bluuebunny/crossref_metadata_2025 huggingface dataset:
The product of the Klebsiella aerogenes nac (nitrogen assimilation control) gene is sufficient for activation of the hut operons and repression of the gdh operon
Authors: Schwacha A , Bender R A
Published: November 2016

In Klebsiella aerogenes, the formation of a large number of enzymes responds to the quality and quantity of the nitrogen source provided in the growth medium, and this regulation requires the action of the nitrogen regulatory (NTR) system in every case known. Nitrogen regulation of several operons requires not only the NTR system, but also NAC, the product of the nac gene, raising the question of whether the role of NAC is to activate operons directly or by modifying the specificity of the NTR system. We isolated an insertion of the transposon Tn5tac1 which puts nac gene expression under the control of the IPTG-inducible tac promoter rather than the nitrogen-responsive nac promoter. When IPTG was present, cells carrying the tac-nac fusion activated NAC-dependent operons and repressed NAC-repressible operons independent of the nitrogen supply and even in the absence of an active NTR system. Thus, NAC is sufficient to regulate operons like hut (encoding histidase) and gdh (encoding glutamate dehydrogenase), confirming the model that the NTR system activates nac expression and NAC activates hut and represses gdh. Activation of urease formation occurred at a lower level of NAC than that required for glutamate dehydrogenase repression, and activation of histidase formation required still more NAC.
Two glucose transport systems in Bacillus licheniformis
Authors: Tangney M , Priest F G , Mitchell W J
Published: November 2016

Bacillus licheniformis NCIB 6346 showed active accumulation of glucose which was inhibited by agents which affect the transmembrane proton gradient. Phosphotransferase (PTS) activity, identified as phosphoenolpyruvate-dependent phosphorylation of glucose, was found in cell extracts but could not be demonstrated in cells permeabilized with toluene when assays were conducted at pH 6.6. The same was true for mannitol and fructose phosphotransferase activities. Cells grown on fructose accumulated glucose at a slower rate than glucose-grown cells, and extracts prepared from them did not contain glucose PTS activity. Examination of the effects of analogs on glucose uptake and phosphorylation showed that 2-deoxyglucose was not a PTS substrate, but did markedly inhibit glucose uptake, with stronger inhibition in cells grown on fructose. Glucose accumulation by whole cells grown on glucose became less sensitive to the uncoupler tetrachlorosalicylanilide (TCS) as the pH was raised from 6.6 to 8.0, while in fructose-grown cells TCS was equally effective across this pH range. PTS activity was exhibited by toluene-treated cells at pH 7.5 and above, although the system itself in extracts was not affected by pH in the range of 5.0 to 8.0. The results are consistent with the presence of two glucose transport systems, one a PTS and the other operating by an alternative mechanisms, and suggest that the PTS in B. licheniformis may be regulated in a pH-dependent manner.
Heat shock-induced axenic growth of Bdellovibrio bacteriovorus
Authors: Gordon R F , Stein M A , Diedrich D L
Published: November 2016

The bdellovibrios are obligately predatory bacteria that attack other gram-negative bacteria. They grow only in the periplasmic space of prey unless they mutate to forms that can grow axenically. A culture medium that promoted enhanced growth of prey-independent bdellovibrios was developed. The ability of this medium to support the growth of prey-dependent bdellovibrios was tested under transcription-altering conditions. This approach tested the hypothesis that the inability to grow prey-dependent bdellovibrios in artificial media was rooted in both nutritional and transcriptional signal deficiencies. It was assumed that nutritional deficiencies had been resolved and that empirically applied artificial signals may evoke the expression of genes required for axenic growth of bdellovibrios. Prey-dependent bdellovibrios could be grown in PPYE medium (0.1% proteose peptone 3 and 0.03% Bacto yeast extract adjusted to pH 7.0 and supplemented with 3 mM MgCl2 and 2 mM CaCl2 after autoclaving) after heat shock, and subsequent rounds of growth occurred after additional heat shocks. Heat shock may have generated or simulated signals normally derived from prey.
Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostats under NaCl stress
Authors: Olz R , Larsson K , Adler L , Gustafsson L
Published: November 2016

