bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription
Authors: Sanders D A , Gillece-Castro B L , Burlingame A L , Koshland D E
Published: November 2016

The NtrC transcription factor is a member of a family of homologous prokaryotic regulatory proteins that participate in the transduction of extracellular and nutritional signals. It has been demonstrated that the phosphate group from a histidine residue of the phosphorylated NtrB protein autokinase is transferred to the NtrC protein. Phosphorylation of the NtrC protein is transient and activates its transcriptional enhancement activity. We have investigated the site of phosphorylation of the Salmonella typhimurium NtrC protein and find that it is an aspartate residue (Asp-54) that is found within a sequence conserved in all of the members of the family of regulatory proteins. We propose that this phosphorylation is an NtrC protein histidine phosphatase catalytic intermediate. This conclusion suggests that the NtrC family should be viewed not as kinase substrates but as enzymes that can catalyze the hydrolysis of their activated forms in a concentration-independent fashion. They are similar in this sense to eukaryotic signal-transducing GTPases.
Evidence that GroEL, not sigma 32, is involved in transcriptional regulation of the Vibrio fischeri luminescence genes in Escherichia coli
Authors: Dolan K M , Greenberg E P
Published: November 2016

In Escherichia coli, transcription of the inducible Vibrio fischeri luminescence operon, luxICDABE, has been reported to require sigma 32, the product of rpoH. Consistent with previous studies, we report that an E. coli delta rpoH mutant, KY1601 containing luxICDABE and luxR, which codes for the activator of luxICDABE transcription on a plasmid (pJE202), was weakly luminescent. Transformation of this E. coli strain with a plasmid containing rpoH under the control of the tac promoter resulted in high levels of cellular luminescence. However, the level of expression of the pJE202 luxICDABE was also high in E. coli 1603, a delta rpoH mutant with a second-site mutation that resulted in sigma 32-independent overexpression of the groE operon. Apparently, sigma 32 is not directly required for the transcription of luxICDABE in E. coli but is required for sufficient expression of groE, which is in turn required for the transcription of luxICDABE. This conclusion is supported by the finding that E. coli groE mutants containing pJE202 were weakly luminescent. In the E. coli delta rpoH mutant KY1601, the sigma 32 requirement for the transcription of luxICDABE was partially compensated for by the addition of saturating concentrations of the inducer to the culture medium and largely compensated for when cells were transformed with a luxR overexpression vector. These data support the hypothesis that sigma 32 is not required for transcription of luxICDABE. Rather, it appears that the products of groE are required for the folding of LuxR into an active protein, like they are for the folding of several other proteins.
Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation
Authors: Batchelor R A , Alifano P , Biffali E , Hull S I , Hull R A
Published: November 2016

In this article, we report on the nucleotide sequences of the rol genes of Escherichia coli O75 and Salmonella typhimurium LT2. The rol gene in E. coli was previously shown to encode a 36-kDa protein that regulates size distribution of the O-antigen moiety of lipopolysaccharide. The E. coli and S. typhimurium rol gene sequences consist of 978 and 984 nucleotides, respectively. The homology between the nucleotide sequences of these two genes was found to be 68.9%. Both the E. coli rol and S. typhimurium rol genes are transcribed counter to the histidine operon and code for deduced polypeptides of 325 and 327 amino acids, respectively. The S. typhimurium rol gene was previously identified to encode a protein of unknown function and to share a transcription termination region with his. The homology between these deduced polypeptide sequences was observed to be 72%. A complementation test was performed in which the S. typhimurium rol gene was placed in trans with an E. coli plasmid (pRAB3) which encodes the O75 rfb gene cluster and not rol. The protein expressed from the S. typhimurium rol gene was found to regulate the distribution of the O75 O polysaccharide on the lipopolysaccharide of the host strain, E. coli S phi 874. The mechanism of Rol action may be independent of O antigen subunit structure, and its presence may be conserved in members of the family Enterobacteriaceae and other gram-negative bacilli that express O polysaccharides on their surface membrane.
NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri
Authors: Cuypers H , Viebrock-Sambale A , Zumft W G
Published: November 2016

