bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the cell wall of the sheathed methanogen Methanospirillum hungatei GP1 as an S layer
Authors: Firtel M , Southam G , Harauz G , Beveridge T J
Published: November 2016

The cell wall of Methanospirillum hungatei GP1 is a labile structure that has been difficult to isolate and characterize because the cells which it encases are contained within a sheath. Cell-sized fragments, 560 nm wide by several micrometers long, of cell wall were extracted by a novel method involving the gradual drying of the filaments in 2% (wt/vol) sodium dodecyl sulfate and 10% (wt/vol) sucrose in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 10 mM EDTA. The surface was a hexagonal array (a = b = 15.1 nm) possessing a helical superstructure with a ca. 2.5 degrees pitch angle. In shadowed relief, the smooth outer face was punctuated with deep pits, whereas the inner face was relatively featureless. Computer-based two-dimensional reconstructed views of the negatively stained layer demonstrated 4.0- and 2.0-nm-wide electron-dense regions on opposite sides of the layer likely corresponding to the openings of funnel-shaped channels. The face featuring the larger openings best corresponds to the outer face of the layer. The smaller opening was encircled by a stalk-like mass from which 2.2-nm-wide protrusions were resolved. The cell wall in situ was degraded at pH 9.6 at 56 degrees C but was unaffected at pH 7.4 at the same temperature. The cell wall was composed of two nonglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of small (< 10-kDa) sheath precursor proteins.
Analysis of Streptococcus pyogenes promoters by using novel Tn916-based shuttle vectors for the construction of transcriptional fusions to chloramphenicol acetyltransferase
Authors: Geist R T , Okada N , Caparon M G
Published: November 2016

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcriptional regulation in Streptococcus pyogenes and other gram-positive bacteria. Designated the pVIT vectors (vectors for integration into Tn916), the vectors are small, stable plasmids in Escherichia coli to facilitate the fusion of promoters from cloned S. pyogenes genes to a promoterless gene which encodes chloramphenicol acetyltransferase. The vectors each contain one or more small regions of Tn916 to direct the integration of the transcriptional fusion into the transposon via homologous recombination following transformation of S. pyogenes or other suitable gram-positive hosts. Integration can be monitored by the inactivation or replacement of an antibiotic resistance determinant in modified derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activity. In addition, since integration is into loci that do not disrupt the conjugative transpositional functions of Tn916, the vectors are useful for analysis of regulation in strains that are difficult or impossible to transform and can be introduced into these strains by conjugation following transformation of an intermediate host. The promoters for the genes which encode both the M protein and protein F of S. pyogenes were active in pVIT vectors, as was the region which controls transcription of mry, a trans-acting positive regulator of M protein expression. However, neither of the two characterized promoters for mry demonstrated activity when independently analyzed in pVIT-generated partial diploid strains, suggesting that regulation of mry is more complex than predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous other bacterial species.
The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase
Authors: Summers R G , Wendt-Pienkowski E , Motamedi H , Hutchinson C R
Published: November 2016

Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase.
An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide
Authors: Russo T A , Singh G
Published: November 2016

We are studying an O4/K54/H5 Escherichia coli bacteremic isolate (CP9) as a model pathogen for extraintestinal infection. Its group 2, K54 capsular polysaccharide is an important virulence determinant and confers serum resistance. In this study the effect of the group 1 capsule regulators, RcsA, RcsB, and Lon protease, on the regulation of CP9's capsular polysaccharides was assessed. It was established that in the presence of multicopy rcsA or with disruption of lon, CP9 can be induced to produce a group 1 capsule. RcsA, RcsB, and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (cl1.29::TnphoA and cl1.137::TnphoA) were used to evaluate the effects of these regulators on group 2 K54 capsule production. Disruption of lon resulted in 1.9-fold (TR293 [cl1.29::TnphoA lon-146]) and 3.4-fold (TR1373 [cl1.137::TnphoA lon-146]) decreases in fusion activity at 28 degrees C, relative to the baseline level. However, decreases in fusion activity at 42 degrees C were only 1.2- and 1.4-fold, respectively. Inactivation of both lon and rcsA or lon and rcsB restored fusion activity to baseline levels at 28 degrees C, but only a partial restoration of activity was seen at higher temperatures. To assess whether these differences in fusion activity reflected a functional change in capsule production, the effects of 80% normal human serum (NHS) were tested against CP9 and TR93 (lon-146). Since the group 2 K54 capsule protects against the bactericidal activity of 80% NHS, a decrease in its production results in an increase in serum sensitivity. Viable counts of CP9 increased 10-fold in 80% NHS over 3 h at 28 degrees C, as expected. In contrast to CP9, TR93 (lon-146) incurred a 10-fold loss in viability under the same conditions. The levels of RcsA are increased in TR93 (lon 146) as consequence of lon disruption; therefore, these results in conjunction with the cl1::TnphoA protein fusion data establish RcsA as a negative regulator of the group 2 K54 capsular polysaccharide. Furthermore, these results also suggest existence of another Lon-sensitive negative regulator of group 2 K54 capsule production, which is active higher temperatures.
Location of IS200 on the genomic cleavage map of Salmonella typhimurium LT2
Authors: Sanderson K E , Sciore P , Liu S L , Hessel A
Published: November 2016

