bluuebunny/crossref_metadata_2025 huggingface dataset:
Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP
Authors: Fraimow H S , Greenman J B , Leviton I M , Dougherty T J , Miller M H
Published: November 2016

Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects. Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force. However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E. coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential. Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells. Absence of delta psi in these cells was confirmed by absence of [3H]tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine). Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay. Unlike unacclimatized CL2 cells treated with CCCP, these cells transported [3H]tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport. These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi. delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content. Despite maintenance of normal ATP, there was no transport of [3H] bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides.
Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti
Authors: Rastogi V K , Watson R J
Published: November 2016

A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.
Separation and partial characterization of the enzymes of the toluene-4-monooxygenase catabolic pathway in Pseudomonas mendocina KR1
Authors: Whited G M , Gibson D T
Published: November 2016

The route of toluene degradation by Pseudomonas mendocina KR1 was studied by separating or purifying from toluene-grown cells the catabolic enzymes responsible for oxidation of p-cresol through the ring cleavage step. Enzymatic transformations corresponding to each of the metabolic steps in the proposed degradative pathway were conducted with cell-free preparations. p-Cresol was metabolized by the enzyme p-cresol methylhydroxylase to p-hydroxybenzaldehyde. p-Hydroxybenzaldehyde was further oxidized by partially purified enzyme preparations to p-hydroxybenzoate and subsequently hydroxylated to form protocatechuate. Protocatechuate was then oxidized by ortho ring cleavage.
Site-specific insertion of three structural gene cassettes in transposon Tn7
Authors: Sundström L , Roy P H , Sköld O
Published: November 2016

Transposon Tn7 has been known to carry genes for resistance to trimethoprim and spectinomycin. A poorly expressed streptothricin resistance gene, identical to the sat gene found in transposons Tn1825 and Tn1826, was localized between the two mentioned genes in Tn7. The surroundings of all three resistance genes indicated site-specific insertion of genetic cassettes.
Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36
Authors: Kobayashi T , Kanai H , Hayashi T , Akiba T , Akaboshi R , Horikoshi K
Published: November 2016

A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.
Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins
Authors: Sawers G , Suppmann B
Published: November 2016

The pyruvate formate-lyase (pfl) gene of Escherichia coli is transcribed from seven promoters which are coordinately induced 12- to 15-fold by anaerobiosis. The FNR protein plays a major role in the anaerobic control of this system. A mutation in the fnr gene, however, only reduces anaerobic induction fivefold, indicating that FNR is not the only factor involved in the anaerobic activation process (Sawers and Böck, J. Bacteriol. 171:2485-2498, 1989). The residual anaerobic induction could be shown to be imparted by the transcriptional regulator ArcA; an arcA fnr double mutant was incapable of inducing pfl transcription anaerobically. A mutant strain unable to synthesize the membrane-associated histidine kinase (ArcB) that has been proposed to activate ArcA showed the same phenotype as an arcA mutant strain, indicating that a functional ArcB protein is also required for wild-type, anaerobic pfl transcriptional activation. Nuclease S1 analysis revealed that an arcA mutation abolished anaerobic transcription from promoter 7 and reduced expression from promoter 6 but did not affect transcription from promoters 1 to 5. On the other hand, an fnr mutation prevented anaerobic expression from promoters 6 and 7 and reduced transcription from promoters 1 to 5. These data indicate that both ArcA and FNR are essential for anaerobic activation of promoter 7 transcription, which suggests functional interaction between these proteins.
Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66
Authors: Sára M , Pum D , Sleytr U B
Published: November 2016

We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.
Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene
Authors: Ishimoto K S , Lory S
Published: November 2016

Two regulatory mutants of Pseudomonas aeruginosa, R1 and RA, that affect transcription of the pilin gene were isolated. This was done by introducing a plasmid carrying a fusion of the pilin gene's promoter with the lacZ gene into a bank of P. aeruginosa DNA mutagenized with the transposon Tn5G. The block in pilin expression in these mutants was shown to be at the level of transcription, since these mutants did not synthesize either pilin mRNA or pilin antigen. A restriction fragment derived from the R1 mutant that contains the entire transposon plus flanking chromosomal DNA was cloned and used as a probe to screen a cosmid library of P. aeruginosa DNA. Cosmids that could complement the pilin expression defect in both R1 and RA were isolated. The gene inactivated in R1 was sequenced. This gene, designated pilR, encodes an approximately 50-kDa polypeptide which exhibits significant similarity to the NtrC family of response regulators of the two-component regulatory system. PilR contains the amino-terminal aspartic acid residues which are conserved among the response regulators, suggesting that pilin gene transcription is regulated via a phosphotransfer mechanism in which PilR is phosphorylated by an as yet unidentified protein kinase.
RNase E-dependent cleavages in the 5' and 3' regions of the Escherichia coli unc mRNA
Authors: Patel A M , Dunn S D
Published: November 2016

