bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor
Authors: Martin D W , Holloway B W , Deretic V
Published: November 2016

Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype.
Modulation of acid-induced amino acid decarboxylase gene expression by hns in Escherichia coli
Authors: Shi X , Waasdorp B C , Bennett G N
Published: November 2016

Biodegradative arginine decarboxylase and lysine decarboxylase, encoded by adi and cadA, respectively, are induced to maximal levels when Escherichia coli is grown anaerobically in rich medium at acidic pH. Mutants formed by transposon mutagenesis, namely, GNB725, GNB729, GNB88, GNB824, and GNB837, exhibited considerably elevated expression at pH 8.0 compared with the corresponding parental strain. Southern hybridization and chromosome mapping showed that the above mutants contained a transposon within the hns gene. Several plasmids from an E. coli library able to complement these mutants by restoring normal pH induction were independently isolated and were found to contain the hns gene. These results suggest a role for the DNA-binding protein H-NS in affecting the activation of these acid-induced genes.
Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida, or Staphylococcus aureus
Authors: Kertesz M A , Leisinger T , Cook A M
Published: November 2016

Two-dimensional gel electrophoresis of proteins from Escherichia coli, Pseudomonas putida, and Staphylococcus aureus, grown with methionine or one of a variety of organosulfates and organosulfonates as the sole source of sulfur, showed expression of specific sets of 7 to 14 proteins which were not observed during growth with sulfate or cysteine for all three species or with thiocyanate for P. putida and S. aureus. Under the same conditions, arylsulfatase activity in P. putida and S. aureus was seen to increase by up to 140-fold, suggesting that the proteins induced under these conditions may be involved in sulfur metabolism. We propose that these proteins are members of a sulfate starvation-induced stimulon.
Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes
Authors: Marconi R T , Samuels D S , Garon C F
Published: November 2016

In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.
Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis
Authors: Omori K , Komatsubara S
Published: November 2016

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.
Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes
Authors: Ralph D , McClelland M , Welsh J , Baranton G , Perolat P
Published: November 2016

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.
Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system
Authors: Yu A , Haggård-Ljungquist E
Published: November 2016

Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.
Synthesis of F pilin
Authors: Maneewannakul K , Maneewannakul S , Ippen-Ihler K
Published: November 2016

Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.
Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of biochemical-genetic study
Authors: Grogan D W , Gunsalus R P
Published: November 2016

A genetic approach was used to establish the route of UMP biosynthesis in Sulfolobus acidocaldarius, a member of the hyperthermophilic division (the Crenarchaeota) of the Archaea domain. Pyrimidine auxotrophs of S. acidocaldarius DG6 were isolated by direct selection and by brute-force methods. Enzymatic assay of extracts from wild-type S. acidocaldarius, from pyrimidine auxotrophs, and from phenotypic revertants demonstrated that S. acidocaldarius synthesizes UMP via orotate in six enzymatic steps corresponding to the de novo pathway of other organisms. The results also show that a single carbamoyl phosphate synthetase supplies both the pyrimidine and arginine pathways of this organism. To gain similar insight into pyrimidine salvage pathway(s), prototrophic mutants resistant to toxic pyrimidine analogs were also isolated and characterized. The results suggest that a single class of mutants which had acquired elevated resistance to four different 5-fluoropyrimidines had been isolated. These fluoropyrimidine-resistant mutants appear to have a regulatory defect leading to overproduction of one or more endogenous pyrimidine compounds.
Analysis of 7-substituted sialic acid in some enterobacterial lipopolysaccharides
Authors: Gamian A , Kenne L
Published: November 2016