The regulatory element NosR was identified within the nos region of the denitrification gene cluster of Pseudomonas stutzeri ZoBell (ATCC 14405) and characterized. It is essential for expression of the N2O reductase encoded by nosZ immediately downstream of nosR. The nosR region was initially identified by Tn5 mutagenesis (W. G. Zumft, K. Döhler, and H. Körner, J. Bacteriol. 163:918-924, 1985). It consists of a single open reading frame of 2,172 nucleotides and has the coding capacity for an 81.9-kDa protein. The codon usage for nosR, with its high G + C content of 62.4 mol% and a preference for G or C at the third position, is characteristic for a Pseudomonas gene. Hydropathy analysis classified NosR as an integral membrane protein with at least seven membrane-spanning segments. No similarity to known bacterial regulator proteins was found in a data bank search. However, the C terminus of NosR shows sequence similarity to the cysteine clusters of several 2[4Fe-4S] bacterial ferrodoxins. A monocistronic mRNA for nosZ which allowed us to monitor NosR function was identified. Complementation of Nos- mutant MK418 (nosR::Tn5) with the nosR gene supplied in trans restored nosZ transcription and expression of a catalytically active N2O reductase. In addition to evidence of the requirement for NosR, indirect evidence for involvement of the transcriptional regulator Fnr is presented.
Membrane topology of the Escherichia coli ExbD protein
Authors: Kampfenkel K , Braun V
Published: November 2016

The ExbD protein is involved in the energy-coupled transport of ferric siderophores, vitamin B12, and B-group colicins across the outer membrane of Escherichia coli. In order to study ExbD membrane topology, ExbD-beta-lactamase fusion proteins were constructed. Cells expressing beta-lactamase fusions to residues 53, 57, 70, 76, 78, 80, 92, 121, and 134 of ExbD displayed high levels of ampicillin resistance, whereas fusions to residues 9 and 19 conferred no ampicillin resistance. It is concluded that the only hydrophobic segment of ExbD, encompassing residues 23 to 43, forms a transmembrane domain and that residues 1 to 22 are located in the cytoplasm and residues 44 to 141 are located in the periplasm.
Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites
Authors: Raya R R , Fremaux C , De Antoni G L , Klaenhammer T R
Published: November 2016

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.
New thermosensitive plasmid for gram-positive bacteria
Authors: Maguin E , Duwat P , Hege T , Ehrlich D , Gruss A
Published: November 2016

We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.
Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype
Authors: Cotter T W , Thomashow M F
Published: November 2016

Bdellovibrios invade and grow within the periplasmic space of suitable gram-negative bacteria. Wild-type bdellovibrios are obligately dependent on host cells for growth, but spontaneous host-independent (H-I) mutants that grow axenically on standard rich culture media can be isolated. Such mutants generally retain the ability to grow intraperiplasmically, although the plaques that they produce on lawns of host cells are smaller and more turbid than those produced by wild-type bdellovibrios. Here, we identify the first genetic locus associated with the H-I phenotype: hit (host interaction). We show that three individual H-I mutants suffered mutations at the hit locus and that recombination of wild-type hit sequences into the genomes of the H-I mutants greatly enhanced their plaquing ability. DNA sequence analysis localized the hit mutation in each of the H-I mutants to a 135-bp region of the genome. Mutations at hit may not fully account for the H-I phenotype, however, as recombination of wild-type hit sequences into the genomes of the H-I mutants had little effect on the axenic-growth phenotype of the mutants. Possible explanations for this result and potential roles for the hit locus are discussed.
Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli
Authors: Feng J , Atkinson M R , McCleary W , Stock J B , Wanner B L , Ninfa A J
Published: November 2016

Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRII (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRII, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase glnAp2 promoter.
Gene structure of Enterococcus hirae (Streptococcus faecalis) F1F0-ATPase, which functions as a regulator of cytoplasmic pH
Authors: Shibata C , Ehara T , Tomura K , Igarashi K , Kobayashi H
Published: November 2016

