mutants of Caulobacter crescentus
Authors: Tarleton J C , Ely B
Published: November 2016

Caulobacter crescentus strains requiring isoleucine and valine (ilv) for growth were shown by transduction and pulsed-field gel electrophoresis to contain mutations at one of two unlinked loci, ilvB and ilvD. Other C. crescentus strains containing mutations at a third locus, ilvA, required either isoleucine or methionine for growth. Biochemical assays for threonine deaminase, acetohydroxyacid synthase, and dihydroxyacid dehydratase demonstrated that the ilvA locus encodes threonine deaminase, the ilvB locus encodes acetohydroxyacid synthase, and the ilvD locus encodes dihydroxyacid dehydratase. C. crescentus strains resistant to the herbicide sulfometuron methyl, which is known to inhibit the action of certain acetohydroxyacid synthases in a variety of bacteria and plants, were shown to contain mutations at the ilvB locus, further suggesting that an acetohydroxyacid synthase gene resides at this locus. Two recombinant plasmids isolated in our laboratory, pPLG389 and pJCT200, were capable of complementing strains containing the ilvB and ilvD mutations, respectively. The DNA in these plasmids hybridized to the corresponding genes of Escherichia coli and Serratia marcescens, confirming the presence of ilvB-like and ilvD-like DNA sequences at the ilvB and ilvD loci, respectively. However, no hybridization was observed between any of the other enteric ilv genes and C. crescentus DNA. These results suggest that C. crescentus contains an isoleucine-valine biosynthetic pathway which is similar to the corresponding pathway in enteric bacteria but that only the ilvB and ilvD genes contain sequences which are highly conserved at the DNA level.
Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein
Authors: Krauel V , Heller K J
Published: November 2016

Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene. We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7 promoter. This segment contains an open reading frame that can encode a protein of the appropriate size. Its deduced amino acid sequence does not closely resemble that of any other protein in the database. The sequence upstream of the open reading frame shows typical characteristics of a promoter region with two overlapping, divergently orientated promoters.
Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium
Authors: De Backer O , Colson C
Published: November 2016

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.
Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains
Authors: Eaton R W , Karns J S
Published: November 2016

DNA encoding the catabolism of the s-triazines ammelide and cyanuric acid was cloned from Pseudomonas sp. strain NRRLB-12228 and Klebsiella pneumoniae 99 with, as a probe, a 4.6-kb PstI fragment from a third strain, Pseudomonas sp. strain NRRLB-12227, which also encodes these activities. In strains NRRLB-12228 and 99 the ammelide aminohydrolase (trzC) and cyanuric acid amidohydrolase (trzD) genes are located on identical 4.6-kb PstI fragments which are part of a 12.4-kb DNA segment present in both strains. Strain NRRLB-12227 also carries this 12.4-kb DNA segment, except that a DNA segment of 0.8 to 1.85 kb encoding a third enzyme, ammeline aminohydrolase (trzB), has been inserted next to the ammelide aminohydrolase gene with the accompanying deletion of 1.1 to 2.15 kb of DNA. In addition, the s-triazine catabolic genes are flanked in strain NRRLB-12227 by apparently identical 2.2-kb segments that are not present in the other two strains and that seem to cause rearrangements in adjacent DNA.
Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound
Authors: Xi L , Cho K W , Tu S C
Published: November 2016

Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase alpha and beta subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate that neither the specific activity nor the size of the alpha (362 amino acid residues, Mr 41,389) and beta (324 amino acid residues, Mr 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the alpha and beta subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.
Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes
Authors: Craven M G , Friedman D I
Published: November 2016

The Escherichia coli nusA gene product, known to influence transcription elongation, is essential for both bacterial viability and growth of lambdoid phages. We report the cloning and sequencing of the conditionally lethal nusA10(Cs) allele. Changes from nusA+ were observed at nucleotides 311 and 634. Functional studies showed that both nucleotide changes are necessary for the cold-sensitive phenotype, although bacteria with the change at 634 grew more slowly at 30 degrees C than the nusA+ controls. The mutant nusA10(Cs) gene product is not as active as nusA+ in supporting transcription antitermination mediated by the N regulatory protein of bacteriophage lambda. The change at nucleotide 634 is shown to be solely responsible for this phenotype. Four differences were found between the nusA+ gene that we sequenced and the published nusA sequence. These changes alter the reading frame of nusA in a functionally important domain [as identified by the nusA1 and nusA11(Ts) mutations], resulting in an arginine-rich region that may be involved with RNA binding.
Timing of flagellar gene expression in the Caulobacter cell cycle is determined by a transcriptional cascade of positive regulatory genes
Authors: Ohta N , Chen L S , Mullin D A , Newton A
Published: November 2016

