bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of bacteriophage T7 in a growing plaque
Authors: Yin J
Published: November 2016

The emergence of mutants during the 10(9)-fold amplification of a bacteriophage was spatially resolved in a growing plaque. When wild-type phage T7 was grown on an Escherichia coli host which expressed an essential early enzyme of the phage infection cycle, the T7 RNA polymerase, mutant phage relying on this enzyme appeared in 10(8) phage replications and outgrew the wild type. Spatial resolution of the selection process was achieved by analyzing stab samples taken along a plaque radius. Different mutants were selected at different rates along different radii of the plaque, based on host range and restriction patterns of the isolates. The mutants deleted up to 11% of their genomes, including the gene for their own RNA polymerase. They gained an advantage over the wild type by replicating more efficiently, as determined by one-step growth cultures.
Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes
Authors: Reed K E , Cronan J E
Published: November 2016

Two genes, lipA and lipB, involved in lipoic acid biosynthesis or metabolism were characterized by DNA sequence analysis. The translational initiation site of the lipA gene was established, and the lipB gene product was identified as a 25-kDa protein. Overproduction of LipA resulted in the formation of inclusion bodies, from which the protein was readily purified. Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. Mutants carrying a null mutation in the lipB gene retained a partial ability to synthesize lipoic acid and produced low levels of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities. The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo; however, the growth of both lipA and lipB mutants was supported by either 6-thiooctanoic acid or 8-thiooctanoic acid in place of lipoic acid. These data suggest that LipA is required for the insertion of the first sulfur into the octanoic acid backbone. LipB functions downstream of LipA, but its role in lipoic acid metabolism remains unclear.
Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli
Authors: Cohen S P , Hächler H , Levy S B
Published: November 2016

A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.
Cloning, characterization, and functional expression in Escherichia coli of chaperonin (groESL) genes from the phototrophic sulfur bacterium Chromatium vinosum
Authors: Ferreyra R G , Soncini F C , Viale A M
Published: November 2016

A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library. A 4-kbp SalI DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C. vinosum genes that allowed lambda growth in these mutants. Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles. Expression of the cloned C. vinosum groESL genes in E. coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host. Coexpression in E. coli of C. vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes. These results indicate that the cloned DNA fragment encodes C. vinosum chaperonins, which serve in the assembly process of oligomeric proteins. Phylogenic analysis indicates a close relationship between C. vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria.
Escherichia coli SecB, SecA, and SecY proteins are required for expression and membrane insertion of the bacteriocin release protein, a small lipoprotein
Authors: Oudega B , Mol O , van Ulsen P , Stegehuis F , van der Wal F J , Luirink J
Published: November 2016

The SecB, SecA, and SecY dependency of a small outer membrane lipoprotein in Escherichia coli, the bacteriocin release protein (BRP), was studied. The detrimental effect of BRP expression on the culture turbidity (quasi-lysis) was strongly reduced in the sec mutants. Immunoblotting and radioactive labeling experiments showed that the expression, membrane insertion, and processing of the BRP precursor are dependent on SecB, SecA, and SecY. Labeling experiments with hybrid BRP gene constructs revealed that the mature part of the BRP precursor and not its stable signal sequence is important for its SecB dependency.
A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae
Authors: Mills S D , Jasalavich C A , Cooksey D A
Published: November 2016

Specific induction of the copper resistance operon (cop) promoter from Pseudomonas syringae was measured by beta-galactosidase production from a cop promoter-lacZ fusion. Induction of the cop promoter in P. syringae pv. syringae required trans-acting factors from copper resistance plasmid pPT23D, from which cop was originally cloned. Tn5 mutagenesis of pPT23D was used to localize two complementation groups immediately downstream from copABCD. Cloning and sequencing of the DNA in this region revealed two genes, copR and copS, expressed in the same orientation as the cop operon but from a separate constitutive promoter. The amino acid sequence deduced from these genes showed distinct similarities to known two-component regulatory systems, including PhoB-PhoR and OmpR-EnvZ. In addition, CopR showed strong similarity to copper resistance activator protein PcoR from Escherichia coli. Functional chromosomal homologs to copRS activated the cop promoter, in a copper-inducible manner, in copper-resistant or -sensitive strains of P. syringae pv. tomato and other Pseudomonas species. This implies that copper-inducible gene regulation is associated with a common chromosomally encoded function, as well as plasmid-borne copper resistance, in Pseudomonas spp.
Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5
Authors: Springael D , Kreps S , Mergeay M
Published: November 2016

