bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the defect in the Escherichia coli mutT1 mutator gene
Authors: Bhatnagar S K , Bullions L C , Lew G , Bessman M J
Published: November 2016

With a probe constructed from the wild-type gene, a DNA fragment containing the entire mutT1 mutator gene was isolated and cloned into pUC18. Nucleotide sequence analysis revealed that the mutator defect was most likely due to an IS1 insertion into the wild-type gene.
The DNA adenine methyltransferase (dam+) gene of bacteriophage T4 reverses the mutator phenotype of an Escherichia coli dam mutant
Authors: Hall R M
Published: November 2016

The mutator phenotype of Escherichia coli dam mutants was found to be reversed by introduction of the bacteriophage T4 gene for DNA adenine methyltransferase. This precludes a direct role for the E. coli DNA adenine methyltransferase in mismatch repair, in addition to its role in strand discrimination, as suggested by earlier studies (S. L. Schlagman, S. Hattman, and M. G. Marinus, J. Bacteriol. 165:896-900, 1986).
Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100
Authors: Lundeen S G , Savage D C
Published: November 2016

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.
Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E
Authors: Jonas R M , Peters H K , Haldenwang W G
Published: November 2016

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode sigma E directly. The sequence specifying the first amino acid of sigma E (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigE delta 84) encodes a sigma E-like protein which differs from the "processed product" by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable sigma E. The sigE delta 84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A sigma E-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that sigma E is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigE delta 48) which made an active sigma E-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigE delta 48 product was not processed, but it activated the sigma E -dependent spoIID promoter in vivo. The sigE delta 48 product therefore lost both an essential target for processing and a region which inhibited sigma sigma E activity. Cells which carried sig E delta 48 were Spo-. The basis of the sigE delta 48-dependent defect in sporulation is unknown, but the sigma E delta 48 activity appeared to persist beyond the time in development (4 h after onset sporulation) when wild-type sigma E activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.
Role of autocide AMI in development of Myxococcus xanthus
Authors: Rosenbluh A , Rosenberg E
Published: November 2016

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.
Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708
Authors: Gopal-Srivastava R , Mallonee D H , White W B , Hylemon P B
Published: November 2016

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.
Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui
Authors: Daniel S L , Hsu T , Dean S I , Drake H L
Published: November 2016

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.
lcrR, a low-Ca2(+)-response locus with dual Ca2(+)-dependent functions in Yersinia pestis
Authors: Barve S S , Straley S C
Published: November 2016

The low-Ca2+ response (Lcr) of Yersinia includes a regulatory cascade and a set of virulence-related proteins, one of which is the V antigen. The regulatory genes modulate both bacterial growth and expression of the virulence-related proteins in response to temperature and the presence of Ca2+ and nucleotides. In this study we defined a new Lcr locus, lcrR, in Yersinia pestis KIM. An lcrR mutant, obtained by insertion mutagenesis, failed to grow at 37 degrees C whether Ca2+ was present or not. However, it grew normally in the presence of ATP, showing that the Ca2(+)- and nucleotide-responsive mechanisms are separate in Y. pestis. The lcrR mutant was avirulent in mice, probably due to its compromised growth at 37 degrees C. beta-Galactosidase measurements and Northern (RNA blot) analysis revealed that lcrR transcription was regulated primarily by temperature. The DNA sequence of the lcrR locus contained a single open reading frame of 441 bases that could encode a protein with a molecular weight of 16,470 and a pI of 10.73. Expression of an lcrR-containing clone in Escherichia coli yielded a 16,000-molecular-weight protein. At 37 degrees C, the lcrR mutant strongly expressed V antigen and initiated lcrGVH transcription whether Ca2+ was present or not, indicating that this mutant had lost the transcriptional downregulation of lcrGVH shown by the parent in the presence of Ca2+. In the absence of Ca2+, the mutant failed to express LcrG, even though lcrGVH mRNA initiated upstream of lcrG at the normal sites. These data suggest that the lcrR locus is necessary for the regulation of LcrG expression in the absence of Ca2+. Therefore, this locus has a dual regulatory role in the low-Ca2+ response.
A cyanide-aldehyde complex inhibits bacterial luciferase
Authors: Makemson J C
Published: November 2016

