bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane
Authors: Braun V , Killmann H , Herrmann C
Published: November 2016

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.
Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes
Authors: Hsu D , Shih L M , Zee Y C
Published: November 2016

We have previously shown that the 23S rRNA of Salmonella strains is highly fragmented by specific enzyme cleavages. In this article, we report that 23S rRNA of Salmonella strains is rapidly degraded as the cells enter the stationary phase. More than 90% of the 23S rRNA is degraded when the cells reach the stationary phase. The rate of degradation of 23S rRNA correlated with its degree of fragmentation. This degradation is probably mediated by newly synthesized protein factor(s), since treatment with chloramphenicol or rifampin inhibits the rRNA degradation. We propose that degradation of 23S rRNA is a novel mechanism in the regulation of the bacterial 23S rRNA and ribosome concentration and that this additional regulatory mechanism provides some selective advantage to cells.
Cloning, expression, and molecular characterization of the gene encoding an extremely thermostable [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus
Authors: Heltzel A , Smith E T , Zhou Z H , Blamey J M , Adams M W
Published: November 2016

The gene for ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, sequenced, and expressed in Escherichia coli. The coding region confirmed the determined amino acid sequence. Putative archaeon-type transcriptional regulatory elements were identified. The fdxA gene appears to be an independent transcriptional unit. Recombinant ferredoxin was indistinguishable from the protein purified from P. furiosus in its thermal stability and in the potentiometric and spectroscopic properties of its [4Fe-4S] cluster.
Cloning and characterization of an autonomous replication sequence from Coxiella burnetii
Authors: Suhan M , Chen S Y , Thompson H A , Hoover T A , Hill A , Williams J C
Published: November 2016

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.
Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products
Authors: Darie S , Gunsalus R P
Published: November 2016

While many organisms synthesize delta-aminolevulinate, the precursor of heme, by condensing succinyl-coenzyme A and glycine, others use a glutamate-dependent pathway in which glutamyl-tRNA dehydrogenase catalyzes the rate-determining step. The hemeA gene that encodes this latter enzyme in Escherichia coli has been cloned and sequenced. To examine how its expression is regulated, we constructed hemA-lacZ operon and gene fusions and inserted them into the chromosome in single copy. The effect of aerobic and anaerobic growth conditions and the availability of electron acceptors and various carbon substrates were documented. Use of different types of cell culture medium resulted in a fivefold variation in hemA-lacZ expression during aerobic cell growth. Anaerobic growth resulted in 2.5-fold-higher hemA-lacZ expression than aerobic growth. This control is mediated by the fnr and arcA gene products. Fnr functions as a repressor of hemA transcription during anaerobic cell growth only, whereas the arcA gene product activates hemA gene expression under both aerobic and anaerobic conditions. Integration host factor protein was also shown to be required for control of hemA gene regulation. To determine whether an intermediate or a product of the heme biosynthetic pathway is involved in hemA regulation, hemA-lacZ expression was analyzed in a hemA mutant. Expression was elevated by 20-fold compared with that in a wild-type strain, while the addition of the heme pathway intermediate delta-aminolevulinate to the culture medium restored expression to wild-type levels. These results suggest that the heme pathway is feedback regulated at the level of hemA gene expression, to supply heme as it is required during different modes of cell growth.
Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits
Authors: Brito B , Palacios J M , Hidalgo E , Imperial J , Ruiz-Argüeso T
Published: November 2016

Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.
Use of a phase variation-specific promoter of Myxococcus xanthus in a strategy for isolating a phase-locked mutant
Authors: Laue B E , Gill R E
Published: November 2016

The bacterium Myxococcus xanthus alternates between two colony types distinguished by colony morphology and pigmentation. Because the two phases are interconvertible, this phenomenon has been termed phase variation. In one phase, the colonies are bright yellow, rough, and swarming. In the alternate phase, the colonies are tan and mucoid with smooth edges. During exponential vegetative growth, the populations within a colony reach an equilibrium of approximately 99% yellow and 1% tan cells. Neither the biological function nor the genetic mechanism of phase variation is currently understood. To investigate phase variation, a yellow-phase-specific promoter was identified by Tn5lac mutagenesis. A tan-phase-locked mutant was isolated by a strategy, described in this study, which involved introducing a selectable marker expressed under phase-regulated expression. This was accomplished by a fusion of the cloned yellow-phase-specific promoter to a promoterless kanamycin resistance gene. The defect in the phase-locked mutant, given the designation var-683, caused the rate of switching from the tan to yellow phase to be reduced by at least 10(3)-fold below the wild-type rate of switching. This strain will provide a stable tan population for genetic and biological analysis. Evidence is presented for the existence of a transcriptional regulator which controls the expression of phase-regulated promoters.
Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline
Authors: Boch J , Kempf B , Bremer E
Published: November 2016