The endonucleolytic processing of the unc mRNA encoding the eight subunits of the Escherichia coli F1F0-ATPase was studied. Northern (RNA) blots of mRNA expressed from a plasmid which contained the 3'-terminal portion of the operon including the uncDC sequences revealed, in addition to the expected 2-kb mRNA, a 0.5-kb RNA species which hybridized to an uncC antisense RNA probe. An uncD antisense RNA probe hybridized to only the 2-kb mRNA, implying that the upstream 1.5-kb fragment is rapidly degraded. The 5' end of the 0.5-kb fragment was determined by primer extension analysis to be 11 bases into the coding region of the uncC gene. In RNase E-deficient strains, the amount of the 0.5-kb product was strongly reduced while the levels of the precursor uncDC transcript remained high. Similar RNase E-dependent processing was found in the chromosomally encoded unc mRNA. As this RNase E-dependent cleavage directly inactivates uncC and appears to leave uncD susceptible to degradation, it seems unlikely to play a role in differential expression of the gene products but may be an important event in unc mRNA degradation. RNase E mutants also showed altered processing of the chromosomally encoded unc mRNA in the uncB region near the 5' end. The expected full-length (7-kb) transcript was recognized when RNA from the RNase E-deficient strain was subjected to Northern blot analysis with uncB- and uncC-specific probes. RNA from strains with functional RNase E lacked the 7-kb transcript but had a 6.2-kb mRNA detectable with the uncC but not the uncB probe. RNase E is therefore implicated in multiple cleavages of the unc mRNA.
Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3
Authors: van Heijenoort Y , Gómez M , Derrien M , Ayala J , van Heijenoort J
Published: November 2016

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.
Roles of rpoD, spoIIF, spoIIJ, spoIIN, and sin in regulation of Bacillus subtilis stage II sporulation-specific transcription
Authors: Louie P , Lee A , Stansmore K , Grant R , Ginther C , Leighton T
Published: November 2016

Bacillus subtilis strains containing defects in the sporulation gene spoIIF (kinA), spoIIJ (kinA), or spoIIN (ftsA) cannot transcribe the sigma E-dependent gene spoIID. Results presented here and by other workers demonstrate that the spoIIF, spoIIJ, and spoIIN gene products control spoIID transcription indirectly by coordinating the induction of the spoIIGAB, spoIIE, and spoIIAC operons, which are required for sigma E synthesis and processing. Sporulation competence and spoIIGAB, spoIIE, and spoIIAC transcription were restored in spoIIF, spoIIJ, and spoIIN mutants by introduction of crsA47, a mutation in the major vegetative sigma factor sigma A. crsA mutations are known to restore sporulation in certain spo0 mutants. crsA suppression of kinA and ftsA mutations was achieved through inhibition of the transcription of sin, a gene involved in the selection between several post-exponential-phase cell states. A deletion of sin restored sporulation competence in spoIIF, spoIIJ, or spoIIN mutant strains. A sin deletion was also able to restore sporulation competence in the crsA suppressible stage 0 mutant spo0K141.
Identification of a gene, rgg, which regulates expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii Challis
Authors: Sulavik M C , Tardif G , Clewell D B
Published: November 2016

Streptococcus gordonii Challis was previously shown to give rise to phase variants expressing high (Spp+) or low (Spp-) levels of extracellular glucosyltransferase (GTF) activity. Here, shotgun cloning of an S. gordonii Spp+ chromosomal digest resulted in a chimeric plasmid (pAM5010) able to complement the Spp- phenotype. In addition, introduction of pAM5010 into an Spp+ strain resulted in a 10-fold increase in GTF expression. Deletion analysis of pAM5010 identified a 1.2-kb DNA segment which exhibited the same functional properties as pAM5010. Nucleotide sequence analysis of this region revealed a gene approximately 1 kb in size. The gene was designated rgg. Disruption of the chromosomal rgg gene open reading frame in an Spp+ strain resulted in strain DS512, which displayed an Spp(-)-like phenotype and had 3% of wild-type GTF activity. A plasmid containing the rgg gene was able to complement the DS512 phenotype and significantly increase GTF expression above wild-type levels. Sequence analysis and other data showed that the S. gordonii GTF determinant, designated gtfG, is located 66 bp downstream of the rgg gene. The sequence also revealed interesting inverted repeats which may play a role in the regulation of gtfG. We conclude that rgg positively regulates the expression of GTF and influences expression of the Spp phenotype.
Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin
Authors: Delrue B , Fontaine T , Routier F , Decq A , Wieruszeski J M , Van Den Koornhuyse N , Maddelein M L , Fournet B , Ball S
Published: November 2016

