bluuebunny/crossref_metadata_2025 huggingface dataset:
Deduced amino acid sequence, functional expression, and unique enzymatic properties of the form I and form II ribulose bisphosphate carboxylase/oxygenase from the chemoautotrophic bacterium Thiobacillus denitrificans
Authors: Hernandez J M , Baker S H , Lorbach S C , Shively J M , Tabita F R
Published: November 2016

The cbbL cbbS and cbbM genes of Thiobacillus denitrificans, encoding form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), respectively, were found to complement a RubisCO-negative mutant of Rhodobacter sphaeroides to autotrophic growth. Endogenous T. denitrificans promoters were shown to function in R. sphaeroides, resulting in high levels of cbbL cbbS and cbbM expression in the R. sphaeroides host. This expression system provided high levels of both T. denitrificans enzymes, each of which was highly purified. The deduced amino acid sequence of the form I enzyme indicated that the large subunit was closely homologous to previously sequenced form I RubisCO enzymes from sulfur-oxidizing bacteria. The form I T. denitrificans enzyme possessed a very low substrate specificity factor and did not exhibit fallover, and yet this enzyme showed a poor ability to recover from incubation with ribulose 1,5-bisphosphate. The deduced amino acid sequence of the form II T. denitrificans enzyme resembled those of other form II RubisCO enzymes. The substrate specificity factor was characteristically low, and the lack of fallover and the inhibition by ribulose 1,5-bisphosphate were similar to those of form II RubisCO obtained from nonsulfur purple bacteria. Both form I and form II RubisCO from T. denitrificans possessed high KCO2 values, suggesting that this organism might suffer in environments containing low levels of dissolved CO2. These studies present the initial description of the kinetic properties of form I and form II RubisCO from a chemoautotrophic bacterium that synthesizes both types of enzyme.
Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress
Authors: Malin G , Lapidot A
Published: November 2016

The metabolic responses of a number of Streptomyces strains to osmotic and heat stress were studied by 13C nuclear magnetic resonance spectroscopy. During cell growth in a chemically defined medium supplemented with 0.5 M NaCl, tetrahydropyrimidine derivatives (THPs), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and, to a lesser extent, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], were found to accumulate in a significant amount in all bacteria examined. In addition, when the growth temperature was shifted from 30 to 39 degrees C, the intracellular concentration of THP(A) increased significantly. Moreover, exogenously provided THP(A) or THP(B) or both reversed inhibition of Escherichia coli growth caused by osmotic stress and increased temperature. Although the ability of Streptomyces strains to tolerate high concentrations of NaCl is well known, very little is known about the osmoregulatory strategy in Streptomyces strains. Similarly, the mechanism by which compatible solutes accumulate in a variety of microorganisms is not understood. Our findings suggest the possibility of a novel mechanism of protection of DNA against salt and heat stresses involving the THPs.
Ammonium/urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii
Authors: Jahns T
Published: November 2016

The influence of ammonium and urea on the components of the proton electrochemical potential (delta p) and de novo synthesis of ATP was studied with Bacillus pasteurii ATCC 11859. In washed cells grown at high urea concentrations, a delta p of -56 +/- 29 mV, consisting of a membrane potential (delta psi) of -228 +/- 19 mV and of a transmembrane pH gradient (delta pH) equivalent to 172 +/- 38 mV, was measured. These cells contained only low amounts of potassium, and the addition of ammonium caused an immediate net decrease of both delta psi and delta pH, resulting in a net increase of delta p of about 49 mV and de novo synthesis of ATP. Addition of urea and its subsequent hydrolysis to ammonium by the cytosolic urease also caused an increase of delta p and ATP synthesis; a net initial increase of delta psi, accompanied by a slower decrease of delta pH in this case, was observed. Cells grown at low concentrations of urea contained high amounts of potassium and maintained a delta p of -113 +/- 26 mV, with a delta psi of -228 +/- 22 mV and a delta pH equivalent to 115 +/- 20 mV. Addition of ammonium to such cells resulted in the net decrease of delta psi and delta pH without a net increase in delta p or synthesis of ATP, whereas urea caused an increase of delta p and de novo synthesis of ATP, mainly because of a net increase of delta psi. The data reported in this work suggest that the ATP-generating system is coupled to urea hydrolysis via both an alkalinization of the cytoplasm by the ammonium generated in the urease reaction and a net increase of delta psi that is probably due to an efflux of ammonium ions. Furthermore, the findings of this study show that potassium ions are involved in the regulation of the intracellular pH and that ammonium ions may functionally replace potassium to a certain extent in reducing the membrane potential and alkalinizing the cytoplasm.
Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein
Authors: Saxild H H , Andersen L N , Hammer K
Published: November 2016