The energetics and accumulation of solutes in Saccharomyces cerevisiae were investigated for cells grown aerobically in a chemostat under NaCl stress and glucose limitation. Changed energy requirements in relation to external salinity were examined by energy balance determinations performed by substrate and product analyses, with the latter including heat measurements by microcalorimetry. In both 0 and 0.9 M NaCl cultures, the catabolism was entirely respiratory at the lowest dilution rates tested but shifted to a mixed respiratory-fermentative metabolism at higher dilution rates. This shift occurred at a considerably lower dilution rate for salt-grown cells. The intracellular solute concentrations, as calculated on the basis of intracellular soluble space determinations, showed that the internal Na+ concentration increased from about 0.02 molal in basal medium to about 0.18 molal in 0.9 M NaCl medium, while intracellular K+ was maintained around 0.29 molal despite the variation in external salinity. The intracellular glycerol concentration increased from below 0.05 molal at low salinity to about 1.2 molal at 0.9 M NaCl. The concentrations of the internal solutes, however, changed insignificantly with growth rate and energy metabolism. The additional maintenance energy expenditure for growth at 0.9 M NaCl was, depending on the growth rate, 14 to 31% of the total energy requirement for growth at 0 M NaCl. Including the energy conserved in glycerol, the total additional energy demand for growth at 0.9 M NaCl corresponded to 28 to 51% of the energy required for growth at 0 M NaCl.
Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions
Authors: Sára M , Sleytr U B
Published: November 2016

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).
Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti
Authors: Robinson J B , Bauer W D
Published: November 2016

The relationship between chemotaxis and transport of C4 dicarboxylic acids was analyzed with Rhizobium meliloti dct mutants defective in one or all of the genes required for dicarboxylic acid transport. Succinate, malate, and fumarate were moderately potent chemoattractants for wild-type R. meliloti and appeared to share a common chemoreceptor. While dicarboxylate transport is inducible, taxis to succinate was shown to be constitutive. Mutations in the dctA and dctB genes both resulted in the reduction, but not elimination, of chemotactic responses to succinate, indicating that transport via DctA or chemosensing via DctB is not essential for C4 dicarboxylate taxis, although they appear to contribute to it. Mutations in dctD and rpoN genes did not affect taxis to succinate. Aspartate, which is also transported by the dicarboxylate transport system, elicited strong chemotactic responses via a chemoreceptor distinct from the succinate-malate-fumarate receptor. Taxis to aspartate was unaltered in dctA and dctB mutants but was considerably reduced in both dctD and rpoN mutants, indicating that aspartate taxis is strongly dependent on elements responsible for transcriptional activation of dctA. Methylation and methanol release experiments failed to show a significant increase in methyl esterification of R. meliloti proteins in response to any of the attractants tested.
5-Aminolevulinic acid availability and control of spectral complex formation in hemA and hemT mutants of Rhodobacter sphaeroides
Authors: Neidle E L , Kaplan S
Published: November 2016