Sialic acid-containing lipopolysaccharides (LPS) were isolated from six bacterial strains of the family Enterobacteriaceae. Sialic acid was released from permethylated LPS by methanolysis, and partially O-methylated N-acetyl-N-methyl-neuraminic acid methyl ester methyl glycosides were analyzed by gas-liquid chromatography-electron ionization mass spectrometry. It was proved that all LPS contain N-acetylneuraminic acid (NeuAc). The occurrence of 7-substituted NeuAc in Escherichia coli serotypes O24 and O56 and in Citrobacter freundii O37 LPS was documented. The LPS preparations also contained terminal NeuAc. LPS of E. coli O104 had exclusively 4-substituted sialic acid. The remaining LPS studied, namely, from Salmonella toucra O48 and Hafnia alvei 2, had 4-linked and terminally localized NeuAc residues.
DNA binding and bending are necessary but not sufficient for Fis-dependent activation of rrnB P1
Authors: Gosink K K , Ross W , Leirmo S , Osuna R , Finkel S E , Johnson R C , Gourse R L
Published: November 2016

The Escherichia coli Fis protein binds to three sites in the upstream activation region of the rrnB P1 promoter and enhances transcription 5- to 10-fold in vivo. In this report, we investigate the mechanism of Fis-dependent activation of transcription. We show that stimulation of rrnB P1 transcription by Fis can occur on linear DNA templates and does not require DNA upstream of the promoter-proximal Fis site I. Mutants of Fis defective for Hin-mediated recombination have been isolated previously and have defined an N-terminal domain required for DNA inversion by Hin in addition to the C-terminal domain which is required for DNA binding. Several of these mutants were found to be defective in stimulation of rrnB P1 transcription in vivo and in vitro. Activation-defective mutants fall into three classes: those that fail to bind to the upstream activation region, those that bind but fail to bend the DNA normally, and those that bind and bend but still fail to activate transcription. We conclude that it is unlikely that Fis functions by simply bringing upstream sequences or bound factors into the proximity of RNA polymerase to activate transcription. Rather, the data are most easily interpreted in terms of transcription activation by direct interactions between Fis and RNA polymerase, requiring precise positioning of the two proteins facilitated by bending of the DNA binding site.
Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators
Authors: Shingler V , Bartilson M , Moore T
Published: November 2016

The catabolic plasmid pVI150 of Pseudomonas sp. strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives. The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence. Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN). Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter. Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR. The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter. Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters. The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively. The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators. This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.
Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus
Authors: Henze U , Sidow T , Wecke J , Labischinski H , Berger-Bächi B
Published: November 2016

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.
Cloning, nucleotide sequence, and regulatory analysis of the Lactococcus lactis dnaJ gene
Authors: van Asseldonk M , Simons A , Visser H , de Vos W M , Simons G
Published: November 2016

The dnaJ gene of Lactococcus lactis was isolated from a genomic library of L. lactis NIZO R5 and cloned into pUC19. Nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids. The deduced amino acid sequence showed homology to the DnaJ proteins of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis, and Clostridium acetobutylicum. The level of the dnaJ monocistronic mRNA increased approximately threefold after heat shock. The transcription initiation site of the dnaJ gene was determined and appeared to be preceded by a typical gram-positive vegetative promoter sequence (TTGCCA-17 bp-TAAAAT). Upstream of the promoter region, an inverted repeat is located that is identical to those detected upstream of heat shock genes of other gram-positive organisms. A transcriptional fusion between the dnaJ expression signals and a usp45-amyS secretion cassette caused a significant increase in alpha-amylase activity after heat shock induction. Deletion mutagenesis showed that the inverted repeat is involved in heat shock regulation of the dnaJ gene. The conservation of this palindromic sequence in gram-positive heat shock genes suggests a common regulatory pathway distinct from the system used in gram-negative bacteria.
Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli
Authors: Klein W , Boos W
Published: November 2016

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITre of the phosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). The apparent Km (16 microM) of trehalose uptake is low. Since trehalose is a good source of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharide trehalose [O-alpha-D-glucosyl(1-1)-alpha-D-glucoside] has no problems diffusing through the outer membrane at high enough rates to allow full growth, particularly at low substrate concentrations. Here we show that induction of the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of all maltose genes, as well as lamB mutants that lack only the lambda receptor (maltoporin), still grow on trehalose at the usual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehalose uptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system is induced by trehalose to about 30% of the fully induced level reached when grown in the presence of maltose in a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30% level of maximal expression is sufficient for maximal trehalose utilization, since there is no difference in the concentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with the wild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)]. In contrast, when the expression of the lambda receptor is reduced to less than 20% of the maximal level, trehalose uptake becomes less efficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lacking amylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltose system.
The patB gene product, required for growth of the cyanobacterium Anabaena sp. strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains
Authors: Liang J , Scappino L , Haselkorn R
Published: November 2016