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.
Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX)
Authors: Stoorvogel J , van Bussel M J , Tommassen J , van de Klundert J A
Published: November 2016

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The ompX gene was located by insertions of the gamma delta sequence into the recombinant plasmid. The polarity of the gene was determined by in vitro transcription and translation of the gamma delta-containing plasmids. The nucleotide sequence of the ompX gene was elucidated by using both inverted terminal repeats of the gamma delta sequence as starting points for M13 dideoxy sequencing. The gene was found to encode a precursor of the OmpX protein consisting of 172 amino acid residues with a molecular mass of 18.6 kDa. The protein contains an N-terminal signal sequence of 23 amino acid residues. The exact cleavage point was established by sequencing the N-terminal part of the mature protein. The OmpX protein has several characteristics in common with outer membrane proteins of gram-negative bacteria. The protein is rather hydrophilic and is devoid of long hydrophobic stretches. On the basis of these results, we present a model for the OmpX protein folding in an outer membrane.
Segmental message stabilization as a mechanism for differential expression from the Zymomonas mobilis gap operon
Authors: Eddy C K , Keshav K F , An H , Utt E A , Mejia J P , Ingram L O
Published: November 2016

In Zymomonas mobilis, three- to fourfold more glyceraldehyde-3-phosphate dehydrogenase protein than phosphoglycerate kinase is needed for glycolysis because of differences in catalytic efficiency. Consistent with this requirement, higher levels of glyceraldehyde-3-phosphate dehydrogenase were observed with two-dimensional polyacrylamide gel electrophoresis. The genes encoding these enzymes (gap and pgk, respectively) form a bicistronic operon, and some form of regulation is required to provide this differential expression. Two transcripts were observed in Northern RNA analyses with segments of gap as a probe: a more abundant 1.2-kb transcript that contained gap alone and a 2.7-kb transcript that contained both genes. Based on the relative amounts of these transcripts, the coding regions for glyceraldehyde-3-phosphate dehydrogenase were calculated to be fivefold more abundant than those for phosphoglycerate kinase. Assuming equal translational efficiency, this is sufficient to provide the observed differences in expression. Operon fusions with lacZ provided no evidence for intercistronic terminators or attenuation mechanisms. Both gap operon messages were very stable, with half-lives of approximately 16 min (1.2-kb transcript) and 7 min (2.7-kb transcript). Transcript mapping and turnover studies indicated that the shorter gap message was a stable degradation product of the full-length message. Thus differential expression of gap and pgk results primarily from increased translation of the more stable 5' segment of the transcript containing gap. The slow turnover of the messages encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase is proposed as a major feature contributing to the high level of expression of these essential enzymes.
Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium
Authors: Valli K , Gold M H
Published: November 2016

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dichlorophenol (I). The pathway for the degradation of 2,4-dichlorophenol (I) was elucidated by the characterization of fungal metabolites and of oxidation products generated by purified lignin peroxidase and manganese peroxidase. The multistep pathway involves the oxidative dechlorination of 2,4-dichlorophenol (I) to yield 1,2,4,5-tetrahydroxybenzene (VIII). The intermediate 1,2,4,5-tetrahydroxybenzene (VIII) is ring cleaved to produce, after subsequent oxidation, malonic acid. In the first step of the pathway, 2,4-dichlorophenol (I) is oxidized to 2-chloro-1,4-benzoquinone (II) by either manganese peroxidase or lignin peroxidase. 2-Chloro-1,4-benzoquinone (II) is then reduced to 2-chloro-1,4-hydroquinone (III), and the latter is methylated to form the lignin peroxidase substrate 2-chloro-1,4-dimethoxybenzene (IV). 2-Chloro-1,4-dimethoxybenzene (IV) is oxidized by lignin peroxidase to generate 2,5-dimethoxy-1,4-benzoquinone (V), which is reduced to 2,5-dimethoxy-1,4-hydroquinone (VI). 2,5-Dimethoxy-1,4-hydroquinone (VI) is oxidized by either peroxidase to generate 2,5-dihydroxy-1,4-benzoquinone (VII) which is reduced to form the tetrahydroxy intermediate 1,2,4,5-tetrahydroxybenzene (VIII). In this pathway, the substrate is oxidatively dechlorinated by lignin peroxidase or manganese peroxidase in a reaction which produces a p-quinone. The p-quinone intermediate is then recycled by reduction and methylation reactions to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This unique pathway apparently results in the removal of both chlorine atoms before ring cleavage occurs.
A newly identified DNA replication terminus site, TerE, on the Escherichia coli chromosome
Authors: Hidaka M , Kobayashi T , Horiuchi T
Published: November 2016