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.
osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli
Authors: Yim H H , Villarejo M
Published: November 2016

A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.
Activation of Bacillus subtilis transcription factor sigma B by a regulatory pathway responsive to stationary-phase signals
Authors: Boylan S A , Rutherford A , Thomas S M , Price C W
Published: November 2016

Alternative transcription factor sigma B of Bacillus subtilis controls a stationary-phase regulon induced under growth conditions that do not favor sporulation. Little is known about the metabolic signals and protein factors regulating the activity of sigma B. The operon containing the sigma B structural gene has the gene order orfV-orfW-sigB-rsbX, and operon expression is autoregulated positively by sigma B and negatively by the rsbX product (rsbX = regulator of sigma B). To establish the roles of the orfV and orfW products, orfV and orfW null and missense mutations were constructed and tested for their effects on expression of the sigma B-dependent genes ctc and csbA. These mutations were tested in two contexts: in the first, the sigB operon was under control of its wild-type, sigma B-dependent promoter, and in the second, the sigB operon promoter was replaced by the inducible Pspac promoter. The principal findings are that (i) the orfV (now called rsbV) product is a positive regulator of sigma B-dependent gene expression; (ii) the orfW (now called rsbW) product is a negative regultor of such expression; (iii) sigma B is inactive during logarithmic growth unless the rsbW product is absent; (iv) the rsbX, rsbV, and rsbW products have a hierarchical order of action; and (v) both the rsbV and rsbW products appear to regulate sigma B activity posttranslationally. There are likely to be at least two routes by which information can enter the system to regulate sigma B: via the rsbX product, and via the rsbV and rsbW products.
Proline biosynthesis in Saccharomyces cerevisiae: analysis of the PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase
Authors: Brandriss M C , Falvey D A
Published: November 2016

The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC 1.5.1.2], which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive. It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system. Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source. The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.
Dissection of the Salmonella typhimurium genome by use of a Tn5 derivative carrying rare restriction sites
Authors: Wong K K , McClelland M
Published: November 2016

A polylinker with rare restriction sites was introduced into a mini-Tn5 derivative. These sites include M.XbaI-DpnI (TCTAGATCTAGA), which is rare in most bacterial genomes, SwaI (ATTTAAAT) and PacI (TTAATTAA), which are rare in G+C-rich genomes, NotI (GCGGCCGC) and SfiI (GGCCN5GGCC), which are rare in A+T-rich genomes, and BlnI (CCTAGG), SpeI (ACTAGT), and XbaI (TCTAGA), which are rare in the genomes of many gram-negative bacteria. This Tn5(pfm) (pulsed-field mapping) transposon carries resistance to chloramphenicol and kanamycin to allow selection in a wide variety of background genomes. This Tn5(pfm) was integrated randomly into the Salmonella typhimurium and Serratia marcescens genomes. Integration of the new rare SwaI, PacI, BlnI, SpeI, and XbaI sites was assayed by restriction digestion and pulsed-field gel electrophoresis. Tn5(pfm) constructs could be valuable tools for pulsed-field mapping of gram-negative bacterial genomes by assisting in the production of physical maps and restriction fragment catalogs. For the first applications of a Tn5(pfm), we bisected five of the six largest BlnI fragments in the S. typhimurium genome, bisected the linearized 90-kb pSLT plasmid, and used Tn5(pfm) and Tn10 to trisect the largest BlnI fragment.
Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase
Authors: Shen B , Summers R G , Gramajo H , Bibb M J , Hutchinson C R
Published: November 2016

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.
An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase
Authors: Mirel D B , Lustre V M , Chamberlin M J
Published: November 2016

Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase. Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro. Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site. Direct evidence linking these genes to motility functions in B. subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al. (A. R. Zuberi, C. Ying, H. M. Parker, and G. W. Ordal, J. Bacteriol. 172:6841-6848, 1990), to within this mot. operon. Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B. subtilis. These current findings support our earlier suggestions that sigma D in B. subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions. Sigma F, the enteric homolog of sigma D, controls similar functions in E. coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family.
The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth
Authors: Tsuchimoto S , Nishimura Y , Ohtsubo E
Published: November 2016

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.
Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior
Authors: McBride M J , Köhler T , Zusman D R
Published: November 2016