Enterococcus hirae (formerly Streptococcus faecalis) ATCC 9790 has an F1F0-ATPase which functions as a regulator of the cytoplasmic pH but does not synthesize ATP. We isolated four clones which contained genes for c, b, delta, and alpha subunits of this enzyme but not for other subunit genes. It was revealed that two specific regions (upstream of the c-subunit gene and downstream of the gamma-subunit gene) were lost at a specific site in the clones we isolated, suggesting that these regions were unstable in Escherichia coli. The deleted regions were amplified by polymerase chain reaction, and the nucleotide sequences of these regions were determined. The results showed that eight genes for a, c, b, delta, alpha, gamma, beta, and epsilon subunits were present in this order. Northern (RNA) blot analysis showed that these eight genes were transcribed to one mRNA. The i gene was not found in the upper region of the a-subunit gene. Instead of the i gene, this operon contained a long untranslated region (240 bp) whose G + C content was only 30%. There was no typical promoter sequence such as was proposed for E. coli, suggesting that the promoter structure of this species is different from that of E. coli. Deduced amino acid sequences suggested that E. hirae H(+)-ATPase is a typical F1F0-type ATPase but that its gene structure is not identical to that of other bacterial F1F0-ATPases.
In vitro activation of dinitrogenase reductase from the cyanobacterium Anabaena variabilis (ATCC 29413)
Authors: Böhm I , Halbherr A , Smaglinski S , Ernst A , Böger P
Published: November 2016

Nitrogenase of the heterocystous cyanobacterium Anabaena variabilis was inactivated in vivo (S. Reich, H. Almon, and P. Böger, FEMS Microbiol. Lett. 34:53-56, 1986). Partially purified and modified (inactivated) dinitrogenase reductase (Fe-protein) of such cells was reactivated by isolated membrane fractions of A. variabilis or of Rhodospirillum rubrum, and acetylene reduction was measured. Reactivation requires ATP, Mg2+, and Mn2+. The activating principle is localized in the heterocyst and was found effective only when prepared from cells exhibiting active nitrogenase. It also restores the activity of modified Fe-protein from R. rubrum.
The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders
Authors: Cook D M , Farrand S K
Published: November 2016

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.
Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source
Authors: Garzón A , Li J , Flores A , Casadesus J , Stewart V
Published: November 2016

Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the Klebsiella pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization.
Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures
Authors: Willimsky G , Bang H , Fischer G , Marahiel M A
Published: November 2016

A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing.
Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae
Authors: Bakker D , Willemsen P T , Willems R H , Huisman T T , Mooi F R , Oudega B , Stegehuis F , de Graaf F K
Published: November 2016

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.
vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri
Authors: Tobe T , Sasakawa C , Okada N , Honma Y , Yoshikawa M
Published: November 2016

Shigellae, the causative agents of bacillary dysentery, are capable of adhering to and invading epithelial cells and spreading into adjacent cells. A chromosomal mutant of Shigella flexneri 2a YSH6000 with reduced invasive capacity was isolated by Tn5 insertion mutagenesis. The linkage of the mutant phenotype to the Tn5 insertion was determined by P1 phage transduction. The site of the Tn5 insertion was assigned to a NotI chromosomal restriction map, confirming that the virulence-associated locus, designated vacB, is a new locus on the chromosome. In the vacB mutant, production of the four plasmid-encoded virulence antigens, IpaB, -C, and -D and VirG, decreased to a low level compared with that in the wild type. In contrast, levels of transcription of the operons for virG, ipa, region-3.4, region-5, virF, and virB on the large plasmid, as determined by Northern dot blotting, were unaffected in the vacB mutant. Furthermore, transcriptional activation of the ipa operon by exploiting a tac promoter could not restore the vacB mutant to production of the same levels of the IpaB, -C, and -D proteins as those in the wild type, indicating that the vacB locus is involved in expression of the vir genes on the large plasmid at the posttranscriptional level. Cloning followed by nucleotide sequencing of the vacB region showed it to contain a 2,280-bp open reading frame encoding an 86.9-kDa protein located 669 bp downstream from the 3' end of the open reading frame for the purA gene. Disruption of the vacB gene of other serotypes of Shigella spp. and enteroinvasive Escherichia coli (EIEC) resulted in reduced expression of virulence phenotypes, indicating that the vacB gene encodes a novel type of virulence-associated gene required for the full expression of the virulence phenotype of Shigella spp. and EIEC.
Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8
Authors: Fernández-Abalos J M , Sánchez P , Coll P M , Villanueva J R , Pérez P , Santamaría R I
Published: November 2016

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.
Reevaluation of envelope profiles and cytoplasmic ultrastructure of mycobacteria processed by conventional embedding and freeze-substitution protocols
Authors: Paul T R , Beveridge T J
Published: November 2016

The cell envelope architectures and cytoplasmic structures of Mycobacterium aurum CIPT 1210005, M. fortuitum, M. phlei 425, and M. thermoresistible ATCC 19527 were compared by conventional embedding and freeze-substitution methods. To ascertain the integrity of cells during each stage of the processing regimens, [1-14C]acetate was incorporated into the mycolic acids of mycobacterial walls, and the extraction of labeled mycolic acids was monitored by liquid scintillation counting. Radiolabeled mycolic acids were extracted by both processing methods; however, freeze-substitution resulted in the extraction of markedly less radiolabel. During conventional processing of cells, most of the radiolabel was extracted during the dehydration stage, whereas postsubstitution washes in acetone yielded the greatest loss of radiolabel during freeze-substitution. Conventional embedding frequently produced cells with condensed fibrous nucleoids and occasional mesosomes. Their cell walls were relatively thick (approximately 25 nm) but lacked substance. Freeze-substituted cells appeared more robust, with well-dispersed nucleoids and ribosomes. The walls of all species were much thinner than those of their conventionally processed counterparts, but these stained well, which was an indication of more wall substance; the fabric of these walls, in particular the plasma membrane, appeared highly condensed and tightly apposed to the peptidoglycan. Some species possessed a thick, irregular outer layer that was readily visualized in the absence of exogenous stabilizing agents by freeze-substitution. Since freeze-substituted mycobacteria retained a greater percentage of mycolic acids in their walls, and probably other labile wall and cytoplasmic constituents, we believe that freeze-substitution provides a more accurate image of structural organization in mycobacteria than that achieved by conventional procedures.
Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403
Authors: Le Bourgeois P , Lautier M , Mata M , Ritzenthaler P
Published: November 2016

A combined physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403 was determined. We constructed a restriction map for the NotI, ApaI, and SmaI enzymes. The order of the restriction fragments was determined by using the randomly integrative plasmid pRL1 and by performing indirect end-labeling experiments. The strain IL1403 chromosome was found to be circular and 2,420 kb in size. A total of 24 chromosomal markers were mapped on the chromosome by performing hybridization experiments with gene probes for L. lactis and various other bacteria. Integration of pRC1-derived plasmids via homologous recombination allowed more precise location of some lactococcal genes and allowed us to determine the orientation of these genes on the chromosome. Recurrent sequences, such as insertion elements and rRNA gene (rrn) clusters, were also mapped. At least seven copies of IS1076 were present and were located on 50% of the chromosome. In contrast, no copy of ISS1RS was detected. Six ribosomal operons were found on the strain IL1403 chromosome; five were located on 16% of the chromosome and were transcribed in the same direction. A comparison of the physical maps of L. lactis subsp. lactis IL1403 and DL11 showed that these two strains are closely related and that the variable regions are located mainly near the rrn gene clusters. In contrast, despite major restriction pattern dissimilarities between L. lactis IL1403 and MG1363, the overall genetic organization of the genome seems to be conserved between these two strains.
DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III
Authors: Lifsics M R , Lancy E D , Maurer R
Published: November 2016

In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.
The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators
Authors: Higgins D E , Nazareno E , DiRita V J
Published: November 2016

Virulence gene expression in Vibrio cholerae is postulated to involve ToxR-dependent activation of the toxT gene followed by ToxT activation of virulence genes, including several of those involved in biogenesis of the toxin-coregulated pilus. ToxR is a transmembrane, DNA-binding protein which is a member of the OmpR subclass of two-component activator systems in bacteria. Data presented in this report demonstrate that ToxT is similar to the AraC family of transcriptional activators identified in a variety of gram-negative bacteria. The toxT open reading frame begins approximately 200 nucleotides from the end of the tcpF gene, which is part of a cluster of genes responsible for production of the toxin-coregulated pilus. Accumulation of toxT specific mRNA is ToxR dependent and is modulated by environmental conditions that modulate expression of the regulon. Within the intergenic region between tcpF and toxT is a potential stem-loop structure of an unusual nature which may play a role in regulating expression of toxT mRNA. Experiments with tcpF and toxT cloned behind a strong, constitutive promoter suggest that the two genes can be cotranscribed, but Northern (RNA) blot analysis of V. cholerae suggests that if they are, steady-state levels of their messages may be controlled by a posttranscriptional mechanism. Possible mechanisms for ToxR-dependent expression of toxT are discussed.
Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization
Authors: Geber A , Williamson P R , Rex J H , Sweeney E C , Bennett J E
Published: November 2016

In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast.
Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein
Authors: Chang C H , Winans S C
Published: November 2016

VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.
A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp
Authors: Ishizuka H , Horinouchi S , Kieser H M , Hopwood D A , Beppu T
Published: November 2016

A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.
Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis
Authors: Shrimankar P , Stordal L , Maurer R
Published: November 2016

The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination.
Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12
Authors: Mandal N , RajBhandary U L
Published: November 2016

We show that the metY locus which specifies tRNA(2fMet) in Escherichia coli K-12 specifies tRNA(1fMet) in E. coli B. This conclusion is based on results of Southern blot analysis of E. coli B and K-12 DNAs and on polymerase chain reaction amplification, cloning, and sequencing of an approximately 200-bp region of DNA corresponding to the metY loci of E. coli B and E. coli K-12. We also show that the metY locus of E. coli B is transcriptionally active. E. coli strains transformed with the multicopy plasmid vector pUC19 carrying the metY locus of E. coli B overproduce tRNA(1fMet) in E. coli B and E. coli K-12 in contrast to strains transformed with pUC19 carrying the corresponding locus from E. coli K-12, which overproduce tRNA(2fMet).
The carboxy-terminal 14 amino acids of phage lambda N protein are dispensable for transcription antitermination
Authors: Franklin N C
Published: November 2016

The analogous N proteins encoded by lambdoid bacteriophages lambda, 21, and 22 are very different in amino acid sequence, except at their carboxy-terminal ends. Since N lambda remains functional despite the deletion of most of its terminal region of homology to N21, that region of homology cannot represent a region of conserved function.
Nonrandom F-plasmid replication in Escherichia coli K-12
Authors: Koppes L J
Published: November 2016

Both the autonomous and chromosomally integrated F plasmids were found to replicate in a nonrandom fashion after a density transfer from heavy medium ([13C]glucose, 15NH4) to light medium ([12C]glucose, 14NH4). The data are consistent with the hypothesis that both the chromosome and the F plasmid are replicated in a cell cycle-specific manner. Thus, these data support the proposal (J. D. Keasling, B. O. Palsson, and S. Cooper, J. Bacteriol. 173:2673-2680, 1991) that plasmids replicate in a cell cycle-specific manner.
Fan-shaped ejections of regularly arranged murosomes involved in penicillin-induced death of staphylococci
Authors: Giesbrecht P , Kersten T , Wecke J
Published: November 2016

Electron microscopic research into the murosomes of staphylococci has shown that the number of murosomes involved in penicillin-induced death varies depending on the experimental conditions employed. With 0.1 micrograms of penicillin G per ml, only 1 of a total of about 20 murosomes, the "killing murosome," completely perforated the pressure-stabilized peripheral cell wall during a three-step process. This strictly localized event was mainly attributed to a mechanical effect being comparable to the process of aneurysm formation. Wall perforation was also considered to mark the very moment of penicillin-induced death ("nonlytic killing event"), while bacteriolysis started only postmortem. By varying the osmolarity of the growth medium, the number of murosomes involved in penicillin-induced killing increased considerably, which resulted in the ejection of a fan-shaped row of murosomes at the second division plane. These data are compatible with the finding that, in untreated or chloramphenicol-treated staphylococci, the activation of the murosomes resulted in (i) the formation of regularly arranged "blebs" on the cell surface, containing traces of disintegrated wall material, and (ii) the subsequent liberation of the murosomes lying underneath, leaving behind their former sites in the peripheral wall as a row of regularly arranged "pores" in every division plane. The number, distribution, and positioning of these blebs corresponded with those of the pores and the original murosomes. The significance of wall autolysins liberated from the first division plane for penicillin-induced wall perforation at the second division plane is discussed.
A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation
Authors: Bobik T A , Ailion M , Roth J R
Published: November 2016