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).
Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA
Authors: Gouesbet G , Trautwetter A , Bonnassie S , Wu L F , Blanco C
Published: November 2016

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.
Thermostable chemotaxis proteins from the hyperthermophilic bacterium Thermotoga maritima
Authors: Swanson R V , Sanna M G , Simon M I
Published: November 2016

An expressed sequence tag homologous to cheA was previously isolated by random sequencing of Thermotoga maritima cDNA clones (C. W. Kim, P. Markiewicz, J. J. Lee, C. F. Schierle, and J. H. Miller, J. Mol. Biol. 231: 960-981, 1993). Oligonucleotides complementary to this sequence tag were synthesized and used to identify a clone from a T. maritima lambda library by using PCR. Two partially overlapping restriction fragments were subcloned from the lambda clone and sequenced. The resulting 5,251-bp sequence contained five open reading frames, including cheA, cheW, and cheY. In addition to the chemotaxis genes, the fragment also encodes a putative protein isoaspartyl methyltransferase and an open reading frame of unknown function. Both the cheW and cheY genes were individually cloned into inducible Escherichia coli expression vectors. Upon induction, both proteins were synthesized at high levels. T. maritima CheW and CheY were both soluble and were easily purified from the bulk of the endogenous E. coli protein by heat treatment at 80 degrees C for 10 min. CheY prepared in this way was shown to be active by the demonstration of Mg(2+)-dependent autophosphorylation with [32P]acetyl phosphate. In E. coli, CheW mediates the physical coupling of the receptors to the kinase CheA. The availability of a thermostable homolog of CheW opens the possibility of structural characterization of this small coupling protein, which is among the least well characterized proteins in the bacterial chemotaxis signal transduction pathway.
Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity
Authors: Peschel A , Götz F
Published: November 2016

The lantibiotic epidermin is produced by Staphylococcus epidermidis Tü3298. The known genes involved in epidermin biosynthesis and regulation are organized as operons (epiABCD and epiQP) that are encoded on the 54-kb plasmid pTü32. Here we describe the characterization of a DNA region that mediates immunity and increased epidermin production, located upstream of the structural gene epiA. The sequence of a 2.6-kb DNA fragment revealed three open reading frames, epiF, -E, and -G, which may form an operon. In the cloning host Staphylococcus carnosus, the three genes mediated an increased tolerance to epidermin, and the highest level of immunity (sevenfold) was achieved with S. carnosus carrying epiFEG and epiQ. The promoter of the first gene, epiF, responded to the activator protein EpiQ and contained a palindromic sequence similar to the EpiQ binding site of the epiA promoter, which is also activated by EpiQ. Inactivation of epiF, -E, or -G resulted in the complete loss of the immunity phenotype. An epidermin-sensitive S. epidermidis Tü3298 mutant was complemented by a DNA fragment containing all three genes. When the epiFEG genes were cloned together with plasmid pTepi14, containing the biosynthetic genes epiABCDQP, the level of epidermin production was approximately fivefold higher. The proteins EpiF, -E, and -G are similar in deduced sequence and proposed structure to the components of various ABC transporter systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while EpiE and -G have six alternating hydrophobic regions and very likely constitute the integral membrane domains. When EpiF was overproduced in S. carnosus, it was at least partially associated with the cytoplasmic membrane. A potential mechanism for how EpiFEG mediates immunity is discussed.
Exchange of precursor-specific elements between Pro-sigma E and Pro-sigma K of Bacillus subtilis
Authors: Carlson H C , Lu S , Kroos L , Haldenwang W G
Published: November 2016