A mutant of Anabaena sp. strain PCC 7120, called PAT-2, that grows poorly under nitrogen-fixing conditions, has been isolated. The heterocysts of the mutant strain develop much more slowly than those of the wild type and are spaced more closely in an older culture of the mutant than in the wild type. The wild-type gene that complements the mutation in PAT-2, called patB, was isolated and characterized. The predicted 529-amino-acid PatB protein contains a region very similar to the Fe4S4 bacterial-type ferredoxins near its N terminus and a helix-turn-helix motif near its C terminus. This pattern of domains resembles those of transcriptional regulators in both prokaryotes and eukaryotes. The mutation in strain PAT-2 is the deletion of G at nucleotide 1342 in the patB gene, resulting in the loss of a 62-amino-acid fragment from the C terminus of the PatB protein, including the helix-turn-helix motif.
Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA
Authors: Christiansen G , Pedersen L B , Koehler J E , Lundemose A G , Birkelund S
Published: November 2016

The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d. In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible in the host. Following induction, the E. coli cells were lysed gently. Gel filtration of the lysate revealed comigration of DNA and Hc1 in the voided volume. Electron microscopy revealed the DNA to be complexed with protein in large aggregates, often in the form of spherical bodies. Purified recombinant Hc1 maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA. Hc1 alone is thus able to package DNA into condensed spherical bodies.
Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation
Authors: Suen W C , Spain J C
Published: November 2016

The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.
The final step of peptidoglycan subunit assembly in Escherichia coli occurs in the cytoplasm
Authors: Bupp K , van Heijenoort J
Published: November 2016

The murG gene of Escherichia coli encodes the N-acetylglucosaminyltransferase responsible for the final step in the formation of the lipid-linked disaccharide-pentapeptide subunit of peptidoglycan. Using trypsin to probe maxicell spheroplasts, we show that this enzyme is peripherally associated with the inner face of the cytoplasmic membrane. Therefore, the peptidoglycan subunit is completely assembled before it traverses the cytoplasmic membrane.
Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface
Authors: Fujino T , Béguin P , Aubert J P
Published: November 2016

The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome. Three open reading frames (ORFs) belonging to two operons were detected. They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively. The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each. Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface. Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function. However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome. Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T. Fujino, P. Béguin, and J.-P. Aubert, FEMS Microbiol. Lett. 94:165-170, 1992). These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.
Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1
Authors: Southam G , Firtel M , Blackford B L , Jericho M H , Xu W , Mulhern P J , Beveridge T J
Published: November 2016

Methanospirillum hungatei GP1 possesses paracrystalline cell envelope components including end plugs and a sheath formed from stacked hoops. Both negative-stain transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) distinguished the 2.8-nm repeat on the outer surface of the sheath, while negative-stain TEM alone demonstrated this repeat around the outer circumference of individual hoops. Thin sections revealed a wave-like outer sheath surface, while STM showed the presence of deep grooves that precisely defined the hoop-to-hoop boundaries at the waveform nodes. Atomic force microscopy of sheath tubes containing entrapped end plugs emphasized the end plug structure, suggesting that the sheath was malleable enough to collapse over the end plugs and deform to mimic the shape of the underlying structure. High-resolution atomic force microscopy has revised the former idea of end plug structure so that we believe each plug consists of at least four discs, each of which is approximately 3.5 nm thick. PT shadow TEM and STM both demonstrated the 14-nm hexagonal, particulate surface of an end plug, and STM showed the constituent particles to be lobed structures with numerous smaller projections, presumably corresponding to the molecular folding of the particle.
A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators
Authors: Ueda K , Miyake K , Horinouchi S , Beppu T
Published: November 2016

Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1.
Cloning and sequencing of the Escherichia coli panB gene, which encodes ketopantoate hydroxymethyltransferase, and overexpression of the enzyme
Authors: Jones C E , Brook J M , Buck D , Abell C , Smith A G
Published: November 2016