In the photosynthetic bacterium Rhodobacter sphaeroides, two genes, hemA and hemT, each encode a distinct 5-aminolevulinic acid (ALA) synthase isozyme (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2292-2303, 1993). This enzyme catalyzes the first and rate-limiting step in a branched pathway for tetrapyrrole formation, leading to the biosynthesis of hemes, bacteriochlorophylls, and corrinoids. In an attempt to determine the functions of hemA and hemT, mutant strains were constructed with specific chromosomal disruptions. These chromosomal disruption allowed hemA and hemT to be precisely localized on the larger and smaller of two R. sphaeroides chromosomes, respectively. Mutants carrying a single hemA or hemT disruption grew well without the addition of ALA, whereas a mutant, HemAT1, in which hemA and hemT had both been inactivated required exogenous ALA for growth. The growth rates, ALA synthase enzyme levels, and the amounts of bacteriochlorophyll-containing intracytoplasmic membrane spectral complexes of all strains were compared. Under photosynthetic growth conditions, the levels of bacteriochlorophyll, carotenoids, and B800-850 and B875 light-harvesting complexes were significantly lower in the Hem mutants than in the wild type. In the mutant strains, available bacteriochlorophyll appeared to be preferentially targeted to the B875 light-harvesting complex relative to the B800-850 complex. In strain HemAT1, the amount of B800-850 complex varied with the concentration of ALA added to the growth medium, and under conditions of ALA limitation, no B800-850 complexes could be detected. In the Hem mutants, there were aberrant transcript levels corresponding to the puc and puf operons encoding structural polypeptides of the B800-850 and B875 complexes. These results suggest that hemA and hemT expression is coupled to the genetic control of the R. sphaeroides photosynthetic apparatus.
Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa
Authors: Exley G E , Colandene J D , Garrett R H
Published: November 2016

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.
Choline transport activity in Staphylococcus aureus induced by osmotic stress and low phosphate concentrations
Authors: Kaenjak A , Graham J E , Wilkinson B J
Published: November 2016

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from [14C]choline was accumulated as [14C]glycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na+ dependent and had a Km of 46 microM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaC1 concentrations.
The Rhodobacter capsulatus chlorin reductase-encoding locus, bchA, consists of three genes, bchX, bchY, and bchZ
Authors: Burke D H , Alberti M , Hearst J E
Published: November 2016

The bchA locus of Rhodobacter capsulatus codes for the chlorin reductase enzyme in the bacteriochlorophyll synthesis pathway. Previous work has suggested that this locus might encompass a single gene. We have sequenced the bchA locus and found it to contain three coding segments, which we designate bchX, bchY, and bchZ. Each coding segment contains its own translational initiation sequence and follows codon utilization patterns consistent with those of previously published R. capsulatus genes. When various regions of the bchA locus and flanking sequences were subcloned into an expression vector and expressed in Escherichia coli, the three coding segments were all expressed as separate peptides. Finally, conservation of amino acid sequences between bchX and a subunit of the protochlorophyllide reductase (bchL, 34% identity) and the nitrogenase Fe protein (nifH, 30 to 37% identity) suggests structural and mechanistic commonalities among all three proteins.
bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants
Authors: Burke D H , Alberti M , Hearst J E
Published: November 2016

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.
Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea
Authors: McTavish H , Fuchs J A , Hooper A B
Published: November 2016

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.
The Ada protein is a class I transcription factor of Escherichia coli
Authors: Sakumi K , Igarashi K , Sekiguchi M , Ishihama A
Published: November 2016

The methylated Ada protein of Escherichia coli, a regulatory protein for the adaptive response, binds to a target DNA from positions -62 to -31 upstream of the ada gene and facilitates the binding of RNA polymerase to the promoter. Mutant RNA polymerases consisting of C-terminal-deleted alpha subunits are virtually inactive in response to activation by the Ada protein. Thus, we conclude that the Ada protein is a class I transcription factor which requires the C-terminal region of the RNA polymerase alpha subunit for transcription activation.
Tyrosine phosphate in a- and b-type flagellins of Pseudomonas aeruginosa
Authors: Kelly-Wintenberg K , South S L , Montie T C
Published: November 2016

Both a- and b-type purified flagellins from a number of Pseudomonas aeruginosa strains grown in radiolabeled phosphate were shown to be phosphorylated. Analysis of partial acid-hydrolyzed flagellar filaments revealed that 32Pi was in phosphotyrosine. Three 32P-phosphopeptides apparently are common to a- and b-type flagellins, but a fourth peptide was found only in b-type hydrolysates. P. aeruginosa PAK flagellin, containing only two tyrosines, both in the variable region, was readily labeled and gave the same peptide pattern as flagellins containing additional tyrosines. Data showing that a- and b-type flagellins gave positive immunoblots with antiphosphotyrosine monoclonal antibody and that release of P(i) by alkaline phosphatase occurred indicated that unmodified tyrosine phosphate exists in flagellin.
The dilution rate affects the outer membrane protein and lipopolysaccharide composition of Haemophilus influenzae type b grown under iron limitation
Authors: Langford P R , Moxon E R
Published: November 2016