sigma E and sigma K are sporulation-specific sigma factors of Bacillus subtilis that are synthesized as inactive proproteins. Pro-sigma E and pro-sigma K are activated by the removal of 27 and 20 amino acids, respectively, from their amino termini. To explore the properties of the precursor-specific sequences, we exchanged the coding elements for these domains in the sigma E and sigma K structural genes and determined the properties of the resulting chimeric proteins in B. subtilis. The pro-sigma E-sigma K chimera accumulated and was cleaved into active sigma K, while the pro-sigma K-sigma E fusion protein failed to accumulate and is likely unstable in B. subtilis. A fusion of the sigE "pro" sequence to an unrelated protein (bovine rhodanese) also formed a protein that was cleaved by the pro-sigma E processing apparatus. The data suggest that the sigma E pro sequence contains sufficient information for pro-sigma E processing as well as a unique quality needed for sigma E accumulation.
Multiple sigma factor genes in Brevibacterium lactofermentum: characterization of sigA and sigB
Authors: Oguiza J A , Marcos A T , Malumbres M , Martín J F
Published: November 2016

Four rpoD hybridizing signals have been identified in the chromosome of Brevibacterium lactofermentum. Two rpoD-like genes, sigA and sigB, have been cloned and sequenced, and they encode principal sigma factors of the RNA polymerase. The deduced amino acid sequences of SigA and SigB showed very high similarities to those of Mycobacterium smegmatis MysA and MysB proteins, respectively, and also to those of HrdB proteins from different Streptomyces species. SigA and SigB maintain the conserved motifs of sigma 70-like principal sigma factors. sigB is closely linked to the dtxR gene (encoding a repressor of iron-regulated promoters homologous to the diphtheria toxin repressor from Corynebacterium diphtheriae.
Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon
Authors: Eberl L , Christiansen G , Molin S , Givskov M
Published: November 2016

The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.
Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis
Authors: Shen Z , Byers D M
Published: November 2016

We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II.
Pyruvate carboxylase from Rhizobium etli: mutant characterization, nucleotide sequence, and physiological role
Authors: Dunn M F , Encarnación S , Araíza G , Vargas M C , Dávalos A , Peralta H , Mora Y , Mora J
Published: November 2016

Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs.
Functional analysis of the Pseudomonas aeruginosa autoinducer PAI
Authors: Passador L , Tucker K D , Guertin K R , Journet M P , Kende A S , Iglewski B H
Published: November 2016

A series of structural analogs of the Pseudomonas aeruginosa autoinducer [PAI, N-3-oxo-dodecanoyl homoserine lactone] were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR. The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity. Replacement of the ring O by S in the homoserine lactone moiety can be tolerated. Tritium-labelled PAI ([3H]PAI) was synthesized and used to demonstrate the association of [3H]PAI with cells overexpressing LasR. The PAI analogs were also tested for their ability to compete with [3H]PAI for binding of LasR. Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity. The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of [3H]PAI. All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI.
Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA
Authors: Kok R G , Nudel C B , Gonzalez R H , Nugteren-Roodzant I M , Hellingwerf K J
Published: November 2016

The extracellular lipase (LipA) produced by Acinetobacter calcoaceticus BD413 is required for growth of the organism on triolein, since mutant strains that lack an active lipase fail to grow with triolein as the sole carbon source. Surprisingly, extracellular lipase activity and expression of the structural lipase gene (lipA), the latter measured through lacZ as a transcriptional reporter, are extremely low in triolein cultures of LipA+ strains. The explanation for this interesting paradox lies in the effect of fatty acids on the expression of lipA. We found that long-chain fatty acids, especially, strongly repress the expression of lipA, thereby negatively influencing the production of lipase. We propose the involvement of a fatty acyl-responsive DNA-binding protein in regulation of expression of the A. calcoaceticus lipBA operon. The potential biological significance of the observed physiological competition between expression and repression of lipA in the triolein medium is discussed. Activity of the extracellular lipase is also negatively affected by proteolytic degradation, as shown in in vitro stability experiments and by Western blotting (immunoblotting) of concentrated supernatants of stationary-phase cultures. In fact, the relatively high levels of extracellular lipase produced in the early stationary phase in media which contain hexadecane are due only to enhanced stability of the extracellular enzyme under those conditions. The rapid extracellular degradation of LipA of A. calcoaceticus BD413 by an endogenous protease is remarkable and suggests that proteolytic degradation of the enzyme is another important factor in regulating the level of active extracellular lipase.
A two-component response regulator, gltR, is required for glucose transport activity in Pseudomonas aeruginosa PAO1
Authors: Sage A E , Proctor W D , Phibbs P V
Published: November 2016