The Caulobacter crescentus flagellar (fla) genes are organized in a regulatory hierarchy in which genes at each level are required for expression of those at the next lower level. To determine the role of this hierarchy in the timing of fla gene expression, we have examined the organization and cell cycle regulation of genes located in the hook gene cluster. As shown here, this cluster is organized into four multicistronic transcription units flaN, flbG, flaO, and flbF that contain fla genes plus a fifth transcription unit II.1 of unknown function. Transcription unit II.1 is regulated independently of the fla gene hierarchy, and it is expressed with a unique pattern of periodicity very late in the cell cycle. The flaN, flbG, and flaO operons are all transcribed periodically, and flaO, which is near the top of the hierarchy and required in trans for the activation of flaN and flbG operons, is expressed earlier in the cell cycle than the other two transcription units. We have shown that delaying flaO transcription by fusing it to the II.1 promoter also delayed the subsequent expression of the flbG operon and the 27- and 25-kDa flagellin genes that are at the bottom of the regulatory hierarchy. Thus, the sequence and timing of fla gene expression in the cell cycle are determined in large measure by the positions of these genes in the regulatory hierarchy. These results also suggest that periodic transcription is a general feature of fla gene expression in C. crescentus.
Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor
Authors: Kinashi H , Shimaji-Murayama M
Published: November 2016

SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with lambda exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein.
In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites
Authors: Chang M , Crawford I P
Published: November 2016

Expression of the trpBA gene pair of Pseudomonas aeruginosa is regulated by the endogenous level of indoleglycerol phosphate (InGP) and the trpI gene product. The TrpI protein binds to the -77 to -32 region of the trpBA promoter. This region is divisible into two sites: site I, which is protected by TrpI in the presence and absence of InGP; and site II, which is protected by TrpI only in the presence of InGP. Recently, the trpI gene was subcloned into an expression vector and the protein was overproduced in Escherichia coli. The TrpI protein was purified to 80 to 95% purity. The molecular weight of native TrpI protein is estimated to be 129,000 by gel exclusion chromatography, and therefore it is likely a tetramer composed of 31,000-dalton monomers. Gel retardation assays with the purified TrpI protein demonstrated that InGP increases the affinity of TrpI for sites I and II approximately 17- and 14-fold, respectively. Binding of TrpI to site I is site II independent. However, the protein has low intrinsic affinity for site II and its binding to site II is site I dependent. Therefore, binding of TrpI to site II probably requires its interaction with a second TrpI molecule at site I.
Genetic and biochemical characterization of a Pseudomonas solanacearum gene cluster required for extracellular polysaccharide production and for virulence
Authors: Cook D , Sequeira L
Published: November 2016

Infection of host plants by Pseudomonas solanacerum results in wilting, which is thought to be due largely to the occlusion of xylem vessels by the P. solanacearum extracellular polysaccharide (EPS) that primarily consists of N-acetylgalactosamine (GalNAc). By means of Tn3 mutagenesis, we identified a 6.5-kb gene cluster that contains five complementation units required for EPS production and virulence in this bacterium. There was positive correlation between the amount of EPS produced in culture and (i) in planta growth and (ii) virulence. Based on analysis of beta-glucuronidase-gene fusions, these genes are expressed both in broth cultures and in planta and may be constitutive. Both wild-type and mutant strains contained similar amounts of UDP-GalNAc, the predicted primary substrate for EPS synthesis. Thus, the EPS mutants we obtained should be useful in the analysis of steps in the assembly of the polysaccharide and how this process is related to virulence.
The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli
Authors: Gustafsson C , Lindström P H , Hagervall T G , Esberg K B , Björk G R
Published: November 2016

The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.
Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp
Authors: Muriana P M , Klaenhammer T R
Published: November 2016

Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.
Purification and properties of two membrane alkaline phosphatases from Bacillus subtilis 168
Authors: Sugahara T , Konno Y , Ohta H , Ito K , Kaneko J , Kamio Y , Izaki K
Published: November 2016

Two alkaline phosphatases were extracted from the membranes of Bacillus subtilis 168 stationary-phase cells and purified as homogeneous proteins by hydroxyapatite column chromatography. Alkaline phosphatases I and II differed in several properties such as subunit molecular weight, substrate specificity, thermostability, Km, pH stability, and peptide maps.
Genes for 7S RNAs can replace the gene for 4.5S RNA in growth of Escherichia coli
Authors: Brown S
Published: November 2016

4.5S RNAs of eubacteria and 7S RNAs of archaebacteria and eukaryotes exist in a hairpin conformation. The apex of this hairpin displays structural and sequence similarities among both 4.5S and 7S RNAs. Furthermore, a hyphenated sequence of 16 nucleotides is conserved in all eubacterial 4.5S RNAs examined. In this article I report that 7S RNAs that contain this 16-nucleotide sequence are able to replace 4.5S RNAs and permit growth of Escherichia coli.
Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO
Authors: Hoshino T , Kose-Terai K , Uratani Y
Published: November 2016

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.