When Haemophilus influenzae type b was grown under iron limitation in continuous culture, the dilution rate affected the outer membrane protein and lipopolysaccharide composition. Investigations of the effect of the reduced availability of iron or other environmental parameters on these surface components should be controlled for growth rate.
Molecular cloning, sequencing, and transcriptional analysis of the groESL operon from Bacillus stearothermophilus
Authors: Schön U , Schumann W
Published: November 2016

Using a gene probe of the Bacillus subtilis groEL gene, a 7.3-kb HindIII fragment of chromosomal DNA of Bacillus stearothermophilus was cloned. Sequencing of 2,309 bp led to the detection of two open reading frames in the order groES groEL. Primer extension studies revealed one potential transcription start site preceding the groESL operon, which was activated upon temperature upshift. Northern blot (RNA) analysis resolved two mRNA species with lengths of 2.2 and 1.5 kb; RNA slot-blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 7 min postinduction followed by a decrease. The 9-bp inverted repeat characteristic of many gram-positive bacteria was found within the 5' leader region of the mRNA. The groESL operon of B. stearothermophilus could complement E. coli groES(Ts) and groEL(Ts) mutants for growth at high temperature and for propagation of phage lambda.
Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris
Authors: Ielpi L , Couso R O , Dankert M A
Published: November 2016

Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.
Functional organization of the glnB-glnA cluster of Azospirillum brasilense
Authors: de Zamaroczy M , Paquelin A , Elmerich C
Published: November 2016

The functional organization of the glnB-A cluster of Azospirillum brasilense, which codes for the PII protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the sigma 70 and sigma 54 types, respectively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription of glnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PII polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PII plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, nifA, and point mutant strains in glnA were analyzed. NtrB and NtrC are not involved in the regulation of glnBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PII acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif- null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nifA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions in A. brasilense. It is therefore most likely that PII is required for NifA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimiar to previously described consensus sites for sigma factors.
Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores
Authors: Sanchez-Salas J L , Setlow P
Published: November 2016

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.
Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I
Authors: Matson S W , Nelson W C , Morton B S
Published: November 2016

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.
The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli
Authors: Gibbs P E , Kilbey B J , Banerjee S K , Lawrence C W
Published: November 2016

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.
A role for the Clp protease in activating Mu-mediated DNA rearrangements
Authors: Shapiro J A
Published: November 2016

Bacteriophage Mu, one of the best-characterized mobile genetic elements, can be used effectively to answer fundamental questions about the regulation of biochemical machinery for DNA rearrangement. Previous studies of Mu virulence have implicated the Clp protease in repressor inactivation (V. Geuskens, A. Mhammedi-Alaoui, L. Desmet, and A. Toussaint, EMBO J. 13:5121-5127, 1992). These studies were extended by analyzing the phenotypic consequences of clp alleles in two Escherichia coli systems: (i) the periodic replication of Mudlac transposons in colonies and (ii) the action of a Mu prophage in forming araB-lacZ coding sequence fusions. The clpP::CM mutation, which removes the proteolytic subunit of Clp protease, caused a drastic reduction in Mu activity in both systems. The clpA::Tn10 mutation, which removes a regulatory subunit of Clp protease, altered the timing of Mu activity in both systems. A clpA deletion reduced the extent of Mudlac replication in colonies. These results point to temporal changes in Clp proteolysis of the Mucts62 repressor as a key molecular event in the regulation of one class of genomic change in E. coli.
Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp
Authors: Smith C J , Parker A C
Published: November 2016

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.
Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry
Authors: Gibson B W , Melaugh W , Phillips N J , Apicella M A , Campagnari A A , Griffiss J M
Published: November 2016

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.
Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family
Authors: Soppa J , Duschl J , Oesterhelt D
Published: November 2016