A 729-bp open reading frame (gltR) was identified in Pseudomonas aeruginosa PAO1 that encodes a product homologous to the two-component response regulator family of proteins. Disruption of gltR caused loss of glucose transport activity. Restoration of gltR resulted in wild-type levels of glucose transport. These findings indicate that gltR is required for expression of the glucose transport system in P. aeruginosa.
Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction system recognizing AGCT
Authors: Voelker L L , Dybvig K
Published: November 2016

Mycoplasma arthritidis is a rat pathogen causing a severe polyarthritis. The study of its pathogenic mechanisms has been hampered by the lack of genetic systems for use with M. arthritidis. Described here are procedures for genetic transformation of M. arthritidis and conjugal transfer of Tn916 from an enterococcal donor to M. arthritidis. The location of Tn916 insertion sites in the mycoplasmal chromosome was random, suggesting that Tn916 may be useful as an insertional mutagen in this organism. Additionally, a restriction and modification system was identified which presented a strong barrier to gene transfer. For transformation, the restriction system was circumvented by using DNA that was modified in vitro with the appropriate site-specific methylase (AluI).
2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45
Authors: Lendenmann U , Spain J C
Published: November 2016

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.
Characterization of the helper proteins for the assembly of tail fibers of coliphages T4 and lambda
Authors: Hashemolhosseini S , Stierhof Y D , Hindennach I , Henning U
Published: November 2016

Assembly of tail fibers of coliphage T4 requires the action of helper proteins. In the absence of one of these, protein 38 (p38), p37, constituting the distal part of the long tail fiber, fails to oligomerize. In the absence of the other, p57, p34 (another component of the long tail fiber), p37, and p12 (the subunit of the short tail fiber) remain unassembled. p38 can be replaced by the Tfa (tail fiber assembly) protein (pTfa) of phage lambda, which has the advantage of remaining soluble even when produced in massive amounts. The mechanisms of action of the helpers are unknown. As a first step towards elucidation of these mechanisms, p57 and pTfa have been purified to homogeneity and have been crystallized. The identity of gene 57 (g57), not known with certainty previously, has been established. The 79-residue protein p57 represents a very exotic polypeptide. It is oligomeric and acidic (an excess of nine negative charges). It does not contain Phe, Trp, Tyr, His, Pro, and Cys. Only 25 N-terminal residues were still able to complement a g57 amber mutant, although with a reduced efficiency. In cells overproducing the protein, it assumed a quasi-crystalline structure in the form of highly ordered fibers. They traversed the cells longitudinally (and thus blocked cell division) with a diameter approaching that of the cell and with a hexagonal appearance. The 194-residue pTfa is also acidic (an excess of 13 negative charges) and is likely to be dimeric.
Characterization of the genetic locus encoding Haemophilus influenzae type b surface fibrils
Authors: St Geme J W , Cutter D , Barenkamp S J
Published: November 2016

Haemophilus influenzae is a common gram-negative pathogen that initiates infection by colonizing the upper respiratory tract epithelium. In previous work, we reported the isolation of a locus involved in expression of short, thin surface fibrils by H. influenzae type b and presented evidence that surface fibrils promote attachment to human epithelial cells. In the present study, we determined that the fibril locus is composed of one long open reading frame, designated hsf, which encodes a protein (Hsf) with a molecular mass of approximately 240 kDa. The derived amino acid sequence of the hsf product demonstrated 81% similarity and 72% identity to a recently identified nontypeable H. influenzae adhesin referred to as Hia. In experiments with a panel of eight cultured cell lines, the Hsf and Hia proteins were found to confer the same binding specificities, suggesting that hsf and hia are alleles of the same locus. Southern analysis and mutagenesis studies reinforced this conclusion. Further investigation revealed that an hsf homolog is ubiquitous among encapsulated H. influenzae strains and is present in a subset of nontypeable Haemophilus strains as well. We speculate that the hsf gene product plays an important role in the process of respiratory tract colonization by H. influenzae.
Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24
Authors: Ogasawara W , Kobayashi G , Okada H , Morikawa Y
Published: November 2016

Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyze Gly-Phe-p-nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1' positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.
Apramycin resistance as a selective marker for gene transfer in mycobacteria
Authors: Paget E , Davies J
Published: November 2016

We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis.
Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12
Authors: García-Martínez J , Martínez-Murcia A , Antón A I , Rodríguez-Valera F
Published: November 2016

Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.
Characterization of exochelins of Mycobacterium avium: evidence for saturated and unsaturated and for acid and ester forms
Authors: Wong D K , Gobin J , Horwitz M A , Gibson B W
Published: November 2016

Mycobacterium avium secretes iron-binding siderophores called exochelins. The exochelins from M. avium have previously been reported to have unsaturated side chains that terminate in carboxylic acid. In contrast, our data show the side chains to be both saturated and unsaturated and to terminate with either a carboxylate or methyl ester.
The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins
Authors: Yuan J , He S Y
Published: November 2016

Pseudomonas syringae pv. tomato DC3000 produces seven to eight major extracellular proteins (EXPs) in a minimal medium inducing hrp genes. Using a polyclonal antibody against DC3000 EXPs, we have determined that the production and secretion of five EXPs (EXP-60, EXP-45, EXP-43, EXP-22, and EXP-10) are under the control of the Hrp regulation and secretion system.
Immunochemical characterization of O polysaccharides composing the alpha-D-rhamnose backbone of lipopolysaccharide of Pseudomonas syringae and classification of bacteria into serogroups O1 and O2 with monoclonal antibodies
Authors: Ovod V , Rudolph K , Knirel Y , Krohn K
Published: November 2016

Murine monoclonal antibodies (MAbs) reacting with Pseudomonas syringae lipopolysaccharide (LPS) O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1-2)D-Rha(alpha1] and [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1] were generated and used for immunochemical analysis and for serological classification of the bacteria. A total of 195 of 358 P. syringae strains tested representing 21 pathovars were shown to share a common epitope, 1a, and were classified into serogroup O1. All strains with pathovars aptata, glycinea, japonica, phaseolicola, and pisi, most of the strains with pathovars atrofaciens and striafaciens, and half of the strains with pathovar syringae were classified into serotypes O1a', O1b, O1c, and O1d within serogroup O1. Serogroup-specific epitope 1a was inferred to be related to the (alpha1-2)D-Rha(alpha1-3) site of the OPS backbone. The serotype-specific epitopes 1b, 1c, 1d, and 1a' were inferred as relating to the immunodominant lateral (alpha1-3)D-Rha, (beta1-4)D-GlcNAc, and (alpha1-4)D-Fuc substituents and backbone-located site (alpha1-3)D-Rha(alpha1-2), respectively, of OPSs that share the common tetra-D-rhamnose repeats in the backbone. A total of 7.3% of the strains studied, all with pathovars morsprunorum and lapsa, were classified as serotypes O2a and O2d within serogroup 02. Serotype-specific epitope 2a was inferred as being related to the backbone-located site D-Rha(alpha1-3)D-Rha and epitope 2d to the immunodominant lateral (alpha1-4)D-Fuc residue of OPS consisting of tri-D-rhamnose repeats in the backbone. Epitope 2d alternated with 2a within the same LPS molecule and did not cross-react with epitope 1d. Serotypes O2a and O2d were observed in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain. The serogroup O1-specific MAb Ps1a reacted weakly but definitely with all strains from serogroup 02. We propose serological formulas for serogroups O1 and 02 as well as for individual strains within these serogroups.
Duplication of pilus gene complexes of Haemophilus influenzae biogroup aegyptius
Authors: Read T D , Dowdell M , Satola S W , Farley M M
Published: November 2016