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain.
The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus
Authors: Yu T W , Chen C W
Published: November 2016

The melC operon of Streptomyces antibioticus is unstable, undergoing frequent spontaneous deletions. All the delta melC mutants analyzed also lost 2-kb V1 DNA, which contained two open reading frames (ORFs) homologous to ORF4 (a putative oxidoreductase gene) and ORF5 (a putative AraC-type regulatory gene) of Tn4811. The two ORFs may constitute an accessory unit of a different transposon.
MsmE, a lipoprotein involved in sugar transport in Streptococcus mutans
Authors: Sutcliffe I C , Tao L , Ferretti J J , Russell R R
Published: November 2016

Metabolic labelling by [14C]palmitic acid showed that growth of Streptococcus mutans LT11 in raffinose, an inducer of the msm operon, resulted in increased production of a 45-kDa lipoprotein corresponding to MsmE, which is believed to be a sugar-binding protein. MsmE was also labelled when an msmE clone was expressed in Escherichia coli. The presence of a lipid anchor on MsmE provides a likely explanation of how the sugar-binding protein component of the msm binding protein-dependent multiple sugar transport system is retained at the cell surface.
Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication
Authors: Weaver K E , Clewell D B , An F
Published: November 2016

A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.
Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase
Authors: Meinke A , Gilkes N R , Kilburn D G , Miller R C , Warren R A
Published: November 2016

Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.
Mechanism of silicate binding to the bacterial cell wall in Bacillus subtilis
Authors: Mera M U , Beveridge T J
Published: November 2016

To investigate the chemical mechanism of silicate binding to the surface of Bacillus subtilis, we chemically modified cell wall carboxylates to reverse their charge by the addition of an ethylenediamine ligand. For up to 9 weeks, mixtures of Si, Al-Fe-Si, and Al-Fe-Si plus toxic heavy metals were reacted with these cells for comparison with control cells and abiotic solutions. In general, more Si and less metal were bound to the chemically modified surfaces, thereby showing the importance of an electropositive charge in cell walls for fine-grain silicate mineral development. The predominant reaction for this development was the initial silicate-to-amine complexation in the peptidoglycan of ethylenediamine-modified and control cell walls, although metal ion bridging between electronegative sites and silicate had an additive effect. The binding of silicate to these bacterial surfaces can thus be described as outer sphere complex formation because it occurs through electrostatic interaction.
Phenotypic revertant mutations of a new OmpR2 mutant (V203Q) of Escherichia coli lie in the envZ gene, which encodes the OmpR kinase
Authors: Harlocker S L , Rampersaud A , Yang W P , Inouye M
Published: November 2016

The Escherichia coli ompR2 allele ompR472 contains a valine-to-methionine point mutation at position 203, resulting in an OmpF-constitutive OmpC- outer membrane phenotype. In the present study, OmpR residue V-203 was replaced with glutamine (V203Q mutation), resulting in the same outer membrane phenotype. However, unlike the OmpFc OmpC- phenotype conferred by the OmpR(V203M) mutant protein, the OmpFc OmpC- phenotype produced by the OmpR(V203Q) mutation was suppressed by the envZ11(T247R) allele. Additional suppressors of OmpR(V203Q) were isolated by random mutagenesis. All suppressor mutations were found in the envZ gene and conferred an OmpC+ OmpF- phenotype in the presence of the wild-type ompR. These envZ11-like mutations mapped to a region different from those previously reported and were incapable of suppressing the ompR(V203M) allele. Our results indicate that while methionine or glutamine replacements could cause similar effects on OmpF and OmpC expression, they conferred different abilities on the mutant proteins to be suppressed by envZ.
Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5
Authors: Ligozzi M , Pittaluga F , Fontana R
Published: November 2016

Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized.
Amino acid transport in the thermophilic anaerobe Clostridium fervidus is driven by an electrochemical sodium gradient
Authors: Speelmans G , Poolman B , Konings W N
Published: November 2016

Amino acid transport was studied in membranes of the peptidolytic, thermophilic, anaerobic bacterium Clostridium fervidus. Uptake of the negatively charged amino acid L-glutamate, the neutral amino acid L-serine, and the positively charged amino acid L-arginine was examined in membrane vesicles fused with cytochrome c-containing liposomes. Artificial ion diffusion gradients were also applied to establish the specific driving forces for the individual amino acid transport systems. Each amino acid was driven by the delta psi and delta mu Na+/F and not by the Z delta pH. The Na+ stoichiometry was estimated from the amino acid-dependent 22Na+ efflux and Na(+)-dependent 3H-amino acid efflux. Serine and arginine were symported with 1 Na+ and glutamate with 2 Na+. C. fervidus membranes contain Na+/Na+ exchange activity, but Na+/H+ exchange activity could not be demonstrated.
Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement
Authors: Goldberg M B , Bârzu O , Parsot C , Sansonetti P J
Published: November 2016

Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.
Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides
Authors: Thanabalu T , Berry C , Hindley J
Published: November 2016

Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified. The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied. Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself. Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C. quinquefasciatus larvae. Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae. Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells. Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C. quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C. quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae.
AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB
Authors: Fuge E K , Farr S B
Published: November 2016

Dinucleotide AppppA (5',5'''-P1,P4-diadenosine tetraphosphate) is rapidly synthesized in Escherichia coli cells during heat shock. apaH mutants lack AppppN hydrolase activity and, therefore, contain constitutively levels of AppppA, which affect several cellular processes. However, the precise role of AppppA remains undetermined. Photo-crosslinking experiments with radioactively labelled azido-AppppA have shown that a number of proteins, including heat shock proteins DnaK and GroEL, specifically bind to AppppA. Several other unidentified proteins (C40, C45, and E89) also bind strongly to AppppA. In this work, we have identified the AppppA-binding protein E89 as heat shock protein ClpB. In addition, since ClpB belongs to a family of proteins implicated in proteolysis, we have examined the effects of apaH mutants on protein degradation. Constitutively elevated levels of AppppA stimulate lon-independent proteolysis only in heat-shocked cells. We also show that overproduction of ClpB from a plasmid rescues apaH mutants from sensitivity to killing by heat.
Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea
Authors: McTavish H , LaQuier F , Arciero D , Logan M , Mundfrom G , Fuchs J A , Hooper A B
Published: November 2016

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.
Expression of gonococcal transferrin-binding protein 1 causes Escherichia coli to bind human transferrin
Authors: Cornelissen C N , Biswas G D , Sparling P F
Published: November 2016

The gene for gonococcal transferrin-binding protein 1 (TBP1) was cloned behind an inducible promoter in Escherichia coli. The resultant strain was capable of binding human transferrin with the same specificity as that of the gonococcus. E. coli expressing TBP1 did not internalize transferrin-bound iron or grow on transferrin as a sole iron source.
Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase
Authors: Sun D , Setlow P
Published: November 2016

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.
Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis
Authors: Chen L , Coleman W G
Published: November 2016