The panB gene from Escherichia coli, encoding the first enzyme of the pantothenate biosynthesis pathway, ketopantoate hydroxymethyltransferase (KPHMT), has been isolated by functional complementation of a panB mutant strain with an E. coli genomic library. The gene is 792 bp long, encoding a protein of 264 amino acids with a predicted M(r) of 28,179. The identity of the gene product as ketopantoate hydroxymethyltransferase was confirmed by purification of the enzyme protein, which was overexpressed approximately 50-fold in the mutant harboring the gene on a high-copy-number plasmid. The N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence, as was its mass, determined by electrospray mass spectrometry. Upstream of the panB gene is an incomplete open reading frame encoding a protein of 220 amino acids, which shares sequence similarity to fimbrial precursor proteins from other bacteria. Northern (RNA) analysis showed that the panB gene is likely to be cotranscribed with at least one other gene but that this is not the putative fimbrial protein, since no transcripts for this gene could be detected.
Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382
Authors: Meletzus D , Bermphol A , Dreier J , Eichenlaub R
Published: November 2016

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype.
KatF (sigma S) synthesis in Escherichia coli is subject to posttranscriptional regulation
Authors: Loewen P C , von Ossowski I , Switala J , Mulvey M R
Published: November 2016

A transcriptional fusion of katF to the lacZ gene was expressed at increasingly higher levels throughout the exponential phase, but a translational fusion was expressed at low levels during exponential-phase growth and was induced 160-fold during the transition to stationary phase, implicating a posttranscriptional mechanism in the regulation of KatF synthesis. Mutational analyses suggested that the initiation codon of katF is the second ATG in the previously identified open reading frame.
Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range
Authors: Prieto M A , Perez-Aranda A , Garcia J L
Published: November 2016

The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12. This gene was located near the pacA gene coding for penicillin G acylase. The hydroxylase has a molecular mass of 59,000 Da, uses NADH as a cosubstrate, and was tentatively classified as a 4-hydroxyphenylacetic acid hydroxylase, albeit it exhibited a rather broad substrate specificity acting on different monohydric and dihydric phenols. E. coli W, C, and B as well as Klebsiella pneumoniae M5a1 and Kluyvera citrophila ATCC 21285 (a penicillin G acylase-producing strain) but not E. coli K-12 contained sequences homologous to hpaB. Our results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W.
Highly selective binding of nascent polypeptides by an Escherichia coli chaperone protein in vivo
Authors: Kumamoto C A , Francetić O
Published: November 2016

Chaperone proteins bind to newly synthesized polypeptides and assist in various assembly reactions. The Escherichia coli chaperone protein SecB binds precursors of exported proteins and assists in export. In vitro, SecB can bind to many unfolded proteins. In this report, we demonstrate that SecB binding in vivo is highly selective; the major polypeptides that are bound by SecB are nascent precursors of the exported proteins maltose-binding protein (MBP), LamB, OmpF, and OmpA. These results support the hypothesis that the primary physiological function of SecB is to stimulate protein export. By interacting with nascent polypeptides, SecB probably stimulates their cotranslational association with the membrane-bound protein translocation apparatus.
A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities
Authors: Lopez P , Lacks S A
Published: November 2016

A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.
Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination
Authors: Diehl P , McFadden B A
Published: November 2016

By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic activity by ca. 50- and 14-fold, respectively, and the nonconservative changes, K193E and K193L, result in assembled tetrameric protein that is completely inactive. The K193H and K193R mutations also increase the Km of the enzyme by five- and twofold, respectively. These results indicate that the cationic and/or acid-base character of K193 is essential for isocitrate lyase activity. In addition to the noted effects on enzyme activity, the effects of the mutations on growth of JE10, an E. coli strain which does not express isocitrate lyase, were observed. Active isocitrate lyase is necessary for E. coli to grow on acetate as the sole carbon source. It was found that a mutation affecting the activity of isocitrate lyase similarly affects the growth of E. coli JE10 on acetate when the mutated plasmid is expressed in this organism. Specifically, the lag time before growth increases over sevenfold and almost twofold for E. coli JE10 expressing the K193H and K193R isocitrate lyase variants, respectively. In addition, the rate of growth decreases by almost 40-fold for E. coli JE10 cells expressing form K193H and ca. 2-fold for those expressing the K193R variants. Thus, the onset and rate of E. coli growth on acetate appears to depend on isocitrate lyase activity.
Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth
Authors: Tojo N , Inouye S , Komano T
Published: November 2016