Brazilian purpuric fever (BPF) is a recently described pediatric septicemia caused by a strain of Haemophilus influenzae biogroup aegyptius. The pilus specified by this bacterium may be important in BPF pathogenesis, enhancing attachment to host tissue. Here, we report the cloning of two haf (for H. influenzae biogroup aegyptius fimbriae) gene clusters from a cosmid library of strain F3031. We sequenced a 6.8-kb segment of the haf1 cluster and identified five genes (hafA to hafE). The predicted protein products, HafA to HafD, are 72, 95, 98, and 90% similar, respectively, to HifA to HifD of the closely related H. influenzae type b pilus. Strikingly, the putative pilus adhesion, HifE, shares only 44% identity with HafE, suggesting that the proteins may differ in receptor specificity. Insertion of a mini-gammadelta transposon in the hafE gene eliminated hemadsorption. The nucleotide sequences of the haf1 and haf2 clusters are more than 99% identical. Using the recently published sequence of the H. influenzae Rd genome, we determined that the haf1 complex lies at a unique position in the chromosome between the pmbA gene and a hypothetical open reading frame, HI1153. The location of the haf2 cluster, inserted between the purE and pepN genes, is analogous to the hif genes on H. influenzae type b. BPF fimbrial phase switching appears to involve slip-strand mispairing of repeated dinucleotides in the pilus promoter. The BPF-associated H. influenzae biogroup aegyptius pilus system generally resembles other H. influenzae, but the possession of a second fimbrial gene cluster, which appears to have arisen by a recent duplication event, and the novel sequence of the HafE adhesin may be significant in the unusual pathogenesis of BPF.
GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp
Authors: Hill D R , Belbin T J , Thorsteinsson M V , Bassam D , Brass S , Ernst A , Böger P , Paerl H , Mulligan M E , Potts M
Published: November 2016

The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity. In N. commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 micromol of O2 mg of chlorophyll a(-1) h(-1). Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP+ oxidoreductase (PetH or FNR). A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN. glbN was present in five Nostoc strains in a single copy. Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp. strain PCC 7120, provided no hybridization signals with a glbN probe. A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii. The nifU-nifH intergenic region of Nostoc sp. strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N. commune UTEX 584. Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired. Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N. commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN. This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp. strain PCC 7942. In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts. Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase.
The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes
Authors: Barbour A G , Carter C J , Bundoc V , Hinnebusch J
Published: November 2016

A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.
Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus
Authors: Udo H , Inouye M , Inouye S
Published: November 2016

Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus. The effect of Pkn2 overexpression on development of M. xanthus was examined by expressing pkn2 under the control of a kanamycin promoter. Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain. Overexpressed Pkn2 was located almost exclusively in the membrane fraction, suggesting that Pkn2 is a transmembrane receptor-type protein Ser/Thr kinase. The PKm/pkn2 strain formed fruiting bodies more slowly than the wild-type strain, in contrast to a Pkn2 deletion strain, the delta pkn2 strain, which developed faster than the wild-type strain. However, spore production was reduced in both the PKm/pkn2 and delta pkn2 strains. These data suggest that Pkn2 functions as a negative regulator for fruiting-body formation and that the proper level of Pkn2 is necessary for maximum myxospore yield.
Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases
Authors: Silakowski B , Pospiech A , Neumann B , Schairer H U
Published: November 2016

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. For analyzing this process, mutants defective in fruiting body formation have been induced by transposon mutagenesis using a Tn5-derived transposon. About 800 bp upstream of the transposon insertion of mutant AP182 which inactivates a gene (fbfB) involved in fruiting, a further gene (fbfA) needed for fruiting body formation was detected. Inactivation of fbfA leads to mutants which form only non-structured clumps instead of the wild-type fruiting body. The mutant phenotype of fbfA mutants can be partially suppressed by mixing the mutant cells with cells of some independent mutants defective in fruiting body formation. The fbfA gene is transcribed after 8 h of development as determined by measuring the induction of beta-galactosidase activity of a fbfA-delta(trp)-lacZ fusion gene and by Northern (RNA) analysis using an insertion encoding a stable mRNA. The predicted polypeptide FbfA shows a homology of about 30% to NodC of rhizobia, an N-acetylglucosamine-transferase which is involved in the synthesis of the sugar backbone of lipo-oligosaccharides. These induce the formation of the root nodules in the Papilionaceae. Besides the predicted molecular mass of 45.5 kDa, the hydropathy profile reveals a structural relationship to the NodC polypeptide.
Regulation of CO2 assimilation in Ralstonia eutropha: premature transcription termination within the cbb operon
Authors: Schäferjohann J , Bednarski R , Bowien B
Published: November 2016