Locations of six Tn10s, closely linked to each of the six IS200s on the genomic cleavage map of Salmonella typhimurium LT2, were determined by digestion with XbaI and BlnI and separation of the fragments by pulsed-field gel electrophoresis; the locations were then further defined by P22-mediated joint transduction. The orientation of each IS200 with respect to its linked Tn10 was determined by Southern blotting. The locations of IS200-I, IS200-III, and IS200-V were confirmed to be close to sufD, melB, and purC, as previously indicated. IS200-II is jointly transduced with cysG. IS200-IV is near fliA; the linked Tn10 is inserted in fli, making the strain nonmotile. IS200-VI is jointly transduced with aspC but not with aroA. IS200 is transposed to a seventh site in some strains, while remaining in the other six locations described above. These data indicate that genome analysis by pulsed-field gel electrophoresis can locate the positions of Tn10s with accuracy sufficient to predict P22-mediated joint transduction.
Identification of the Shiga toxin A-subunit residues required for holotoxin assembly
Authors: Haddad J E , Jackson M P
Published: November 2016

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.
Patch length of localized repair events: role of DNA polymerase I in mutY-dependent mismatch repair
Authors: Radicella J P , Clark E A , Chen S , Fox M S
Published: November 2016

In vivo experiments with heteroduplex lambda genomes show that the MutY mismatch repair system of Escherichia coli defines an average repair tract that is shorter than 27 nucleotides and longer than 9 nucleotides and extends 3' from the corrected adenine. The phenotype of a mutant defective in DNA polymerase I shows that this enzyme plays a significant, though not an essential, role in the in vivo repair of apurinic sites generated by this system. Evidence is presented that in the absence of polymerase I the repair tracts are modestly longer than in the polA+ extending in the 5' direction from the corrected adenine, suggesting a role for another DNA polymerase.
Determinants of the quantity of the stable SecY complex in the Escherichia coli cell
Authors: Taura T , Baba T , Akiyama Y , Ito K
Published: November 2016

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZ alpha fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.
A 3.9-kb DNA region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose
Authors: Köplin R , Wang G , Hötte B , Priefer U B , Pühler A
Published: November 2016

By mutational analysis it was found that a 3.9-kb SmaI-XhoII DNA fragment of Xanthomonas campestris pv. campestris is involved in lipopolysaccharide (LPS) biosynthesis. LPS samples isolated from different mutants carrying mutations in the 3.9-kb SmaI-XhoII DNA fragment exhibited banding patterns in silver-stained sodium dodecyl sulfate-polyacrylamide gels markedly different from that of the wild-type LPS. Moreover, comparison of the monosaccharide composition obtained by high-performance anion-exchange chromatography with pulsed amperometric detection of LPS purified from wild-type Xanthomonas campestris pv. campestris B100 and from mutants with mutations in the 3.9-kb SmaI-XhoII DNA fragment revealed a lack of rhamnose moieties in the mutant LPS. Sequence analysis of this DNA fragment revealed four open reading frames (ORFs), designated ORF302, ORF183, ORF295, and ORF351. The deduced amino acid sequences of these ORFs showed a high degree of homology to the deduced amino acid sequences of the rfbC, rfbD, rfbA, and rfbB genes of Salmonella typhimurium LT2, which have been shown to encode a set of enzymes responsible for conversion of glucose 1-phosphate to dTDP-rhamnose.
Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning
Authors: Ingmer H , Cohen S N
Published: November 2016

RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division.
Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated
Authors: Hoe N P , Goguen J D
Published: November 2016

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.
Transcriptional regulation of the Bacillus thuringiensis subsp. thompsoni crystal protein gene operon
Authors: Brown K L
Published: November 2016

The two predominant polypeptides of the Bacillus thuringiensis subsp. thompsoni crystal are encoded by the cry40 and cry34 genes. These crystal protein genes are located in an operon. Western analysis (immunoblotting) demonstrated that the operon promoter activity was located in the region upstream of the cry40 gene. The Cry34 protein was expressed only when the upstream promoter region was present. The crystal protein genes are the only cistrons in the operon, and they are expressed during sporulation, with the highest transcript levels detected early in sporulation (1.5 to 3 h after the onset of sporulation). Transcription initiates from two adjacent sites located 84 and 85 bases upstream of the cry40 translational start codon. The B. thuringiensis subsp. thompsoni crystal protein gene operon promoter aligned with other crystal protein gene promoters, which are activated from early to midsporulation and transcribed in vitro by the B. thuringiensis RNA polymerase E sigma 35.
Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins
Authors: Gandhi A J , Perussia B , Goldfine H
Published: November 2016

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.
Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster
Authors: Thariath A , Socha D , Valvano M A , Viswanatha T
Published: November 2016

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of a broad-range disulfide reductase from Streptomyces clavuligerus and its possible role in beta-lactam antibiotic biosynthesis
Authors: Aharonowitz Y , Av-Gay Y , Schreiber R , Cohen G
Published: November 2016

Streptomyces clavuligerus is a potent producer of penicillin and cephalosporin antibiotics. A key step in the biosynthesis of these beta-lactam compounds is the cyclization of the thiol tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). However, bis-ACV, the oxidized disulfide form of the tripeptide, is not a substrate for IPNS. We show here that S. clavuligerus possesses an NADPH-dependent disulfide reductase of broad substrate specificity that efficiently catalyzes the reduction of disulfide bonds in bis-ACV and in other low-molecular-weight disulfide containing compounds and proteins. The disulfide reductase comprises two protein components, a 70-kDa reductase consisting of two identical subunits, and a 12-kDa heat-stable protein reductant. The structural and functional properties of the disulfide reductase resemble those of the thioredoxin class of oxidoreductases. When the disulfide reductase system is coupled with IPNS, it quantitatively converts bis-ACV to isopenicillin N. These findings suggest that the disulfide reductase may play a role in the biosynthesis of penicillins and cephalosporins in streptomycetes. We also show here that S. clavuligerus lacks glutathione reductase and have previously reported that Streptomyces species do not contain glutathione. This disulfide reductase may therefore be important in determining the thiol-disulfide redox balance in streptomycetes.
Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro
Authors: Newlands J T , Gaal T , Mecsas J , Gourse R L
Published: November 2016

The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.
Presence of two independent chromosomes in the Brucella melitensis 16M genome
Authors: Michaux S , Paillisson J , Carles-Nurit M J , Bourg G , Allardet-Servent A , Ramuz M
Published: November 2016

Mapping the restriction fragments of the Brucella melitensis 16M genome with a new restriction endonuclease, PacI, which cut the DNA into only eight fragments, indicated that this species contains two unique and independent replicons of about 2,100 and 1,150 kb. Pulsed-field gel electrophoresis of intact DNA revealed two bands migrating the expected distances. These replicons were identified as two unique and independent chromosomes by the presence of rRNA operons and genes for heat shock proteins mapping to separate replicons.
Stability and asymmetric replication of the Bacillus subtilis 168 chromosome structure
Authors: Itaya M
Published: November 2016