A genetically defined mutation, designated rfa-2, results in altered lipopolysaccharide (LPS) biosynthesis. rfa-2 mutants produce a core-defective LPS that contains lipid A and a single sugar moiety, 2-keto-3-deoxyoctulosonic acid, in the LPS core region. Such LPS core-defective or deep-rough (R) mutant structures were previously designated chemotype Re. Phenotypically, rfa-2 mutants exhibit increased permeability to a number of hydrophilic and hydrophobic agents. By restriction analyses and complementation studies, we clearly defined the rfa-2 gene on a 1,056-bp AluI-DraI fragment. The rfa-2 gene and the flanking rfa locus regions were completely sequenced. Additionally, the location of the rfa-2 gene on the physical map of the Escherichia coli chromosome was determined. The rfa-2 gene encodes a 36,000-dalton polypeptide in an in vivo expression system. N-terminal analysis of the purified rfa-2 gene product confirmed the first 24 amino acid residues as deduced from the nucleotide sequence of the rfa-2 gene coding region. By interspecies complementation, a Salmonella typhimurium rfaC mutant (LPS chemotype Re) is transformed with the E. coli rfa-2+ gene, and the transformant is characterized by wild-type sensitivity to novobiocin (i.e., uninhibited growth at 600 micrograms of novobiocin per ml) and restoration of the ability to synthesize wild-type LPS structures. On the basis of the identity and significant similarity of the rfa-2 gene sequence and its product to the recently defined (D. M. Sirisena, K. A. Brozek, P. R. MacLachlan, K. E. Sanderson, and C. R. H. Raetz, J. Biol. Chem. 267:18874-18884, 1992), the S. typhimurium rfaC gene sequence and its product (heptosyltransferase 1), the E. coli K-12 rfa-2 locus will be designated rfaC.
Escherichia coli shows two types of behavioral responses to osmotic upshift
Authors: Li C , Adler J
Published: November 2016

Behavioral responses to osmotic upshift were characterized by temporal assays of free-swimming cells of Escherichia coli. Small osmotic upshifts (200 to 300 mosM) elicited tumble responses which were chemotaxis dependent, while large osmotic upshifts (400 to 500 mosM) elicited stopping followed by pseudotumbling which was chemotaxis independent.
Molecular cloning and characterization of the Candida albicans enolase gene
Authors: Mason A B , Buckley H R , Gorman J A
Published: November 2016

A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene.
Negative regulation of sigma 54-dependent dctA expression by the transcriptional activator DctD
Authors: Labes M , Finan T M
Published: November 2016

In Rhizobium meliloti, the presence of the C4-dicarboxylate transport protein DctA is required for symbiotic N2 fixation in alfalfa root nodules. Expression of dctA is inducible and is mediated by a sensor and activator gene pair encoded by dctB and dctD. In the presence of C4-dicarboxylates, the DCTB sensor protein is believed to phosphorylate and activate DCTD, which in turn activates transcription at the sigma 54-dependent dctA promoter. Here, we present evidence that in addition to activating dctA transcription, DCTD can also repress expression of dctA. By employing an ntrC allele, ntrC283, whose product appears to activate dctA transcription independently of DCTD, we found that while ntrC283 leads to constitutive dctA expression in the absence of dctB and dctD, in a dctB+ dctD+ ntrC283 background high-level expression of dctA occurred in succinate but not in glucose-grown cells. This result suggested that in uninduced cells, inactive DCTD binds to the dctA promoter and prevents its activation by NTRC283. Consistent with the latter interpretation was the observation that overexpression of DCTD from a plasmid promoter prevents dctA expression and results in a Dct- phenotype. Moreover the Dct- phenotype resulting from the overexpression of dctD was dominant to ntrC283. Results from studies of the ability of ntrC283 to suppress the Dct- phenotype of dctB alleles, together with the finding that the Fix- phenotype of a particular dctB allele was dctD dependent, suggest that in particular dctB alleles, sufficient dctD transcription occurs such that the resulting inactive DCTD prevents activation of dctA transcription by NtrC283 or alternate symbiotic regulators. The latter suggestion is supported by the observation that in symbiosis, R. meliloti strains in which DCTD was overexpressed formed nodules which failed to fix nitrogen.
Nucleotide sequence analysis of the termini and chromosomal locus involved in site-specific integration of the streptococcal conjugative transposon Tn5252
Authors: Vijayakumar M N , Ayalew S
Published: November 2016

