bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli
Authors: Sun T P , Webster R E
Published: November 2016

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.
Functional genes for cellobiose utilization in natural isolates of Escherichia coli
Authors: Hall B G , Faunce W
Published: November 2016

The genes for utilization of cellobiose are normally cryptic in both laboratory strains and natural isolates of Escherichia coli. A survey of natural isolates of E. coli reveals that functional genes for cellobiose utilization, while rare, are present. The fraction of E. coli that utilized cellobiose ranged from less than 0.01% in human fecal samples to 7% in fecal samples obtained from horses. Samples obtained from sheep, cows, dogs, and pigs contained 0.1 to 0.5% cellobiose-positive E. coli. Neither the previously identified cel genes nor the bgl genes from E. coli K-12 were expressed during growth on cellobiose by any of the 14 naturally occurring Cel+ isolates that were tested. All of the naturally occurring Cel+ isolates possessed a cel operon, but all were deleted for the major portion of the bgl operon. The functional cel+ genes from these natural isolates differed from the mutationally activated cel+ genes obtained in earlier studies in that (i) the mutationally activated cel+ genes were temperature sensitive, while the functional genes were not, and (ii) transport of cellobiose was inducible in the strains carrying functional cel+ genes, while it was expressed constitutively in strains carrying mutationally activated genes.
Clonal diversity and host distribution in Bordetella bronchiseptica
Authors: Musser J M , Bemis D A , Ishikawa H , Selander R K
Published: November 2016

A total of 303 isolates of Bordetella bronchiseptica recovered from 11 host species were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 21 distinctive multilocus genotypes (electrophoretic types) were distinguished on the basis of allele profiles at the enzyme loci. The population structure of B. bronchiseptica is clonal, and its genetic diversity is limited in comparison with most other pathogenic bacteria, perhaps reflecting a relatively recent origin of the species. Electrophoretic types mark clones which are, in many cases, nonrandomly associated with host species. Clones differing only slightly in overall chromosomal genetic character may have pronounced differences in virulence potential. There was considerable variation among individual clones and clone families in degree of host specificity and among various species of hosts in the diversity of clones causing disease. The diversity of clones infecting dogs was an order of magnitude greater than that of clones infecting pigs. Most bordetellosis in pigs in the United States and Japan was found to be caused by strains of a single multilocus genotype.
Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila
Authors: Howard S P , Garland W J , Green M J , Buckley J T
Published: November 2016

The gene for the hole-forming toxin aerolysin from Aeromonas hydrophila was sequenced. Although most of the sequence seems unrelated to that of Staphylococcus aureus alpha-toxin, both proteins are very hydrophilic, and they each contain a nearly identical string of 10 amino acids.
Gene location affects expression level in Salmonella typhimurium
Authors: Schmid M B , Roth J R
Published: November 2016

Directed translocation of an expressed gene (hisD) provided strains to test for position effects on gene expression in Salmonella typhimurium. The predominant position effect seems to be caused by the gene dosage of a chromosomal site; the orientation of the translocated genes did not affect expression, and no deviant positions were found.
Physical and genetic characterization of the glucitol operon in Escherichia coli
Authors: Yamada M , Saier M H
Published: November 2016

The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A. Sancar and W. D. Rupp, Proc. Natl. Acad. Sci. USA 76:3144-3148, 1979). We subcloned the gut operon by using paCYC184, pACYC177, and pBR322. The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes. The repressor of the gut operon seemed to be encoded in the region downstream from the operon. The gene products of the gut operon were identified by using maxicells. The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity
Authors: Kanemoto R H , Ludden P W
Published: November 2016

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions.
Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices
Authors: Götz R , Schmitt R
Published: November 2016

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.
Influence of the catBCE sequence on the phenotypic reversion of a pcaE mutation in Acinetobacter calcoaceticus
Authors: Doten R C , Gregg L A , Ornston L N
Published: November 2016