To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences. We found a Ter site, which we named TerE, located at 23.2 min on the linkage map. TerE inhibits only counterclockwise DNA replication. Macroscopically, five Ter sites are located in a periodic arrangement on the genome.
RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins
Authors: Gao J G , Gussin G N
Published: November 2016

The activities of RNA polymerases (RNAPs) from Pseudomonas aeruginosa and Pseudomonas syringae were compared with that of Escherichia coli RNAP. All three enzymes are able to initiate transcription at the trpBA promoter of P. aeruginosa and at the coliphage lambda promoters, pRM and pRE, in response to heterospecific activators (TrpI protein, repressor, and cII protein, respectively). However, both Pseudomonas polymerases have less stringent requirements for promoter recognition in the absence of activators than does E. coli RNAP.
NarK enhances nitrate uptake and nitrite excretion in Escherichia coli
Authors: DeMoss J A , Hsu P Y
Published: November 2016

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.
Genetic analysis of the flaA locus of Bacillus subtilis
Authors: Hauser P M , Crabb W D , Fiora M G , Scoffone F , Galizzi A
Published: November 2016

We isolated two clones of recombinant lambda bacteriophage with overlapping inserts of Bacillus subtilis chromosomal DNA corresponding to part of the flaA locus. The flaA4 and flaA15 mutations were localized on the physical map by marker rescue experiments. The flaA locus and the flaB (sigD) gene were mapped in transduction crosses, and the order glnA polC flaB flaA was determined. FlaB was linked to polC in transformation crosses.
Critical sequences in the core of the P1 plasmid replication origin
Authors: Brendler T , Abeles A , Austin S
Published: November 2016

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.
Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein
Authors: Ball C A , Johnson R C
Published: November 2016

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.
The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin
Authors: Brunen M , Engelhardt H , Schmid A , Benz R
Published: November 2016

The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.
Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites
Authors: Ponniah S , Endres R O , Hasty D L , Abraham S N
Published: November 2016

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.
Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1
Authors: Kukor J J , Olsen R H
Published: November 2016

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol.
The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases
Authors: Massidda O , Rossolini G M , Satta G
Published: November 2016

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.
Role of two flagellin genes in Campylobacter motility
Authors: Guerry P , Alm R A , Power M E , Logan S M , Trust T J
Published: November 2016

Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity. The flaA gene is transcribed from a o-28 promoter, and the flaB gene from a o-54 promoter. Gene replacement mutagenesis techniques were used to generate flaA+ flaB and flaA flaB+ mutants. Both gene products are capable of assembling independently into functional filaments. A flagellar filament composed exclusively of the flaA gene product is indistinguishable in length from that of the wild type and shows a slight reduction in motility. The flagellar filament composed exclusively of the flaB gene product is severely truncated in length and greatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products are required for a fully active flagellar filament. Although the wild-type flagellar filament is a heteropolymer of the flaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surface exposed along the length of the wild-type filament.
Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro
Authors: Tovar-Rojo F , Setlow P
Published: November 2016

alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.
Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure
Authors: Tipples G , McClarty G
Published: November 2016

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.
Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica
Authors: Michiels T , Vanooteghem J C , Lambert de Rouvroit C , China B , Gustin A , Boudry P , Cornelis G R
Published: November 2016