The 47-kb, broad-host-range, streptococcal conjugative transposon Tn5252 is capable of site-specific integration into the pneumococcal chromosome. We present the nucleotide sequence of the terminal regions of the transposon and its target site in the pneumococcal genome. No inverted repeats were found at the termini of the transposon. A 72-bp region of the target was present on either side following the insertion of Tn5252 and appeared to serve as a signal for its integration and excision. The data suggest that the left copy of the 72-bp segment was a part of the conjugative element, the crossover point of integration was nonrandom within this region, and the mechanism of insertion could resemble that of the site-specific temperate phages.
Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases
Authors: Drechsel H , Thieken A , Reissbrodt R , Jung G , Winkelmann G
Published: November 2016

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.
Specific regions of Escherichia coli OmpF protein involved in antigenic and colicin receptor sites and in stable trimerization
Authors: Fourel D , Mizushima S , Bernadac A , Pagès J M
Published: November 2016

Four different mutations were obtained by selecting for resistance to colicin N and screening for continued production of the OmpF protein of Escherichia coli. Two of them also conferred resistance to colicin A. The substitutions C for R-168 (R168C) and E284K caused the loss of the E21 epitope, while the transition G285D altered the E18, E19, and E20 antigenic sites. The substitution G119D drastically affected the stability of the trimeric conformation.
Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes
Authors: Mitchell W J , Reizer J , Herring C , Hoischen C , Saier M H
Published: November 2016

Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.
Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films
Authors: Pum D , Weinhandl M , Hödl C , Sleytr U B
Published: November 2016

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.
Altered muropeptide composition in Staphylococcus aureus strains with an inactivated femA locus
Authors: de Jonge B L , Sidow T , Chang Y S , Labischinski H , Berger-Bachi B , Gage D A , Tomasz A
Published: November 2016

Tn551 inactivation of femA, a factor involved in methicillin resistance of Staphylococcus aureus, caused the production of peptidoglycan in which the fraction of monoglycyl- and serine-containing muropeptides was increased at the expense of pentaglycyl muropeptides. femA mutants have a specific block in the biosynthesis of pentaglycine cross bridges after the addition of the first glycine residue.
Cell division and transcription of ftsZ
Authors: Smith R W , Masters M , Donachie W D
Published: November 2016

For normal cell division, the ftsZ gene must be transcribed from a number of promoters that are located within the proximal upstream genes (ddlB, ftsQ, and ftsA). We show that the main promoters have identical responses to changes in growth rate, i.e., under all conditions, the frequency of transcription per septum formed is approximately constant and independent of cell size or growth rate per se. We also show that transcription from these promoters is independent of stationary-phase transcription factor sigma s.
The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein
Authors: Wray L V , Atkinson M R , Fisher S H
Published: November 2016

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.
Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP
Authors: Dai K , Mukherjee A , Xu Y , Lutkenhaus J
Published: November 2016

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.
The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi
Authors: Brzoska P , Rimmele M , Brzostek K , Boos W
Published: November 2016

sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H. Schweizer, M. Argast, and W. Boos, J. Bacteriol. 150:1154-1163, 1982). In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi. Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system. This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P. Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system. Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P. However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system. Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM. Cells had to be starved for Pi in order to observe this inhibition. We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate.
Structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth, sporulation, and extracellular enzyme production
Authors: Nishiguchi M , Honda K , Amikura R , Nakamura K , Yamane K
Published: November 2016