In the facultatively chemoautotrophic bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), most genes required for CO2 assimilation via the Calvin cycle are organized within two highly homologous cbb operons located on the chromosome and on megaplasmid pHG1, respectively, of strain H16. These operons are subject to tight control exerted by a promoter upstream of the 5'-terminal cbbL gene that is regulated by the activator CbbR. The existence of subpromoters within the operons was now excluded, as determined with lacZ operon fusions to suitable cbb gene fragments in the promoter-probe vector pBK. Nevertheless, marked differential expression of the promoter-proximal ribulose-1,5-bisphosphate carboxylase-oxygenase genes cbbLS and the remaining distal genes occurs within the operons. Computer analysis revealed a potential stem-loop structure immediately downstream of cbbS that was suspected to be involved in the differential gene expression. Nuclease S1 mapping identified a major 3' end and a minor 3' end of the relatively stable cbbLS partial transcript just downstream of this structure. Moreover, operon fusions containing progressively deleted stem-loop structures showed that the structure primarily caused transcriptional termination downstream of cbbS rather than increased the segmental stability of the cbbLS transcript. Premature transcription termination thus represents an important mechanism leading to differential gene expression within the cbb operons.
TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4
Authors: Haase J , Kalkum M , Lanka E
Published: November 2016

TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4. The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes. TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide. The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency. An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed. TrbK localizes predominately to the cytoplasmic membrane. Its function depends on presence in the recipient cell but not in the donor cell. TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system. The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets.
Transcription-independent DNA translocation of bacteriophage T7 DNA into Escherichia coli
Authors: García L R , Molineux I J
Published: November 2016

Penetration of wild-type T7 DNA into the host cell occurs in two steps. The phage particle ejects a few hundred base pairs of the left end of the genome into the host. Translocation of the remainder of the DNA is then coupled to transcription. In a normal infection, transcription-coupled translocation of wild-type T7 DNA is initiated at the major A1, A2, and A3 promoters for Escherichia coli RNA polymerase. At 37 degrees C, various deletion mutants lacking these three promoters grow at the same efficiency as wild-type T7 because the minor B promoter is efficiently transferred from the phage head into the cell. As the temperature of the phage infection decreases, the latent periods of (A1, A2, A3)- phages increase relative to that of wild-type T7; nevertheless, (A1, A2, A3)- phages have normal plating efficiencies at reduced temperatures. Lengthening of the latent period at low temperatures is due to a delay in transferring the complete (A1, A2, A3)- genome into the host cell. The (A1, A2, A3)- phages eject the leading end of their genome into the host, but at low temperature, insufficient DNA is transferred into the cell to allow RNA polymerase immediate access the B promoter. However, by an inefficient transcription-independent process, the B promoter eventually translocates into the cell. Mutant derivatives of (A1, A2, A3)- phages that have growth profiles at low temperatures similar to that of wild-type T7 have been isolated. The mutations allow both (A1, A2, A3)- and (A1, A2, A3)+ phages to translocate their entire genomes into the cell by a transcription-independent mechanism. The mutations are located in gene 16, a gene that encodes a component of the internal virion core. We postulate that gp16 is directly involved with the process of DNA translocation from the virion into the cell.
Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2
Authors: Christie G E , Cale S B , Isaksson L A , Jin D J , Xu M , Sauer B , Calendar R
Published: November 2016

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.
N-acetylputrescine as a characteristic constituent of cyanelle peptidoglycan in glaucocystophyte algae
Authors: Pfanzagl B , Allmaier G , Schmid E R , de Pedro M A , Löffelhardt W
Published: November 2016

Cyanelle peptidoglycan from the glaucocystophyte algae Glaucocystis nostochinearum and Cyanoptyche gloeocystis was investigated by high-performance liquid chromatography of muropeptides, supported by matrix-assisted laser desorption-ionization mass spectrometry. The peptidoglycans of both species are modified with N-acetylputrescine, as has been demonstrated for cyanelle peptidoglycan of Cyanophora paradoxa.
Electron microscopic evaluation of a two-step theory of pore formation by streptolysin O
Authors: Sekiya K , Danbara H , Yase K , Futaesaku Y
Published: November 2016

The formation of pores by streptolysin O (SLO) was analyzed in erythrocyte membranes and liposomes by immunoelectron microscopy and electron spectroscopic imaging. The binding of SLO molecules to membranes was temperature independent, while the polymerization of SLO molecules was temperature dependent. Our results also suggest that proteins in erythrocyte membranes are not involved in the formation of SLO rings.