Upon incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic yersiniae release large amounts of pYV plasmid-encoded proteins called Yops that are involved in pathogenesis. Yersinia enterocolitica also expresses an outer membrane protein that is considered an adhesin and called YadA (previously called P1 or YopA). The production of Yops is coordinately regulated by a 20-kb region of the plasmid referred to as the Ca2+ dependence region and containing at least four loci called virA, virB, virC, and virF. The virF gene encodes a key transcriptional activator of yop genes. We have shown here that virF is also required for transcription of yadA and that virB is necessary for full transcription of the yop and yadA genes. In contrast, mutations in genes virA and virC had only a weak influence on the transcription of yop and yadA genes. These mutations did not affect the production of YadA but they completely inhibited the translocation of Yops from the intracellular compartment to the extracellular milieu. We inferred from these data that virA and virC are involved in the specific transport of Yops. We analyzed the 8.5-kb virC region and showed that it is most probably a single operon containing 13 open reading frames called yscA to yscM (for Yop secretion). Protein YscC has a putative signal sequence and shares significant homology with outer membrane proteins involved in the secretion of pullulanase by Klebsiella pneumoniae (PulD) or in the assembly of filamentous bacteriophages (gene IV product). At least the putative products of yscD, yscJ, and yscL were shown to be required for the export of Yops. YscJ turned out to be YlpB, a lipoprotein that we had detected previously. The yscM gene shares homology with yopH, the adjacent gene on the pYV plasmid. Its product does not appear to be necessary for the production of Yops. Transcription of the virC operon was subjected to the same regulation as the yop genes.
Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription
Authors: Eschenlauer A C , Reznikoff W S
Published: November 2016

We isolated three Escherichia coli catabolite gene activator protein mutants that are defective in the positive control of transcription initiation from the lac operon promoter region yet retain negative control of transcription from other promoters. One mutant has a substitution of valine for glutamate at residue 72, which lies in the cyclic AMP binding domain and contacts cyclic AMP. The other two mutants have substitutions of asparagine and cysteine for glycine 162, which lies in a surface-exposed turn of the DNA-binding domain. Surprisingly, although all three mutants can repress the lacP2/P3 promoters through the catabolite gene activator protein target site of lac, none displays strong dominance over the ability of wild-type catabolite gene activator protein to stimulate the lacP1 promoter.
Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators
Authors: Dillard J P , Yother J
Published: November 2016

Difficulties encountered in the cloning of DNA from Streptococcus pneumoniae and other AT-rich organisms into ColE1-type Escherichia coli vectors have been proposed to be due to the presence of a large number of strong promoter-acting sequences in the donor DNA. The use of transcription terminators has been advocated as a means of reducing instability resulting from disruption of plasmid replication caused by strong promoters. However, neither the existence of promoter-acting sequences of sufficient strength and number to explain the reported cloning difficulties nor their role as a source of instability has been proven. As a direct test of the "strong promoter" hypothesis, we cloned random fragments from S. pneumoniae into an E. coli vector containing transcription terminators, identified strong promoter-acting sequences, and subsequently removed the transcription terminators. We observed that terminator removal resulted in reduced copy numbers for the strongest promoter-acting sequences but not in reduced promoter strengths or altered plasmid stabilities. Our results indicate that promoters strong enough to require transcription terminators for plasmid stability are probably rare in S. pneumoniae DNA.
Characterization and expression of the Escherichia coli Mrr restriction system
Authors: Waite-Rees P A , Keating C J , Moran L S , Slatko B E , Hornstra L J , Benner J S
Published: November 2016

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.
Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus
Authors: Ravenscroft N , Walker S G , Dutton G G , Smit J
Published: November 2016

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.
Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein
Authors: An H , Scopes R K , Rodriguez M , Keshav K F , Ingram L O
Published: November 2016

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45 degrees C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.
Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase
Authors: Cameron B , Blanche F , Rouyez M C , Bisch D , Famechon A , Couder M , Cauchois L , Thibaut D , Debussche L , Crouzet J
Published: November 2016

Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.
Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase
Authors: Crouzet J , Levy-Schil S , Cameron B , Cauchois L , Rigault S , Rouyez M C , Blanche F , Debussche L , Thibaut D
Published: November 2016

A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype.