bluuebunny/crossref_metadata_2025 huggingface dataset:
Genes required for cellulose synthesis in Agrobacterium tumefaciens
Authors: Matthysse A G , White S , Lightfoot R
Published: November 2016

A region of the chromosome of Agrobacterium tumefaciens 11 kb long containing two operons required for cellulose synthesis and a part of a gene homologous to the fixR gene of Bradyrhizobium japonicum has been sequenced. One of the cellulose synthesis operons contained a gene (celA) homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum. The same operon also contained a gene (celC) homologous to endoglucanase genes from A. xylinum, Cellulomonas uda, and Erwinia chrysanthemi. The middle gene of this operon (celB) and both the genes of the other operon required for cellulose synthesis (celDE) showed no significant homology to genes contained in the databases. Transposon insertions showed that at least the last gene of each of these operons (celC and celE) was required for cellulose synthesis in A. tumefaciens.
Mechanism of cellulose synthesis in Agrobacterium tumefaciens
Authors: Matthysse A G , Thomas D L , White A R
Published: November 2016

Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.
Thermoregulation of virB transcription in Shigella flexneri by sensing of changes in local DNA superhelicity
Authors: Tobe T , Yoshikawa M , Sasakawa C
Published: November 2016

Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of Shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase-dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription. Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function. The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.
Architecture of the cell envelope of Chlamydia psittaci 6BC
Authors: Everett K D , Hatch T P
Published: November 2016

The cysteine-rich envelope proteins of the elementary body form of chlamydiae are thought to be located in the outer membrane on the basis of their insolubility in the weak anionic detergent N-lauryl sarcosinate (Sarkosyl). We found, however, that the insolubility of the small (EnvA) and the large (EnvB) cysteine-rich proteins of Chlamydia psittaci 6BC in Sarkosyl is dependent on the maintenance of a supramolecular disulfide-cross-linked complex and is unlikely to be a valid indicator of outer membrane location. Consequently, we used other methods to characterize the architecture of the cell envelope of C. psittaci 6BC. We found that disulfide-reduced EnvA, previously shown to be a lipoprotein, segregated into the detergent phase during Triton X-114 partitioning experiments and was recovered from the membrane fraction of elementary bodies lysed by nondetergent means. In contrast, disulfide-reduced EnvB segregated to the aqueous phase in partitioning experiments and was found in the soluble fraction of elementary bodies lysed in the absence of detergents. The hydrophobic affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine labeled the major outer membrane protein and EnvA but did not label EnvB. Treatment of intact elementary bodies of C. psittaci with trypsin had no effect on the cysteine-rich proteins, although the major outer membrane protein was partially degraded. On the basis of these and other observations, we propose that EnvA is anchored to the outer membrane by its lipid moiety, with a hydrophilic peptide portion extending into the periplasm, and that EnvB is located exclusively within the periplasm. We further propose that disulfide-cross-linked polymers of EnvB are the functional equivalent of peptidoglycan, forming a disulfide-cross-linked network with the periplasmic domains of EnvA and other membrane proteins, which accounts for the osmotic stability of elementary bodies.
Activation of the dephosphorylation of nitrogen regulator I-phosphate of Escherichia coli
Authors: Liu J , Magasanik B
Published: November 2016

The transcription of sigma 54 RNA polymerase-dependent nitrogen-regulated genes is activated by nitrogen regulator I (NRI)-phosphate. The kinase NRII is responsible for the phosphorylation of NRI. It has been shown that NRII also has the ability to dephosphorylate NRI-phosphate but only when PII is present at a concentration greatly in excess of that of NRII. We have now shown that glutamate enables PII to stimulate the dephosphorylation of NRI-phosphate when present in equimolar concentration with NRII. This effect of glutamate appears to be a backup control that becomes effective when the normal regulation of PII activity is disabled.
Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa
Authors: Masduki A , Nakamura J , Ohga T , Umezaki R , Kato J , Ohtake H
Published: November 2016

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computer-assisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.
Discrete amplifiable regions (amplicons) in the symbiotic plasmid of Rhizobium etli CFN42
Authors: Romero D , Martínez-Salazar J , Girard L , Brom S , Dávilla G , Palacios R , Flores M , Rodríguez C
Published: November 2016

Frequent tandem amplification of defined regions of the genome, called amplicons, is a common characteristic in the genomes of some Rhizobium species, such as Rhizobium etli. In order to map these zones in a model Rhizobium replicon, we undertook an analysis of the plasticity patterns fostered by amplicons in the pSym (390 kb) of R. etli CFN42. Data presented in this article indicate the presence of four amplicons in pSym, used for the generation of tandem amplifications and deletions. The amplicons are large, ranging from 90 to 175 kb, and they are overlapping. Each amplicon is usually flanked by specific reiterated sequences. Formation of amplifications and deletions requires an active recA gene. All the amplicons detected are concentrated in a zone of roughly one-third of pSym, covering most of the symbiotic genes detected in this plasmid. No amplicons were detected in the remaining two-thirds of pSym. These data support the idea that most of the known symbiotic genes in this plasmid are located in a genomic region that is prone to the formation of frequent tandem amplification.
Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution
Authors: Ruepp A , Müller H N , Lottspeich F , Soppa J
Published: November 2016

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.
Adaptation of Xanthobacter autotrophicus GJ10 to bromoacetate due to activation and mobilization of the haloacetate dehalogenase gene by insertion element IS1247
Authors: van der Ploeg J , Willemsen M , van Hall G , Janssen D B
Published: November 2016

Monobromoacetate (MBA) is toxic for the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 at concentrations higher than 5 mM. Mutants which are able to grow on higher concentrations of MBA were isolated and found to overexpress haloacid dehalogenase, which is encoded by the dhlB gene. In mutant GJ10M50, a DNA fragment (designated IS1247) had copied itself from a position on the chromosome that was not linked to the dhlB region to a site immediately upstream of dhlB, resulting in a 1,672-bp insertion. IS1247 was found to encode an open reading frame corresponding to 464 amino acids which showed similarity to putative transposases from two other insertion elements. In most of the other MBA-resistant mutants of GJ10, IS1247 was also present in one more copy than in the wild type, which had two copies located within 20 kb. After insertion to a site proximal to dhlB, IS1247 was able to transpose itself together with the dhlB gene to a plasmid, without the requirement of a second insertion element being present downstream of dhlB. The results show that IS1247 causes bromoacetate resistance by overexpression and mobilization of the haloacid dehalogenase gene, which mimics steps during the evolution of a catabolic transposon and plasmid during adaptation to a toxic growth substrate.
The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis
Authors: Ogawa K , Akagawa E , Yamane K , Sun Z W , LaCelle M , Zuber P , Nakano M M
Published: November 2016

Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase.
The S-layer from Bacillus stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase
Authors: Egelseer E , Schocher I , Sára M , Sleytr U B
Published: November 2016

The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.
Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus
Authors: Sugai M , Komatsuzawa H , Akiyama T , Hong Y M , Oshida T , Miyake Y , Yamaguchi T , Suginaka H
Published: November 2016

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.
Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation
Authors: Buist G , Kok J , Leenhouts K J , Dabrowska M , Venema G , Haandrikman A J
Published: November 2016

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.
Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus
Authors: McCarter L L
Published: November 2016

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.
A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity
Authors: Hou S Y , Chao Y P , Liao J C
Published: November 2016

The phosphoenolpyruvate carboxykinase in Escherichia coli (encoded by pck) catalyzes the conversion from oxaloacetate (OAA) to phosphoenolpyruvate under gluconeogenic conditions. We report here the characterization of two mutant alleles, pck-51 and pck-53, both of which are point mutations leading to single amino acid changes (D to N at position 268 and G to S at position 284, respectively). Pck51 is an altered-activity mutant that catalyzes the conversion from OAA to pyruvate (OAA decarboxylase activity). This new activity was not detected from the wild-type Pck, and it complements the pck null mutation only in a pps+ background. Pck53 is a reduced-activity mutant that complements the pck null mutation in a strain-dependent fashion.
The amino acid sequence of Lrp is highly conserved in four enteric microorganisms
Authors: Friedberg D , Platko J V , Tyler B , Calvo J M
Published: November 2016

Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.
Genetic evidence that PpsR from Rhodobacter sphaeroides 2.4.1 functions as a repressor of puc and bchF expression
Authors: Gomelsky M , Kaplan S
Published: November 2016

The ppsR gene (R. J. Penfold and J. M. Pemberton, J. Bacteriol. 176:2869-2876, 1994) from Rhodobacter sphaeroides 2.4.1 functions as a transcriptional repressor of puc and bchF expression. The carboxy terminus of PpsR, containing the putative DNA-binding domain, by itself possesses repressor activity. Intact palindromes having the motif TGT-N12-ACA are required for PpsR activity.
Modulation of the allosteric equilibrium of yeast chorismate mutase by variation of a single amino acid residue
Authors: Graf R , Dubaquié Y , Braus G H
Published: November 2016

Chorismate mutase (EC 5.4.99.5) from the yeast Saccharomyces cerevisiae is an allosteric enzyme which can be locked in its active R (relaxed) state by a single threonine-to-isoleucine exchange at position 226. Seven new replacements of residue 226 reveal that this position is able to direct the enzyme's allosteric equilibrium, without interfering with the catalytic constant or the affinity for the activator.
Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy
Authors: Stephens C M , Zweiger G , Shapiro L
Published: November 2016

The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3' to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle.
Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading
Authors: Fukuda I , Suzuki T , Munakata H , Hayashi N , Katayama E , Yoshikawa M , Sasakawa C
Published: November 2016

The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigella flexneri is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that VirG is located at one pole on the surface of the bacterium and secreted into the culture supernatant and that in host cells it is localized along the length of the F-actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface-exposed portion of the VirG protein by limited trypsin digestion of S. flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that the 85-kDa amino-terminal portion of VirG is located on the external side of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it. The VirG cleavage required for release of the 85-kDa protein into the culture supernatant occurred at the Arg-Arg bond at positions 758 to 759. VirG-specific cleavage was observed in Shigella species and enteroinvasive Escherichia coli, which requires an as yet unidentified protease activity governed by the virB gene on the large plasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG function in bacterial spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were capable of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, these mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positive Sereny test. Therefore, the ability to cleave and secrete VirG in Shigella species is not a prerequisite for intracellular spreading.
Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase
Authors: Morris V L , Jackson D P , Grattan M , Ainsworth T , Cuppels D A
Published: November 2016

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant.
Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression
Authors: Barrios H , Fischer H M , Hennecke H , Morett E
Published: November 2016

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.
Physical map of the Treponema denticola circular chromosome
Authors: MacDougall J , Saint Girons I
Published: November 2016

A physical map of the Treponema denticola ATCC 33520 genome was constructed by pulsed-field gel electrophoresis and DNA hybridization. The organism possesses a single, circular chromosome of approximately 3.0 Mbp and a 2.6-kbp circular plasmid, pTD1. The physical map of the A+T-rich genome was constructed with the rare-cutting restriction enzymes AscI, NotI, and SrfI, which have 8-bp G+C-rich recognition sites. The genes flgE, tdpA, and prtB encoding the flagellar hook protein, a 53-kDa immunogenic protein, and chymotrypsinlike protease, respectively, were located on the map. This treponeme was found to have two copies of each of the rRNA genes, as has been found to be the case for both Treponema phagedenis and Treponema pallidum.
The Alcaligenes eutrophus protein HoxN mediates nickel transport in Escherichia coli
Authors: Wolfram L , Friedrich B , Eitinger T
Published: November 2016

HoxN, an integral membrane protein with seven transmembrane helices and a molecular mass of 33.1 kDa, is involved in high-affinity nickel transport in Alcaligenes eutrophus H16. From genetic analyses, it has been concluded that HoxN is a single-component ion carrier. To investigate this assumption, hoxN was introduced into Escherichia coli. The recombinant strain showed significantly enhanced nickel uptake in a short-interval assay. Likewise, growth in the presence of 63NiCl2 yielded a more than 15-fold-increased cellular nickel content. The HoxN-based nickel transport activity could also be demonstrated in a physiological assay: an E. coli strain coexpressing hoxN and the urease operon of Klebsiella aerogenes exhibited urease activity 10-fold greater than that in the strain lacking a functional hoxN. These results strongly suggest that HoxN is sufficient to operate as a nickel permease. Multiple sequence alignment of HoxN and four other bacterial membrane proteins implicated in nickel metabolism revealed two conserved signatures which may play a role in the nickel translocation process.
In vivo definition of an archaeal promoter
Authors: Palmer J R , Daniels C J
Published: November 2016

We have used a plasmid-based transcriptional reporter system to examine the transcriptional effects of 33 single point mutations in the box A region (TATA-like sequence) of the Haloferax volcanii tRNA(Lys) promoter. The most pronounced effects on transcriptional efficiency were found when the nucleotides corresponding to the TATA-like region were altered. Promoters with wild-type or higher levels of transcriptional activity conformed to the general archaeal box A consensus, 5'-T/CTTAT/AA-3'. The preference for a pyrimidine residue in the 5' position of this region and the exclusion of guanine and cytosine in the next four positions in the 3' direction are defining characteristics shared by all efficient archaeal promoters. We have also observed that replacement of a 10-nucleotide purine-rich sequence, located 5' of the H. volcanii tRNA(Lys) box A element, completely abolished transcription from this promoter. These data show that the H. volcanii tRNA(Lys) promoter is dependent on two separate, and essential, sequence elements. The possible functions of these sequences, in view of the recent descriptions of eucaryal-like transcription factors for Archaea, are discussed.
Differences between inner membrane and peptidoglycan-associated PBP1B dimers of Escherichia coli
Authors: Zijderveld C A , Aarsman M E , Nanninga N
Published: November 2016

Earlier studies revealed that PBP1B of Escherichia coli occurred as a monomeric as well as a dimeric form (C.A.L. Zijderveld, M.E.G. Aarsman, T. den Blaauwen, and N. Nanninga, J. Bacteriol. 173:5740-5746, 1991). In this study, the dimer of PBP1B was further analyzed. It appeared that the dimeric form could be divided into two classes. One class, which cofractionated with the cell wall fraction, could be artificially cross-linked to peptidoglycan, indicating a close association with the latter. This class of PBP1B dimers was sensitive to beta-mercaptoethanol. The second class, like the monomeric form of PBP1B, could be isolated with the inner membrane fraction. This dimeric form dissociated in the presence of zinc in combination with beta-mercaptoethanol.
The umpA gene of Escherichia coli encodes phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (lgt) and regulates thymidylate synthase levels through translational coupling
Authors: Gan K , Sankaran K , Williams M G , Aldea M , Rudd K E , Kushner S R , Wu H C
Published: November 2016

Using a combination of biochemical, physical, and genetic techniques, we have shown that the umpA gene of Escherichia coli is allelic with the lgt (phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) of Salmonella typhimurium. These genes are essential for the viability of the respective organism and exhibit 92.8% sequence identity at the amino acid level. In E. coli, lgt and thyA (thymidylate synthase) form an operon. Thymidylate synthase levels are regulated by transcription from the lgt promoter and by translational coupling.
Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene
Authors: Andersen P S , Frees D , Fast R , Mygind B
Published: November 2016

Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids.
The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development
Authors: Plamann L , Li Y , Cantwell B , Mayor J
Published: November 2016

The Myxococcus xanthus asgA gene is one of three known genes necessary for the production of extracellular A-signal, a cell density signal required early in fruiting body development. We determined the DNA sequence of asgA. The deduced 385-amino-acid sequence of AsgA was found to contain two domains: one homologous to the receiver domain of response regulators and the other homologous to the transmitter domain of histidine protein kinases. A kanamycin resistance (Kmr) gene was inserted at various positions within or near the asgA gene to determine the null phenotype. Those strains with the Kmr gene inserted upstream or downstream of asgA are able to form fruiting bodies, while strains containing the Kmr gene inserted within asgA fail to develop. The nature and location of the asgA476 mutation were determined. This mutation causes a leucine-to-proline substitution within a conserved stretch of hydrophobic residues in the N-terminal receiver domain. Cells containing the insertion within asgA and cells containing the asgA476 substitution have similar phenotypes with respect to development, colony color, and expression of an asg-dependent gene. An analysis of expression of a translational asgA-lacZ fusion confirms that asgA is expressed during growth and early development. Finally, we propose that AsgA functions within a signal transduction pathway that is required to sense starvation and to respond with the production of extracellular A-signal.
Sequence, regulation, and functions of fis in Salmonella typhimurium
Authors: Osuna R , Lienau D , Hughes K T , Johnson R C
Published: November 2016

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.
Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Forchhammer K , Tandeau de Marsac N
Published: November 2016

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.
An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria
Authors: Diorio C , Cai J , Marmor J , Shinder R , DuBow M S
Published: November 2016

Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.
Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria
Authors: Liu Y , Golden S S , Kondo T , Ishiura M , Johnson C H
Published: November 2016

To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium.
Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium
Authors: Bäumler A J , Heffron F
Published: November 2016

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.