Bacillus subtilis small cytoplasmic RNA (scRNA; 271 nucleotides) is a member of the signal recognition particle (SRP) RNA family, which has evolutionarily conserved primary and secondary structures. The scRNA consists of three domains corresponding to domains I, II, and IV of human SRP 7S RNA. To identify the structural determinants required for its function, we constructed mutant scRNAs in which individual domains or conserved nucleotides were deleted, and their importance was assayed in vivo. The results demonstrated that domain IV of scRNA is necessary to maintain cell viability. On the other hand, domains I and II were not essential for vegetative growth but were preferentially required for the RNA to achieve its active structure, and assembled ribonucleoprotein between Ffh and scRNA is required for sporulation to proceed. This view is highly consistent with the fact that the presence of domains I and II is restricted to sporeforming B. subtilis scRNA among eubacterial SRP RNA-like RNAs.
Cloning and nucleotide sequence of the cyclic AMP receptor protein-regulated Salmonella typhimurium pepE gene and crystallization of its product, an alpha-aspartyl dipeptidase
Authors: Conlin C A , Håkensson K , Liljas A , Miller C G
Published: November 2016

The Salmonella typhimurium pepE gene, encoding an N-terminal-Asp-specific dipeptidase, has been cloned on pBR328 by complementation of the Asp-Pro growth defect conferred by a pepE mutation. Strains carrying the complementing plasmids greatly overproduce peptidase E. The enzyme has been purified from an extract of such a strain, its N-terminal amino acid sequence has been determined, and crystals suitable for X-ray diffraction have been grown. A new assay using L-aspartic acid p-nitroanilide as a substrate has been used to determine the pH optimum (approximately 7.5) and to test the effect of potential inhibitors. Insertions of transposon gamma delta (Tn1000) into one of the plasmids have been used to localize the gene and as sites for priming sequencing reactions. The nucleotide sequence of a 1,088-bp region of one of these plasmids has been determined. This sequence contains an open reading frame that predicts a 24.8-kDa protein with an N-terminal sequence that agrees with that determined for peptidase E. The predicted peptidase E amino acid sequence is not similar to that of any other known protein. The nucleotide sequence of the region upstream from pepE contains a promoter with a cyclic AMP receptor protein (CRP) site, and the effects of growth medium and of a crp mutation on expression of a pepE-lacZ fusion indicate that pepE is a member of the CRP regulon. The unique specificity of peptidase E and its lack of sequence similarity to any other peptidase suggest that this enzyme may be the prototype of a new class of peptidases. Its regulation by CPR and its specificity suggest that the enzyme may play a role in allowing the cell to use peptide aspartate to spare carbon otherwise required for the synthesis of the aspartate family of amino acids.
Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy
Authors: Radolf J D , Bourell K W , Akins D R , Brusca J S , Norgard M V
Published: November 2016

Freeze-fracture electron microscopy was used to investigate the membrane architectures of high-passage Borrelia burgdorferi B31 and low- and high-passage isolates of B. burgdorferi N40. In all three organisms, fractures occurred almost exclusively through the outer membrane (OM), and the large majority of intramembranous particles were distributed randomly throughout the concave OM leaflet. The density of intramembranous particles in the concave OM leaflet of the high-passage N40 isolate was significantly greater than that in the corresponding leaflet of the low-passage N40 isolate. Also noted in the OMs of all three organisms were unusual structures, designated linear bodies, which typically were more or less perpendicular to the axis of the bacterium. A comparison of freeze-fractured B. burgdorferi and Treponema pallidum, the syphilis spirochete, revealed that the OM architectures of these two pathogens differed markedly. All large membrane blebs appeared to be bounded by a membrane identical to the OM of B. burgdorferi whole cells; in some blebs, the fracture plane also revealed a second bilayer closely resembling the B. burgdorferi cytoplasmic membrane. Aggregation of the lipoprotein immunogens outer surface protein A (OspA) and OspB on the bacterial surface by incubation of B. burgdorferi B31 with specific polyclonal antisera did not affect the distribution of OM particles, supporting the contention that lipoproteins do not form particles in freeze-fractured OMs. The expression of poorly immunogenic, surface-exposed proteins as virulence determinants may be part of the parasitic strategy used by B. burgdorferi to establish and maintain chronic infection in Lyme disease.