Chromosomal DNAs from a number of strains derived from Bacillus subtilis 168 were digested with restriction endonucleases NotI or SfiI, and the locations of chromosomal alterations were compared with the recently constructed standard NotI-SfiI restriction map (M. Itaya and T. Tanaka, J. Mol. Biol. 220:631-648, 1991). In general, the chromosome structure of B. subtilis 168 was found to be stable, as expected from the genetic stability of this species. DNA alterations, typically deletions, are formed in three limited loci on the chromosome. One of these alterations was characterized as a spontaneous deletion formed between rrn operons, and another occurred as a result of prophage SP beta excision. I found that oriC and terC are not located on precisely opposite sides of the chromosome. Replication in the counter clockwise direction was 196 kb longer than replication in the clockwise direction. The characteristic of length difference is not changed by deletion formation.
Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I
Authors: Omori K , Imai Y , Suzuki S , Komatsubara S
Published: November 2016

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).
Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22
Authors: Kinder S A , Holt S C
Published: November 2016

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.
Expression and regulation of Bradyrhizobium japonicum and Xanthobacter flavus CO2 fixation genes in a photosynthetic bacterial host
Authors: Falcone D L , Tabita F R
Published: November 2016

Calvin cycle carbon dioxide fixation genes encoded on DNA fragments from two nonphotosynthetic, chemolithoautotrophic bacteria, Bradyrhizobium japonicum and Xanthobacter flavus, were found to complement and support photosynthetic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion mutant of the purple nonsulfur bacterium Rhodobacter sphaeroides. The regulation of RubisCO expression was analyzed in the complemented R. sphaeroides RubisCO deletion mutant. Distinct differences in the regulation of RubisCO synthesis were revealed when the complemented R. sphaeroides strains were cultured under photolithoautotrophic and photoheterotrophic growth conditions, e.g., a reversal in the normal pattern of RubisCO gene expression. These studies suggest that sequences and molecular signals which regulate the expression of diverse RubisCO genes may be probed by using the R. sphaeroides complementation system.
Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli
Authors: Leonardo M R , Cunningham P R , Clark D P
Published: November 2016

The regulation of the adhE gene, which encodes the trifunctional fermentative acetaldehyde-alcohol dehydrogenase of Escherichia coli, was investigated by the construction of gene fusions and by two-dimensional protein gel electrophoresis. Both operon and protein fusions of adhE to lacZ were induced 10- to 20-fold by anaerobic conditions, and both fusions were repressed by nitrate, demonstrating that regulation is at the level of transcription. Nitrate repression of phi (adhE-lacZ) expression, as well as of alcohol dehydrogenase enzyme activity, was partly relieved by a mutation in narL. Mutations in rpoN or fnr had no effect on the expression of adhE. Two-dimensional protein gels demonstrated that increases in the amount of adhE protein correlated with increases in enzyme activity, demonstrating that induction was due to synthesis of new protein, not to activation of preexisting protein. When oxidized sugar derivatives such as gluconate or glucuronate were used as carbon sources, the anaerobic expression of phi (adhE-lacZ) was greatly reduced, whereas when sugar alcohols such as sorbitol were used, the expression was increased compared with expression when glucose was the carbon source. This observation suggested that induction of phi (adhE-lacZ) might depend on the level of reduced NADH, which should be highest with sorbitol-grown cells and lowest with glucuronate-grown cells. When phi (adhE-lacZ) was present in a strain deleted for the adhE structural gene, anaerobic expression of phi (adhE-lacZ) was approximately 10-fold higher than in an adhE+ strain. Since the presence of alcohol dehydrogenase would serve to decrease NADH levels, this finding again implies that the adhE gene is regulated by the concentration of reduced NAD. Introduction of a pgi (phosphoglucose isomerase) mutation reduced the anaerobic induction of phi(adhE-lacZ) when the cells were grown on glucose, but had little effect on fructose-grown cells. Pyruvate did not overcome the pgi effect, but glycerol 3-phosphate did, which is again consistent with the possibility that adhE expression responds to the level of reduced NAD rather than to a glycolytic intermediate.
Positive regulation of the Escherichia coli glycine cleavage enzyme system
Authors: Wilson R L , Steiert P S , Stauffer G V
Published: November 2016

A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv.
Purification and characterization of SP21, a development-specific protein of the myxobacterium Stigmatella aurantiaca
Authors: Heidelbach M , Skladny H , Schairer H U
Published: November 2016