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.
Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum
Authors: Atkinson M , Allen C , Sequeira L
Published: November 2016

We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.
Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system
Authors: Mueller J P , Bukusoglu G , Sonenshein A L
Published: November 2016

The Bacillus subtilis glucose starvation-inducible transcription units, gsiA and gsiB, were characterized by DNA sequencing, transcriptional mapping, mutational analysis, and expression in response to changes in environmental conditions. The gsiA operon was shown to consist of two genes, gsiAA and gsiAB, predicted to encode 44.9- and 4.8-kDa polypeptides, respectively. The gsiB locus contains a single cistron which encodes a protein of unusual structure; most of its amino acids are arranged in five highly conserved, tandemly repeated units of 20 amino acids. The 5' ends of gsiA and gsiB mRNAs were located by primer extension analysis; their locations suggest that both are transcribed by RNA polymerase containing sigma A. Expression of both gsiA and gsiB was induced by starvation for glucose or phosphate or by addition of decoyinine, but only gsiA was induced by exhaustion of nutrient broth or by amino acid starvation. Regulation of gsiA expression was shown to be dependent upon the two-component signal transduction system ComP-ComA, which also controls expression of genetic competence genes. Mutations in mecA bypassed the dependency of gsiA expression on ComA. Disruption of gsiA relieved glucose repression of sporulation but did not otherwise interfere with sporulation, development of competence, motility, or glucose starvation survival. We propose that gsiA and gsiB are members of an adaptive pathway of genes whose products are involved in responses to nutrient deprivation other than sporulation.
Physical and functional maps of the luminescence gene cluster in an autoinducer-deficient Vibrio fischeri strain isolated from a squid light organ
Authors: Gray K M , Greenberg E P
Published: November 2016

Vibrio fischeri ES114 is an isolate representing the specific bacterial light organ symbiont of the squid Euprymna scolopes. An interesting feature of this strain of V. fischeri is that it is visibly luminous within the light organ of the squid host but is nonluminous when grown under standard laboratory conditions. Luminescence can be restored in laboratory culture, however, by the addition of autoinducer, a species-specific inducer of the V. fischeri luminescence (lux) genes. Most other isolates of V. fischeri produce autoinducer in sufficient quantities to induce luminescence in laboratory culture. We have cloned an 8.8-kb DNA fragment from V. fischeri ES114 that encodes all of the functions necessary for luminescence in Escherichia coli in the absence of exogenous autoinducer. This DNA contains both of the recognized V. fischeri lux regulatory genes, one of which (luxI) directs E. coli to synthesize autoinducer. The organization of the individual lux genes within this DNA fragment appears to be the same as that in the other strains of V. fischeri studied; the restriction map of the V. fischeri ES114 lux DNA has diverged substantially, however, from the largely conserved maps of V. fischeri MJ1 and ATCC 7744. Although E. coli containing the V. fischeri ES114 lux DNA synthesizes considerable amounts of autoinducer, V. fischeri ES114 synthesizes autoinducer only in small amounts, even when transcription of the lux genes, including luxI, is activated by the addition of exogenous autoinducer. Nonetheless, transconjugants of V. fischeri ES114 that contain multicopy plasmids bearing the ES114 lux genes synthesize sufficient autoinducer to induce luminescence. These results suggest that V. fischeri ES11r does not lack a functional luxl, nor is it deficient in the ability to synthesize metabolic precursors for autoinducer synthesis.
Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa
Authors: Ankenbauer R G
Published: November 2016

Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa.
Thin aggregative fimbriae from diarrheagenic Escherichia coli
Authors: Collinson S K , Emödy L , Trust T J , Kay W W
Published: November 2016

Four strains of diarrheagenic Escherichia coli originally isolated from distinct geographic regions were found to produce unusual thin aggregative fimbriae requiring depolymerization in formic acid prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy of native fimbriae and Western blot (immunoblot) analysis of the corresponding 18-kDa fimbrins showed that these E. coli fimbriae were serologically cross-reactive with SEF 17 (Salmonella enteritidis fimbriae with a fimbrin molecular mass of 17 kDa). The E. coli and S. enteritidis fimbrins had similar total amino acid compositions and highly conserved N-terminal amino acid sequences. These results indicate that E. coli and S. enteritidis produce biochemically related, aggregative fimbriae which constitute a new type of intergenerically distributed fimbriae for which we propose the descriptive name GVVPQ fimbriae on the basis of the conserved N-terminal amino acid sequence.
Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase
Authors: Norris U , Karp P E , Fimmel A L
Published: November 2016

Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis. Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation. Membranes prepared from these strains possess basal levels of ATPase activity. In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.
Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene
Authors: Gupta R S , Singh B
Published: November 2016

Heat shock induces the synthesis of a set of proteins in Halobacterium marismortui whose molecular sizes correspond to the known major heat shock proteins. By using the polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of the 70-kDa heat shock protein (HSP70) family, we have successfully cloned and sequenced a gene fragment containing the entire coding sequence for HSP70 from H. marismortui. HSP70 from H. marismortui shows between 44 and 47% amino acid identity with various eukaryotic HSP70s and between 51 and 58% identity with its eubacterial and archaebacterial homologs. On the basis of a comparison of all available HSP70 sequences, we have identified a number of unique sequence signatures in this protein family that provide a clear distinction between eukaryotic organisms and prokaryotic organisms (archaebacteria and eubacteria). The archaebacterial (viz., H. marismortui and Methanosarcina mazei) HSP70s have been found to contain all of the signature sequences characteristic of eubacteria (particularly the gram-positive bacteria), which suggests a close evolutionary relationship between these groups. In addition, detailed analyses of HSP70 sequences that we have carried out have revealed a number of additional novel features of the HSP70 protein family. These include (i) the presence of an insertion of about 25 to 27 amino acids in the N-terminal quadrants of all known eukaryotic and prokaryotic HSP70s except those from archaebacteria and the gram-positive group of bacteria, (ii) significant sequence similarity in HSP70 regions comprising its first and second quadrants from organisms lacking the above insertion, (iii) highly significant similarity between a protein, MreB, of Escherichia coli and the N-terminal half of HSP70s, (iv) significant sequence similarity between the N-terminal quadrant of HSP70 (from gram-positive bacteria and archaebacteria) and the m-type thioredoxin of plant chloroplasts. To account for these and other observations, a model for the evolution of HSP70 proteins involving gene duplication is proposed. The model proposes that HSP70 from archaebacteria (H. marismortui and M. mazei) and the gram-positive group of bacteria constitutes the ancestral form of the protein and that all other HSP70s (viz., other eubacteria as well as eukaryotes) containing the insert have evolved from this ancient protein.
Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production
Authors: Ludovice M , Martin J F , Carrachas P , Liras P
Published: November 2016

The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.
Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide
Authors: Clarke B R , Whitfield C
Published: November 2016

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.
Regulation of transcription of the cell division gene ftsA during sporulation of Bacillus subtilis
Authors: Gholamhoseinian A , Shen Z , Wu J J , Piggot P
Published: November 2016

Three distinct 5' ends of ftsA mRNA were identified by S1 mapping and by primer extension analysis. These are thought to represent three transcription start sites. The transcripts from the downstream and upstream sites were detected throughout growth. The transcript from the middle site was not detected during exponential growth but was detected within 30 min of the start of sporulation, when it was the predominant transcript. Insertion of a cat cassette in the middle promoter, ftsAp2 (p2), did not affect vegetative growth but prevented postexponential symmetrical division and spore formation. Transcription from p2 was dependent on RNA polymerase containing sigma H, and promoter p2 resembled the consensus sigma H promoter. Transcription from p2 did not require expression of the spo0A, spo0B, spo0E, spo0F, or spo0K loci. Northern (RNA) blot analysis indicated that ftsA is cotranscribed with the adjacent ftsZ gene. Multiple promoters provide a mechanism by which essential vegetative genes can be subjected to sporulation control independent of control during vegetative growth. In the case of ftsA,Z, the promoters provide a mechanism to permit septum formation in conditions of nutrient depletion that might be expected to shut down the vegetative division machinery.
The catalytic domain of endoglucanase A from Clostridium cellulolyticum: effects of arginine 79 and histidine 122 mutations on catalysis
Authors: Belaich A , Fierobe H P , Baty D , Busetta B , Bagnara-Tardif C , Gaudin C , Belaich J P
Published: November 2016

Sequence analysis of the endoglucanase EGCCA of Clostridium cellulolyticum indicates the existence of two domains: a catalytic domain extending from residue 1 to residue 376 and a reiterated domain running from residue 390 to 450. A small deletion in the C terminal end of the catalytic domain inactivated the protein. From the analysis of the sequences of 26 endoglucanases belonging to family A, we focused on seven amino acids which were totally conserved in all the catalytic domains compared. The roles of two of these, Arg-79 and His-122, were studied and defined on the basis of the mutants obtained by introducing various substitutions. Our findings suggest that Arg-79 is involved in the structural organization of the protein; the His-122 residue seems to be more essential for catalysis. The role of His-123, which is conserved only in subfamily A4, was also investigated.