The cob operon of Salmonella typhimurium encodes enzymes required for synthesis of adenosyl-cobalamin (vitamin B12). The pdu operon encodes enzymes needed for use of propanediol as a carbon source, including an adenosyl-cobalamin-dependent enzyme, propanediol dehydratase. These two operons both map near min 41 of the S. typhimurium linkage map and are transcribed divergently. Here we report that the cob and pdu operons form a single regulon. Transcription of this regulon is induced by either glycerol or propanediol. The metabolism of these compounds is not required for induction. Propanediol induces the regulon either aerobically or anaerobically during growth on poor carbon sources. Aerobically glycerol induces only if its metabolism is prevented by a mutational block such as a glpK mutation. Under anaerobic conditions, glycerol induces in both glpK+ and glpK mutant strains during growth on poor carbon sources. A new class of mutations, pocR, prevents induction of the cob/pdu regulon by either propanediol or glycerol and causes a Cob- Pdu- phenotype. The pocR gene is located between the cob and pdu operons and appears to encode a trans-acting protein that acts as a positive regulator of both operons. Transcription of the pocR regulatory gene is induced, even without the PocR protein, during aerobic growth on poor carbon sources and during anaerobic respiration. With the functional PocR protein, transcription of the pocR gene is autoinduced by propanediol but not by glycerol. The growth conditions that increase pocR gene expression correlate with growth conditions that allow high induction of the cob/pdu regulon. A model for control of this regulon suggests that the PocR protein is a transcriptional activator of both the cob and pdu operons and that both glycerol and propanediol can individually serve as effectors of the PocR protein. We suggest that global control mechanisms cause variation in the level of the PocR protein; an increased level of the PocR protein permits higher induction by propanediol or glycerol.
The poc locus is required for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium
Authors: Rondon M R , Escalante-Semerena J C
Published: November 2016

In this communication we present evidence that indicates that 1,2-propanediol (1,2-PDL) is a positive effector of the transcription of the cobalamin (vitamin B12) biosynthetic (cob) operon and of the 1,2-PDL utilization (pdu) genes. The stimulatory effects of 1,2-PDL were demonstrated using Mu d-lac transcriptional fusions to cob and pdu. Significantly increased levels of transcription of the cob and pdu operon fusions were measured in cultures grown under both anoxic and highly aerated conditions when 1,2-PDL was present in the culture medium. We have isolated mutants that carry lesions that render both pdu and cob transcription unresponsive to 1,2-PDL. These mutations were mapped to the region between cob and pdu (41 min), and they define the poc locus PDL and cobalamin). The poc locus is required for the positive regulatory effects of 1,2-PDL to be exerted. Complementation of Poc function was achieved in trans by an F' plasmid carrying the poc+ locus, suggesting that at least one diffusible product regulates the expression of cob and pdu in S. typhimurium.
Structural and biochemical characterization of the Escherichia coli argE gene product
Authors: Meinnel T , Schmitt E , Mechulam Y , Blanquet S
Published: November 2016

The DNA sequence of a 2,100-bp region containing the argE gene from Escherichia coli has been determined. The nucleotide sequence of the ppc-argE intergenic region was also solved and shown to contain six tandemly repeated REP sequences. Moreover, the oxyR gene has been mapped on the E. coli chromosome and shown to flank the arg operon. The codon responsible for the translation start of argE was determined by using site-directed mutants. This gene spans 1,400 bp and encodes a 42,350-Da polypeptide. The argE3 allele and a widely used argE amber gene have also been cloned and sequenced. N-Acetylornithinase, the argE product, has been overproduced and purified to homogeneity. Its main biochemical and catalytic properties are described. Moreover, we demonstrate that the protein is composed of two identical subunits. Finally, the amino acid sequence of N-acetylornithinase is shown to display a high degree of identity with those of the succinyldiaminopimelate desuccinylase from E. coli and carboxypeptidase G2 from a Pseudomonas sp. It is proposed that this carboxypeptidase might be responsible for the acetylornithinase-related activity found in the Pseudomonas sp.