Isofunctional beta-ketoadipate:succinyl coenzyme A transferases I and II are encoded by the pcaE and catE genes, respectively, of Acinetobacter calcoaceticus. The genes are under separate transcriptional control and genetically unlinked. Mutations in the pcaE gene result in a p-hydroxybenzoate-negative (POB-) phenotype, whereas catE mutations cause a benzoate-negative (Ben-) phenotype. A. calcoaceticus ADP125 carries the pcaE3125 mutation and gave rise to POB+ revertants with a frequency of 10(-4). A 5.0-kilobase-pair (kb) EcoRI restriction fragment containing the catBCDE genes possesses two SalI restriction sites separated by 1.5 kb. Removal of the DNA between the SalI sites created a deletion removing the terminal 35 base pairs of the catB gene, the 300-base-pair catC gene, and about 1.1 kb of the 1.2-kb catE gene. Transformation of strain ADP125 with the modified EcoRI fragment lacking the SalI segment produced natural transformants containing this designed deletion with a frequency of 20%. The frequency of POB+ phenotypic reversion of the pcaE3125 mutation in these transformants was more than 300-fold lower than the frequency of phenotypic reversion observed in genetic backgrounds containing the catBCE segment. Alleles created by pcaE phenotypic reversion in a wild-type cat genetic background were unlinked to the cat gene cluster, and revertant transferases were expressed inducibly with the pca genes. Alterations in the restriction pattern of the pca gene cluster in several revertants were observed, indicating that multiple sequence changes have occurred in the pca genes during reversion. Growth of the phenotypic revertants under nonselective conditions resulted in loss of either the POB+ phenotype or both the POB+ and Ben+ phenotypes at high frequency. Southern hybridizations revealed that loss of the POB+ of Ben+ phenotype was due to deletion of the entire pca or cat gene cluster, a loss of at least 16 kb in some strains. Revertants isolated in a catBCE deletion background were stable. These results suggest that enhanced phenotypic reversion of pcaE3125 in wild-type cat background is due to repair of the mutation by recombination between the catBCE and pcaE3125 sequences. Genetic instability of the phenotypic revertants may be attributed to deletion of pca and cat sequences by recombination between regions of homology created as a consequence of pcaE repair.
Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis
Authors: Smith H , Bron S , Van Ee J , Venema G
Published: November 2016

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.
Reexamination of the presence and linkage of 3-hydroxybutyryl substituents in the acidic capsular polysaccharide of Rhizobium trifolii 0403
Authors: Hollingsworth R I , Dazzo F B , Mort A J
Published: November 2016

We resolved previous conflicting results concerning the presence of 3-hydroxybutyryl substituents on the extracellular acidic polysaccharide from Rhizobium trifolii 0403. These substituents were indeed present in the polysaccharide and in the oligosaccharide fragments obtained by hydrogen fluoride solvolysis of the extracellular and capsular polysaccharides of the bacteria grown on plates. The 3-hydroxybutyrate substituent could be removed from the polysaccharide by 10 mM sodium deuteroxide without evidence of elimination, indicating that this substituent was ester linked.
Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli
Authors: Sakka K , Watanabe T , Beers R , Wu H C
Published: November 2016

We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.
Purification and characterization of an aminopeptidase from Mycoplasma salivarium
Authors: Shibata K , Watanabe T
Published: November 2016

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.
Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein
Authors: Egner C , Azhderian E , Tsang S S , Radding C M , Chase J W
Published: November 2016

To relate the roles of Escherichia coli SSB in recombination in vivo and in vitro, we have studied the mutant proteins SSB-1 and SSB-113, the variant SSBc produced by chymotryptic cleavage, the partially homologous variant F SSB (encoded by the E. coli sex factor), and the protein encoded by gene 32 of bacteriophage T4. All of these, with the exception of SSB-1, augmented both the initial rate of homologous pairing and strand exchange promoted by RecA protein. From these and related observations, we conclude that SSB stimulates the initial formation of joint molecules by nonspecifically promoting the binding of RecA protein to single-stranded DNA; that SSB plays no role in synapsis of the RecA nucleoprotein filament with duplex DNA; that stimulation of strand exchange by SSB is similarly nonspecific; and that all members of the class of proteins represented by SSB, F SSB, and gene 32 protein may play equivalent roles in making single-stranded DNA more accessible to RecA protein.
Sodium-coupled motility in a swimming cyanobacterium
Authors: Willey J M , Waterbury J B , Greenberg E P
Published: November 2016

The energetics of motility in Synechococcus strain WH8113 were studied to understand the unique nonflagellar swimming of this cyanobacterium. There was a specific sodium requirement for motility such that cells were immotile below 10 mM external sodium and cell speed increased with increasing sodium levels above 10 mM to a maximum of about 15 microns/s at 150 to 250 mM sodium. The sodium motive force increased similarly with increasing external sodium from -120 to -165 mV, but other energetic parameters including proton motive force, electrical potential, the proton diffusion gradient, and the sodium diffusion gradient did not show such a correlation. Over a range of external sodium concentrations, cell speed was greater in alkaline environments than in neutral or acidic environments. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited motility and affected components of sodium motive force but did not affect ATP levels. Cells were motile when incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and arsenate, which decreased cellular ATP to about 2% of control values. The results of this investigation are consistent with the conclusion that the direct source of energy for Synechococcus motility is a sodium motive force and that below a threshold of about -100 mV, cells are immotile.
Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp
Authors: Smith C J , Spiegel H
Published: November 2016