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.
Regulation of sigma B levels and activity in Bacillus subtilis
Authors: Benson A K , Haldenwang W G
Published: November 2016

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.
Genetic evidence that promoter P2 is the physiologically significant promoter for the pyrBI operon of Escherichia coli K-12
Authors: Liu C , Donahue J P , Heath L S , Turnbough C L
Published: November 2016

The pyrBI operon of Escherichia coli K-12 encodes the two nonidentical subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase). Expression of this operon is negatively regulated by pyrimidine availability primarily through UTP-sensitive transcriptional attenuation and, to a lesser extent, at the level of transcriptional initiation. Previous studies indicated that the pyrBI operon was transcribed from tandem sigma 70 promoters designated P1 and P2, with the large majority of transcription initiated at the more downstream promoter P2. To more clearly define the roles of these promoters, mutations that severely impair or inactivate individual promoters were constructed in the chromosomal pyrBI operon, and their effects on ATCase synthesis were measured. In cells grown under conditions of either pyrimidine excess or pyrimidine limitation, more than 99% of all ATCase synthesis was directed by transcripts initiated at promoter P2, indicating that it is the only physiologically significant pyrBI promoter. However, mutations that effectively inactivate promoter P1 caused a 15% reduction in ATCase levels, apparently by inhibiting transcription from promoter P2 by an unknown mechanism. Support for this explanation was provided by the demonstration that little, if any, transcriptional initiation occurred at promoter P1 in a transcriptional fusion vector whereas a high level of transcription was initiated at promoter P2 in an equivalent construction. Our results also provide evidence for pyrimidine-mediated regulation of transcriptional initiation at promoter P2 over a severalfold range and show that cells can grow reasonably well with very low levels of ATCase, apparently because of changes in the concentration of allosteric effectors that increase the specific activity of the enzyme.
Characterization of three fimbrial genes, sefABC, of Salmonella enteritidis
Authors: Clouthier S C , Müller K H , Doran J L , Collinson S K , Kay W W
Published: November 2016

Salmonella enteritidis produces thin, filamentous fimbriae designated SEF14. A 3.9-kb region of a 5.3-kb fragment encoding genes responsible for SEF14 biosynthesis was sequenced and found to contain three genes, sefABC. sefA encoded a novel fimbrin, the structural subunit of SEF14 fimbriae. sefB and sefC encoded proteins homologous to Escherichia coli and Klebsiella pneumoniae fimbrial periplasmic chaperone proteins and fimbrial outer membrane proteins, respectively, and are the first such genes to be characterized from Salmonella spp. in vitro expression directed by the 5.3-kb DNA fragment identified SefA, SefB, and SefC as approximately 14,000-, 28,000-, and 90,000-M(r) proteins, respectively, which correlated with their predicted amino acid sequences. sefB and sefC were not expressed in the absence of sefA. Primer extension analysis of sefABC revealed two major transcription start sites located upstream of sefA. Transcription of sefBC also initiated from the sefA promoter region. Secondary-structure analysis of the mRNA transcript for sefABC predicted the formation of two stable stem-loop structures in the intercistronic region between sefA and sefB indicative of differential regulation of SefA, SefB, and SefC translation. E. coli cells carrying the 5.3-kb DNA fragment of S. enteritidis DNA were unable to assemble distinguishable SEF14 fimbriae; however, immunogold-labelled SEF14 fimbriae were displayed on E. coli clones containing a 44-kb DNA fragment which encompassed the 5.3-kb region. Therefore, sefABC genes make up part of a complex sef operon responsible for the expression and assembly of SEF14 fimbriae.