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.
Determination of the growth rate-regulated steps in expression of the Escherichia coli K-12 gnd gene
Authors: Pease A J , Wolf R E
Published: November 2016

In Escherichia coli K-12 strain W3110, the amount of 6-phosphogluconate dehydrogenase relative to that of total protein, i.e., the specific enzyme activity, increases about threefold during growth in minimal media over the range of growth rates with acetate and glucose as sole carbon sources. Previous work with gnd-lac operon and protein fusion strains indicated that two steps in the expression of the gnd gene are subject to growth rate-dependent control, with at least one step being posttranscriptional. With both Northern (RNA) and slot blot analyses, we found that the amount of gnd mRNA relative to that of total RNA was 2.5-fold higher in cells growing in glucose minimal medium than in cells grown on acetate. Therefore, since the total mRNA fraction of total RNA is essentially independent of the growth rate, the amount of gnd mRNA relative to that of total mRNA increases about 2.5-fold with increasing growth rate. This indicates that most of the growth rate-dependent increase in 6-phosphogluconate dehydrogenase can be accounted for by the growth rate-dependent increase in gnd mRNA level. We measured the decay of gnd mRNA mass in the two growth conditions after blocking transcription initiation with rifampin and found that the stability of gnd mRNA does not change with growth rate. We also used a gnd-lacZ protein fusion to measure the functional mRNA half-life and found that it too is growth rate independent. Thus, the growth rate-dependent increase in the level of gnd mRNA is due to an increase in gnd transcription, and this increase is sufficient to account for the growth rate regulation of the 6-phosphogluconate dehydrogenase level. The dilemma posed by interpretations of the properties of gnd-lac fusion strains and by direct measurement of gnd mRNA level is discussed.
In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli
Authors: Hou Y , Lin Y P , Sharer J D , March P E
Published: November 2016

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.
Translation initiation factor IF1 is essential for cell viability in Escherichia coli
Authors: Cummings H S , Hershey J W
Published: November 2016

Translation initiation factor IF1 is a highly conserved element of the prokaryotic translational apparatus. It has been demonstrated earlier that the factor stimulates in vitro the initiation phase of protein synthesis. However, no mutation in its gene, infA, has been identified, and a role for IF1 in translation has not been demonstrated in vivo. To elucidate the function of IF1 and determine if the protein is essential for cell growth, the chromosomal copy of infA was disrupted. Cell viability is maintained only when infA is expressed in trans from a plasmid, thereby demonstrating that IF1 is essential for cell growth in Escherichia coli. Cells depleted of IF1 exhibit few polysomes, suggesting that IF1 functions in the initiation phase of protein synthesis.
The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses
Authors: Packer H L , Armitage J P
Published: November 2016

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.
TRK2 is not a low-affinity potassium transporter in Saccharomyces cerevisiae
Authors: Ramos J , Alijo R , Haro R , Rodriguez-Navarro A
Published: November 2016

TRK1 and TRK2 encode proteins involved in K+ uptake in Saccharomyces cerevisiae. A kinetic study of Rb+ influx in trk1 TRK2, trk1 TRK2D, and trk1 trk2 mutants reveals that TRK2 shows moderate affinity for Rb+. K(+)-starved trk1 delta TRK2 cells show a low-affinity component accounting for almost the total Vmax of the influx and a moderate-affinity component exhibiting a very low Vmax. Overexpression of TRK2 in trk1 delta TRK2D cells increases the Vmax of the moderate-affinity component, and this component disappears in trk1 delta trk2 delta cells. In contrast, the low-affinity component of Rb+ influx in trk1 delta TRK2 cells is not affected by mutations in TRK2. Consistent with the different levels of activity of the moderate-affinity Rb+ influx, trk1 delta TRK2 cells grow slowly in micromolar K+, trk1 delta TRK2D cells grow rapidly, and trk1 delta trk2 delta cells fail to grow. The existence of a unique K+ uptake system composed of several proteins is also discussed.