Exogenously provided glycine betaine functions as an efficient osmoprotectant for Bacillus subtilis in high-osmolarity environments. This gram-positive soil organism is not able to increase the intracellular level of glycine betaine through de novo synthesis in defined medium (A. M. Whatmore, J. A. Chudek, and R. H. Reed, J. Gen. Microbiol. 136:2527-2535, 1990). We found, however, that B. subtilis can synthesize glycine betaine when its biosynthetic precursor, choline, is present in the growth medium. Uptake studies with radiolabelled [methyl-14C]choline demonstrated that choline transport is osmotically controlled and is mediated by a high-affinity uptake system. Choline transport of cells grown in low- and high-osmolarity media showed Michaelis-Menten kinetics with Km values of 3 and 5 microM and maximum rates of transport (Vmax) of 10 and 36 nmol min-1 mg of protein-1, respectively. The choline transporter exhibited considerable substrate specificity, and the results of competition experiments suggest that the fully methylated quaternary ammonium group is a key feature for substrate recognition. Thin-layer chromatography revealed that the radioactivity from exogenously provided [methyl-14C]choline accumulated intracellularly as [methyl-14C]glycine betaine, demonstrating that B. subtilis possesses enzymes for the oxidative conversion of choline into glycine betaine. Exogenously provided choline significantly increased the growth rate of B. subtilis in high-osmolarity media and permitted its proliferation under conditions that are otherwise strongly inhibitory for its growth. Choline and glycine betaine were not used as sole sources of carbon or nitrogen, consistent with their functional role in the process of adaptation of B. subtilis to high-osmolarity stress.
Dominant negative mutator mutations in the mutS gene of Escherichia coli
Authors: Wu T H , Marinus M G
Published: November 2016

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.
A glucose-activated electron transfer system in the plasma membrane stimulates the H(+)-ATPase in Penicillium cyclopium
Authors: Pönitz J , Roos W
Published: November 2016

Hyphal cells of three fungal species of the genus Penicillium reduced the nonpermeable, external electron acceptor hexabromoiridate IV (HBI IV). In Penicillium cyclopium, the rate of HBI IV reduction by hyphal cells was drastically increased by the addition of beta-glucose. The stimulation showed high specificity for this sugar and did not require its uptake and cellular metabolism. Cell wall oxidases (e.g., glucose oxidase) did not seem to be involved in the reduction of HBI IV, as no measurable H2O2 was formed from added glucose and removal of oxygen had no effect. We propose that there is a glucose-binding component outside the plasma membrane which controls transmembrane electron fluxes in response to external glucose. Reduction of HBI IV was accompanied by rapid acidification of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified with an e-/H+ stoichiometry of > 1. In plasma membrane vesicles containing endogenous electron donors, the membrane-residing fluoroprobe Di-8-ANEPPS reported a transient depolarization of the membrane potential triggered by the external electron acceptor. Inhibitors of ATP-dependent proton pumping enhanced the extent of this depolarization, inhibited the subsequent normalization of membrane potential, and, in whole cells, reduced the amount of redox-triggered proton extrusion. From these and other findings, it is concluded that the observed trans-plasma membrane redox process activates the H(+)-ATPase via membrane depolarization and cytosolic acidification.
Membrane topology of Escherichia coli diacylglycerol kinase
Authors: Smith R L , O'Toole J F , Maguire M E , Sanders C R
Published: November 2016

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.
The control region of the pdu/cob regulon in Salmonella typhimurium
Authors: Chen P , Andersson D I , Roth J R
Published: November 2016

The pdu operon encodes proteins for the catabolism of 1,2-propanediol; the nearby cob operon encodes enzymes for the biosynthesis of adenosyl-cobalamin (vitamin B12), a cofactor required for the use of propanediol. These operons are transcribed divergently from distinct promoters separated by several kilobases. The regulation of the two operons is tightly integrated in that both require the positive activator protein PocR and both are subject to global control by the Crp and ArcA proteins. We have determined the DNA nucleotide sequences of the promoter-proximal portion of the pdu operon and the region between the pdu and cob operons. Four open reading frames have been identified, pduB, pduA, pduF, and pocR. The pduA and pduB genes are the first two genes of the pdu operon (transcribed clockwise). The pduA gene encodes a hydrophobic protein with 56% amino acid identity to a 10.9-kDa protein which serves as a component of the carboxysomes of several photosynthetic bacteria. The pduF gene encodes a hydrophobic protein with a strong similarity to the GlpF protein of Escherichia coli, which facilitates the diffusion of glycerol. The N-terminal end of the PduF protein includes a motif for a membrane lipoprotein-lipid attachment site as well as a motif characteristic of the MIP (major intrinsic protein) family of transmembrane channel proteins. We presume that the PduF protein facilitates the diffusion of propanediol. The pocR gene encodes the positive regulatory protein of the cob and pdu operons and shares the helix-turn-helix DNA binding motif of the AraC family of regulatory proteins. The mutations cobR4 and cobR58 cause constitutive, pocR-independent expression of the cob operon under both aerobic and anaerobic conditions. Evidence that each mutation is a deletion creating a new promoter near the normal promoter site of the cob operon is presented.
Identification of the hutUH operator (hutUo) from Klebsiella aerogenes by DNA deletion analysis
Authors: Osuna R , Schwacha A , Bender R A
Published: November 2016

Expression of Klebsiella aerogenes histidine utilization operons hutUH and hutIG is negatively regulated by the product of hutC. Multiple copies of the hutUH promoter region [hut(P)] present in trans were able to titrate the limited amount of host-encoded hut repressor (HutC). Thus, the hut(P) region contains a specific binding site for HutC. To identify DNA sequences required for HutC titration, we constructed and characterized a set of 40 left-entering and 28 right-entering deletions within a 250-bp DNA sequence containing the hut(P) region. Mutants carrying deletions that altered a unique dyad symmetric sequence, ATGCTTGTATAGACAAGTAT, from -11 to -30 relative to the hutUH promoter (hutUp) were unable to titrate hut repressor; mutants carrying deletions that left this sequence intact retained their ability to titrate hut repressor. Thus, we identify ATGCTTGT ACAAGTAT as the hutUH operator.
Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli
Authors: Bartlett M S , Gourse R L
Published: November 2016

We have extended our previous studies of the DNA sequences required for growth rate-dependent control of rRNA transcription in Escherichia coli. Utilizing a reporter system suitable for evaluation of promoters with low activities, we have found that the core promoter region of rrnB P1 (-41 to +1 with respect to the transcription initiation site) is sufficient for growth rate-dependent control of transcription, both in the presence and in the absence of guanosine 3'-diphosphate 5'-diphosphate (ppGpp). The core promoter contains the -10 and -35 hexamers for recognition by the sigma 70 subunit of RNA polymerase but lacks the upstream (UP) element, which increases transcription by interacting with the alpha subunit of RNA polymerase. It also lacks the binding sites for the positive transcription factor FIS. Thus, the UP element, FIS, and ppGpp are not needed for growth rate-dependent regulation of rRNA transcription. In addition, we find that several core promoter mutations, including -10 and -35 hexamer substitutions, severely reduce rrnB P1 activity without affecting growth rate-dependent control. Thus, a high activity is not a determinant of growth rate regulation of rRNA transcription.
Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants
Authors: Hakenbeck R , Martin C , Dowson C , Grebe T
Published: November 2016

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.
The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes
Authors: Simon G , Méjean V , Jourlin C , Chippaux M , Pascal M C
Published: November 2016

Expression of the Escherichia coli torCAD operon encoding the trimethylamine N-oxide (TMAO) reductase system is induced by both TMAO and anaerobiosis. A torR insertion mutant unable to express the torA gene had previously been isolated. The torR gene was cloned and sequenced. It encodes a 25,000-Da protein which shares homology with response regulators of two-component systems and belongs to the OmpR-PhoB subclass. Overproduction of TorR mimics the presence of the inducer TMAO while the anaerobic control is unchanged, suggesting that TorR mediates only the TMAO induction. The overproduced TorR protein was purified to more than 90%. The torR gene is located just upstream of the torCAD operon, with an opposite transcription direction. The torR-torCAD intergenic region is unusual in that it contains four direct repeats of a 10-nucleotide motif. Part or all of these motifs could be involved in the binding of TorR. The gene encoding the sensor partner does not seem to be adjacent to torR, since the divergent open reading frame found immediately downstream of torR exhibits none of the features of a protein histidine kinase.
BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis
Authors: Bardowski J , Ehrlich S D , Chopin A
Published: November 2016

A fragment of the Lactococcus lactis chromosome containing an open reading frame of 265 codons, denoted bglR, has been characterized. The polypeptide encoded by bglR shares 36 to 30% sequence identity with a family of regulatory proteins including ArbG from Erwinia chrysanthemi, BglG from Escherichia coli, and SacT and SacY from Bacillus subtilis. These regulatory proteins are involved in positive control of the utilization of different sugars by transcription antitermination. For some of these regulatory proteins it has been demonstrated that antitermination is exerted by binding to a conserved RNA sequence, partially overlapping the transcription terminator and thus preventing transcription termination. Upstream of bglR, we identified a transcription terminator whose 5' end was overlapped by a 32-bp sequence, highly homologous to the RNA-binding site that is conserved in other regulatory systems. Constitutive expression of bglR in E. coli increased the expression of a bglG::lacZ transcriptional fusion. The fact that that the expression of BglG is autoregulated in E. coli suggests that BglG and BglR are functionally equivalent. In L. lactis, we observed that (i) the expression of a bglR::lacZ fusion is increased by beta-glucoside sugars, (ii) disruption of bglR impairs growth on some beta-glucosides, and (iii) the expression of bglR is positively autoregulated. Because of these structural and functional similarities between BglR and the transcription antiterminators of the BglG family, we propose that BglR may be the lactococcal counterpart of the E. coli BglG regulator of beta-glucoside utilization.
Molecular analysis and characterization of a broad-host-range plasmid, pEP2
Authors: Zhang Y , Praszkier J , Hodgson A , Pittard A J
Published: November 2016

Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A. J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 was shown to accumulate single-stranded DNA corresponding to the RepA coding strand during its replication in E. coli and C. pseudotuberculosis, suggesting that it may replicate by a rolling circle mechanism. However, RepA has no significant sequence homology with the replication initiator proteins of plasmids known to use this mode of replication.
High-resolution restriction map for a 240-kilobase region spanning 91 to 96 minutes on the Salmonella typhimurium LT2 chromosome
Authors: Wong K K , Wong R M , Rudd K E , McClelland M
Published: November 2016

A hierarchical approach allows the completion of contiguous sets of overlapping clones for small regions of a genome, one at a time rather than tackling the whole genome at once. On the basis of the BlnI restriction map for Salmonella typhimurium LT2, we dissected the chromosome into 21 different fragments by using a Tn5 transposon carrying a BlnI site. Dissected chromosomal fragments were purified by pulsed-field gel electrophoresis and used as probes for sorting a lambda DASHII genomic library of 2,304 primary clones. A total of 129 clones identified as spanning the region from 91 min to 98 min were partly ordered on the basis of the intensity of hybridization with mitomycin-induced Mud-P22 phage DNAs from insertions with pac sites in opposite orientations at 93 min used as probes. Decreased signal intensity with the Mud-P22 probes corresponded to the increased distance of the clone from the site of Mud-P22 insertion and allowed the clones to be placed in two groups from 91 min to 93 min and from 93 min to 98 min and into four intensity categories within the two groups. A member of each category was used to generate a riboprobe from the T3 promoter flanking the insert. This probe identified overlapping clones among the 129 clones. This subchromosomal library was then screened again with riboprobes from nonoverlapping clones. After four cycles of this strategy, a minimal contiguous sequence of 19 partly overlapping clones was selected for restriction mapping. A detailed map of 378 sites for eight restriction enzymes is presented for a region of about 240 kb. Working clockwise, the following genes were placed on this physical map on the basis of their restriction maps: malFEK, lamB, malM, lexA, qor, dnaB, alr, uvrA, proP, pmrB, pmrA, melA, melB, phoN, amiB, mutL, and miaA.
Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis
Authors: Mengin-Lecreulx D , van Heijenoort J
Published: November 2016

The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.
Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis
Authors: Chen M , Nagarajan V
Published: November 2016

The role of positively charged residues at the N termini of signal peptides in protein export has been studied in Bacillus subtilis. Bacillus signal peptides (alkaline protease [Apr] and neutral protease [Npr] from Bacillus amyloliquefaciens) were altered and fused to mature levansucrase (Lvs). The effects of the various alterations on the export of Lvs in B. subtilis were determined. The replacement of positively charged residues with neutral residues in both Apr and Npr signal peptides resulted in a slight defect in the export of Lvs from B. subtilis. Introduction of a negatively charged residue (aspartic acid) at the N terminus of Npr signal peptide blocked the export of Lvs. However, Apr signal peptide with a net charge of -3 (three aspartic acid residues) was still functional.
Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis
Authors: McDonnell G E , Wood H , Devine K M , McConnell D J
Published: November 2016

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter PL, which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.
Overproduction, isolation, and DNA-binding characteristics of Xre, the repressor protein from the Bacillus subtilis defective prophage PBSX
Authors: McDonnell G E , McConnell D J
Published: November 2016

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Lysogeny is maintained by the PBSX-encoded repressor, Xre. The Xre protein was overproduced in Escherichia coli and isolated by affinity chromatography. Gel retardation and DNase I footprinting studies indicated that Xre binds to four sites close to its own gene. These sites overlap putative promoters for xre and a divergent transcriptional unit, containing the middle genes.
Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production
Authors: Nair R V , Papoutsakis E T
Published: November 2016

Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol, 176:871-885, 1994). In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production. The corresponding protein (AAD) appeared as a ca. 96-kDa band in a denaturing protein gel. Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase. These findings suggest that BYDH activity in C. acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.
The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis
Authors: Kawasaki S , Moriguchi R , Sekiya K , Nakai T , Ono E , Kume K , Kawahara K
Published: November 2016

From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with different densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.
Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation
Authors: Bowler L D , Zhang Q Y , Riou J Y , Spratt B G
Published: November 2016

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.
DcrA, a c-type heme-containing methyl-accepting protein from Desulfovibrio vulgaris Hildenborough, senses the oxygen concentration or redox potential of the environment
Authors: Fu R , Wall J D , Voordouw G
Published: November 2016

The amino acid sequence of DcrA from Desulfovibrio vulgaris Hildenborough, a strictly anaerobic, sulfate-reducing bacterium, indicated homology with the methyl-accepting chemotaxis proteins from enteric bacteria (A. Dolla, R. Fu, M. J. Brumlik, and G. Voordouw, J. Bacteriol. 174:1726-1733, 1992). The homology is restricted to the cytoplasmic C-terminal signaling domain. The periplasmic N-terminal sensor domain was found to contain a unique sequence, CHHCH, corresponding to a consensus c-type heme binding site. A pretreated, DcrA-specific polyclonal antiserum, generated against DcrA protein overproduced in Escherichia coli, was used for immunoprecipitation of 35S-labeled DcrA from D. vulgaris and Desulfovibrio desulfuricans G200(pJRFR2), a transconjugant that overexpresses functional DcrA. Labeling of the latter with the heme precursor 5-amino-[4-14C]levulinic acid, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography, confirmed the presence of c-type heme, while labeling with L-[methyl-3H]methionine in the absence of protein synthesis confirmed that DcrA is a methyl-accepting protein. The base liability of the incorporated radioactivity indicated methyl ester formation like that occurring in the methyl-accepting chemotaxis proteins of enteric bacteria. L-[methyl-3H]methionine labeling of D. desulfuricans G200(pJRFR2) under different conditions indicated that methyl labeling of DcrA decreased upon addition of oxygen and increased upon subsequent addition of the reducing agent dithionite. These results indicate that DcrA may serve as a sensor of oxygen concentration and/or redox potential.
Identification of a large family of genes for putative chemoreceptor proteins in an ordered library of the Desulfovibrio vulgaris Hildenborough genome
Authors: Deckers H M , Voordouw G
Published: November 2016

A library of 879 recombinant lambda phages, constructed for the genome of Desulfovibrio vulgaris Hildenborough, has been ordered by restriction fingerprinting. Restriction endonuclease HinfI digestion patterns were entered into a data base and sorted into 87 overlapping groups (contigs), with 19 clones remaining unattached. Eight of ten cloned genes of D. vulgaris, including dcrA, which encodes a transmembrane methyl-accepting protein, were assigned to contigs. Probing of a filter containing the lambda DNAs of the library with the labeled, conserved 3' end of the dcrA gene indicated hybridization to 54 clones distributed over multiple contigs. The presence of 11 additional dcr genes (dcrB to dcrL) was confirmed by direct cycled dideoxy sequencing of positive lambda clones. Since the ordered library provides only partial coverage of the D. vulgaris Hildenborough genome, we estimate that the dcr gene family has 16 members spread throughout the genome, making it the second largest gene family found in prokaryotes.
Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion
Authors: Augustin L B , Jacobson B A , Fuchs J A
Published: November 2016

The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990). We have purified this protein to homogeneity by affinity chromatography and identified it as the E. coli factor for inversion stimulation (Fis). Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription. Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion. The previously reported negative regulatory nature of this site (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions. Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site. Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting. Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA. Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd.
Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli
Authors: Maeda M , Hidaka M , Nakamura A , Masaki H , Uozumi T
Published: November 2016

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.
Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus
Authors: Wu C Y , Alborn W E , Flokowitsch J E , Hoskins J , Unal S , Blaszczak L C , Preston D A , Skatrud P L
Published: November 2016

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.
Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen
Authors: Metzger M , Bellemann P , Bugert P , Geider K
Published: November 2016

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.