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.
Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus
Authors: Hohn T M , Paznokas J L
Published: November 2016

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.
Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli
Authors: Hughes J A , Brown L R , Ferro A J
Published: November 2016

We have developed a new research tool for the study of S-adenosylmethionine (AdoMet) metabolism by cloning the coliphage T3 AdoMet hydrolase (AdoMetase; EC 3.3.1.2) gene into the M13mp8 expression vector. The recombinant bacteriophage clones expressed an AdoMetase activity in Escherichia coli like that found in T3-infected cells. High levels of AdoMetase expression impaired AdoMet-mediated activities such as dam and dcm methylase-directed DNA modifications and the synthesis of spermidine from putrescine. Expression vectors containing the cloned AdoMetase gene thus provide an alternate approach to the use of chemical inhibitors or mutants defective in AdoMet biosynthesis to probe the effect of AdoMet limitation.
Nephelometric determination of turgor pressure in growing gram-negative bacteria
Authors: Koch A L , Pinette M F
Published: November 2016

Gas vesicles were used as probes to measure turgor pressure in Ancylobacter aquaticus. The externally applied pressure required to collapse the vesicles in turgid cells was compared with that in cells whose turgor had been partially or totally removed by adding an impermeable solute to the external medium. Since gram-negative bacteria do not have rigid cell walls, plasmolysis is not expected to occur in the same way as it does in the cells of higher plants. Bacterial cells shrink considerably before plasmolysis occurs in hyperosmotic media. The increase in pressure required to collapse 50% of the vesicles as external osmotic pressure increases is less than predicted from the degree of osmotically inducible shrinkage seen with this organism or with another gram-negative bacterium. This feature complicates the calculation of the turgor pressure as the difference between the collapse pressure of vesicles with and without sucrose present in the medium. We propose a new model of the relationship between turgor pressure and the cell wall stress in gram-negative bacteria based on the behavior of an ideal elastic container when the pressure differential across its surface is decreased. We developed a new curve-fitting technique for evaluating bacterial turgor pressure measurements.
Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and alpha 2-macroglobulin
Authors: Chhatwal G S , Albohn G , Blobel H
Published: November 2016

Binding of 125I-labeled alpha 2-macroglobulin (alpha 2M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native alpha 2M, and those of group C reacted only with alpha 2M-trypsin complex. Binding of alpha 2M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 X 10(-7) M, and the number of binding sites was calculated to be 18,000 per streptococcus. The alpha 2M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native alpha 2M in Western blots (immunoblots). Interaction of purified binding protein with alpha 2M led to a change in the conformation of alpha 2M similar to that obtained by alpha 2M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of alpha 2M is thus a novel feature of alpha 2M reactivity.
DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli
Authors: Joseleau-Petit D , Képès F , Peutat L , D'Ari R , Képès A
Published: November 2016

In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.
Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli
Authors: Chen Y M , Tobin J F , Zhu Y , Schleif R F , Lin E C
Published: November 2016

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.
Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli
Authors: Owen P , Caffrey P , Josefsson L G
Published: November 2016

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed alpha and beta (Mrs of 60,000 and 53,000, respectively). The beta polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70 degrees C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both alpha and beta polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual alpha and beta polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both alpha and beta polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.
Characterization and structure of genes for proteases A and B from Streptomyces griseus
Authors: Henderson G , Krygsman P , Liu C J , Davey C C , Malek L T
Published: November 2016

Protease A and protease B are extracellular proteins which are secreted by Streptomyces griseus. The genes encoding protease A (sprA) and protease B (sprB) were isolated from an S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing sprA and sprB were characterized by hybridization and demonstration of proteolytic activity in Streptomyces lividans. Each DNA sequence contains a large open reading frame with the coding region of the mature protease situated at its carboxy terminus. The amino terminus of each reading frame appears to encode a 38-amino-acid signal peptide followed by a 76- or 78-amino-acid polypeptide, a propeptide, which is joined to the mature protease. Strong homology between the coding regions of the protease genes suggests that sprA and sprB originated by gene duplication.
Degradation of substituted mandelic acids by meta fission reactions
Authors: Sze I S , Dagley S
Published: November 2016