Stigmatella aurantiaca is a gram-negative bacterium with a complex life cycle, including cellular aggregation resulting in the formation of a characteristic three-dimensional structure, the so-called fruiting body. During fruiting and upon chemical induction of sporulation, a major development-specific protein, SP21, is synthesized. SP21 was purified to homogeneity from the membranous fraction of chemically induced spores. Expression of SP21 was studied with an antiserum raised against the purified protein.
Gene replacement, integration, and amplification at the gdhA locus of Corynebacterium glutamicum
Authors: Labarre J , Reyes O , Guyonvarch A , Leblon G
Published: November 2016

Gene replacement and integration in a Corynebacterium glutamicum ATCC 21086 derivative were achieved by transformation with a nonreplicative plasmid that contains the C. glutamicum ATCC 17965 gdhA gene modified by the insertion of an aphIII cartridge. We isolated rare derivatives of the integrative transformants that have higher levels of expression of the integrated plasmid genes than the parent. Different types of such amplified clones were distinguished according to their antibiotic resistance levels, enzyme specific activities, and physical structures. All amplified clones share a structural DNA motif confined to the chromosomal gdhA locus: a variable number (up to 10) of tandem copies of a unit that includes the selected gene and one flanking repeat. A given clone contains subpopulations that differ in the number of repeats of this unit.
A complete physical map of a Bacillus thuringiensis chromosome
Authors: Carlson C R , Kolstø A B
Published: November 2016

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.
Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae
Authors: Graf R , Mehmann B , Braus G H
Published: November 2016

The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway. We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan. Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence. However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution. Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme. We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene. From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms.
Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2
Authors: Locher H H , Poolman B , Cook A M , Konings W N
Published: November 2016

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.
Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5
Authors: Choe M , Reznikoff W S
Published: November 2016

A newly identified anaerobically expressed locus, aeg-46.5, which is located at min 46.5 on Escherichia coli linkage map, was cloned and analyzed. The phenotype of this gene was studied by using a lacZ operon fusion. aeg-46.5 is induced anaerobically in the presence of nitrate in wild-type and narL cells. It is repressed by the narL gene product, as it showed derepressed anaerobic expression in narL mutant cells. We postulate that aeg-46.5 is subject to multiple regulatory systems, activation as a result of anaerobiosis, narL-independent nitrate-dependent activation, and narL-mediated repression. The regulatory region of aeg-46.5 was identified. A 304-bp DNA sequence which includes the regulatory elements was obtained, and the 5' end of aeg-46.5 mRNA was identified. It was verified that the anaerobic regulation of aeg-46.5 expression is controlled on the transcriptional level. Computer analysis predicted possible control sites for the NarL and FNR proteins. The proposed NarL site was found in a perfect-symmetry element. The aeg-46.5 regulatory elements are adjacent to, but divergent from, those of the eco gene.
Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus
Authors: Muñoz-Dorado J , Almaula N , Inouye S , Inouye M
Published: November 2016

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.
Identification of the
Escherichia coli lytB
Gene, Which Is Involved in Penicillin Tolerance and Control of the Stringent Response
Authors: Gustafson Corinne E. , Kaul Sonu , Ishiguro Edward E.
Published: November 2016

The
Escherichia coli lytB
gene, which is involved in penicillin tolerance and control of the stringent response, was identified as a previously described open reading frame designated
orf316
located in the
ileS-lsp
operon (0.4 min on the linkage map).
Two developmentally controlled promoters of Streptomyces coelicolor A3(2) that resemble the major class of motility-related promoters in other bacteria
Authors: Tan H , Chater K F
Published: November 2016