A strain of Acinetobacter lwoffii degraded 4-hydroxymandelic and 4-hydroxy-3-methoxymandelic acids to their corresponding benzoates, which were then hydroxylated by specific monooxygenases to yield, respectively, protocatechuic and 3-O-methylgallic acids; these were substrates for meta fission dioxygenases. The product formed from 3-O-methylgallate underwent slow spontaneous cyclization at pH 7 to release methanol.
Nucleotide sequence of the epidermolytic toxin A gene of Staphylococcus aureus
Authors: O'Toole P W , Foster T J
Published: November 2016

The nucleotide sequence of the eta gene, which codes for the epidermolytic toxin serotype A of Staphylococcus aureus TC16, is reported. The coding sequence of 840 nucleotides specifies a protein which, when secreted, has a predicted molecular weight of 26,950. The sequence of eta and the deduced amino acid sequence of the toxin have been compared with those of epidermolytic toxin serotype B. The coding sequences have 52% identical residues, and the polypeptides have 40% identical residues. Amino acid residues have been conserved in the areas of the proteins which correspond to major hydrophobic domains, whereas the regions likely to specify antigenic determinants occur in hydrophilic sequences that have diverged. The level of expression of epidermolytic toxin A in S. aureus 8325-4 was shown to be dependent on the integrity of a regulatory gene called agr.
Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis
Authors: Medoff G , Painter A , Kobayashi G S
Published: November 2016

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.
Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences
Authors: Baumann P , Baumann L , Bowditch R D , Broadwell A H
Published: November 2016

During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Escherichia coli cloning vector lambda gt11, in which gene products of the inserts may be fused to beta-galactosidase, we isolated 29 bacteriophages which produced peptides-reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a beta-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27- and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43- and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidal crystal protein. The two recombinants of group B and the 23 of group C were all beta-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.
Molecular sieving through S layers of Bacillus stearothermophilus strains
Authors: Sára M , Sleytr U B
Published: November 2016

The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.
Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene
Authors: Pettis G S , McIntosh M A
Published: November 2016

The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin. Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein. A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403. In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration. Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301. These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes.
Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess
Authors: Westby C A , Enderlin C S , Steinberg N A , Joseph C M , Meeks J C
Published: November 2016

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.
Low-affinity potassium uptake system in Bacillus acidocaldarius
Authors: Michels M , Bakker E P
Published: November 2016

Cells of Bacillus acidocaldarius that were grown with 2.7 mM K+ expressed a low-affinity K+ uptake system. The following observations indicate that its properties closely resemble those of the Escherichia coli Trk and Streptococcus faecalis KtrI systems: (i) the B. acidocaldarius system took up K+ with a Km of 1 mM; (ii) it accepted Rb+ (Km of 6 mM; same Vmax as for K+); (iii) it was still active in the presence of low concentrations of sodium; (iv) the observed accumulation ratio of K+ maintained by metabolizing cells was consistent with K+ being taken up via a K+-H+ symporter; and (v) K+ uptake did not occur in cells in which the ATP level was low. Under the latter conditions, the cells still took up methylammonium ions via a system that was derepressed by growth with low levels of ammonium ions, indicating that in the acidophile ammonium (methylammonium) uptake requires a high transmembrane proton motive force rather than ATP.
Tunicamycin inhibition of epispore formation in Saccharomyces cerevisiae
Authors: Weinstock K G , Ballou C E
Published: November 2016

The ascopore wall of Saccharomyces cerevisiae was found to contain more protein, polymeric glucosamine, and beta-glucan than the vegetative cell wall, which was enriched in mannoprotein relative to ascospore walls. Tunicamycin inhibited sporulation, as judged by the absence of refractile ascospores visible by phase-contrast microscopy, but cells completed meiosis, as demonstrated by the presence of multinucleate asci. Such spores lacked the dense outer layer characteristic of normal spores. Thus, the tunicamycin effect was similar to that of glucosamine auxotrophy (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975).
Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza
Authors: Barthelemy Adeline , Ivanov Stoyan , Hassane Maya , Fontaine Josette , Heurtault Béatrice , Frisch Benoit , Faveeuw Christelle , Paget Christophe , Trottein François
Published: November 2016

Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103
+
dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections.

IMPORTANCE
Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today’s treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)—a potent agonist of invariant NKT cells that is currently in clinical development—in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.