Experiments were designed to allow isolation of Streptomyces coelicolor promoters that depend on the whiG sporulation gene, which encodes a putative sigma factor important in the sporulation of aerial hyphae. The strategy, based on earlier evidence that sigma WhiG is limiting for sporulation (K. F. Chater, C. J. Burton, K. A. Plaskitt, M. J. Buttner, C. Méndez, and J. Helmann, Cell 59:133-143, 1989) was to seek DNA fragments that inhibit sporulation in aerial hyphae when present at a high copy number. In a suitable Sau3AI-generated library of DNA from S. coelicolor A3(2), two inserts were found to inhibit sporulation. Both inserts caused expression of the adjacent xylE reporter gene present in the vector in a developmentally normal strain of S. coelicolor, but there was no xylE expression in an otherwise isogenic whiG mutant. S1 nuclease protection experiments were done with RNAs isolated from these plasmid-bearing strains or from the wild-type strain lacking either recombinant plasmid. In each case, an apparent transcription start site was found upstream of an apparent open reading frame (ORF) and just downstream of sequences that resemble consensus features of promoters for motility-related genes in Bacillus subtilis and coliform bacteria. Such promoters depend on sigma factors (sigma D and sigma F, respectively) particularly similar to the deduced whiG gene product. Each of the putative whiG-dependent promoters is within an ORF that is upstream of, and potentially translationally coupled to, the putative whiG-dependent ORF (although use of one of the promoters would necessitate the use of a different start codon, further downstream). Thus, in unknown circumstances, the whiG-dependent ORFs may be expressed from a more remote promoter as part of a complex transcription unit.
Nucleotide sequence of the adi gene, which encodes the biodegradative acid-induced arginine decarboxylase of Escherichia coli
Authors: Stim K P , Bennett G N
Published: November 2016

Arginine decarboxylase (encoded by adi) is induced under conditions of acidic pH, anaerobiosis, and rich medium. The DNA sequence of a 3-kb fragment of the Escherichia coli chromosome encoding biodegradative arginine decarboxylase was determined. This sequence encodes a protein of 755 amino acids with a molecular size of 84,420 daltons. The molecular weight and predicted Adi amino acid composition agree with those found in earlier work. The amino acid sequence of arginine decarboxylase showed homology to those of three other decarboxylases of E. coli: (i) CadA, encoding lysine decarboxylase; (ii) SpeC, encoding biosynthetic ornithine decarboxylase; and (iii) SpeF, encoding biodegradative ornithine decarboxylase and the lysine decarboxylase of Hafnia alvei. Unlike SpeC and SpeF, Adi is not similar to the biosynthetic arginine decarboxylase, SpeA. adi is also dissimilar to cadA and speF in that it does not appear to be part of an operon containing a metabolically related transport protein, indicating that it represents a new type of biodegradative decarboxylase regulation. Transcriptional fusions between fragments upstream of adi and lacZ, primer extension, and site-directed mutagenesis experiments defined the pH-regulated promoter. Deletion analysis of the upstream region and cloning of fragments to make adi::lacZ protein fusion implicated a region beyond an upstream SspI site in pH regulation. Induction of adi in the presence of sublethal concentrations of novobiocin or coumermycin A1, inhibitors of DNA gyrase, was dramatically decreased, indicating that DNA supercoiling is involved in adi expression. These results and those of promoter structure studies indicated that acid regulation of adi may involve a mechanism different from that of acid regulation of cad.
Inhibition of Escherichia coli chemotaxis by omega-conotoxin, a calcium ion channel blocker
Authors: Tisa L S , Olivera B M , Adler J
Published: November 2016

Escherichia coli chemotaxis was inhibited by omega-conotoxin, a calcium ion channel blocker. With Tris-EDTA-permeabilized cells, nanomolar levels of omega-conotoxin inhibited chemotaxis without loss of motility. Cells treated with omega-conotoxin swam with a smooth bias, i.e., tumbling was inhibited.
Implications of Tn5-associated adjacent deletions
Authors: Jilk R A , Makris J C , Borchardt L , Reznikoff W S
Published: November 2016

The prokaryotic transposable element Tn5 has been found to promote the formation of adjacent deletions. The frequency of adjacent deletion formation is much lower than that of normal transposition events. Like normal transposition, however, adjacent deletion formation requires the activity of the transposase protein. The deletions can be divided into two classes, as distinguished by their endpoints. The occurrence of one of the two deletion classes is increased when the frequency of normal transposition is reduced by the introduction of a deletion or a certain base substitution at one of the two outside ends (OEs). The nature of the base substitution at the mutant OE influences the class of deletion found adjacent to the wild-type OE, even though these two ends are about 12 kbp apart. By studying the formation of these deletions, we have gained some insight into the way in which the transposase interacts with the OEs. Our observations suggest that there is a protein-mediated interaction between the two ends, that different end base pairs are involved in different transposition-related processes, and that the adjacent deletions are the result of nonproductive attempts at transposition.