bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1
Authors: Morton T M , Eaton D M , Johnston J L , Archer G L
Published: November 2016

The conjugative transfer genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related genes necessary for conjugative transfer of plasmids in gram-negative species and 32% identity of trsC to a gene required for conjugative mobilization of plasmid pC221 from staphylococci.
Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi
Authors: Hashimoto Y , Li N , Yokoyama H , Ezaki T
Published: November 2016

Plasmid pGBM124, which contains a 14-kb Salmonella typhi chromosomal DNA fragment capable of producing the Vi antigen in Escherichia coli HB101 and ViaB-deleted S. typhi GIFU 10007-3, was studied. We determined the complete nucleotide sequence of this fragment and found 11 open reading frames. Mutagenesis, subcloning, and complementation analysis showed that three genes (vipA, vipB, and vipC) are involved in biosynthesis of the Vi polysaccharide. The putative primary amino acid sequence suggests that both vipA and vipB encode the NAD- or NADP-dependent enzymes to synthesize the nucleotide sugar for the Vi polysaccharide. Five genes (vexA, vexB, vexC, vexD, and vexE) may be involved in translocation of the Vi polysaccharide. Proteins VexA, VexB, VexC, and VexD had moderate similarities to components of group II capsule transporters, and the VexC protein had a putative ATP-binding site. These data indicate that the transport system for the Vi polysaccharide belongs to the ATP-binding cassette transporters. By using the isogenic Vi+ and Vi- strains constructed in this study, we reconfirmed that the Vi antigen is necessary for the serum resistance of S. typhi.
A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells
Authors: Behlau I , Miller S I
Published: November 2016

The Salmonella typhimurium transcriptional regulators, PhoP/PhoQ, induce phoP-activated gene (pag) expression to promote virulence and intracellular survival within macrophages. This response to the macrophage intracellular environment is simulated by phoP/phoQ constitutive mutations (phenotype PhoPc) that increase the expression of pag genes and repress the synthesis of approximately 20 proteins encoded by phoP-repressed genes (prg genes) (S. I. Miller and J. J. Mekalanos, J. Bacteriol. 172:2485-2490, 1990). PhoPc bacteria are attenuated for mouse virulence, suggesting that prg genes are virulence genes. We now report the identification of five unlinked prg loci by use of the transposon TnphoA. In general, medium conditions (i.e., starvation) that activate pag expression repress prg expression. However, variable effects on the PhoP regulon were observed when bacteria were grown under different oxygen tensions (pag and prg genes) or exposed to low pH (prg genes), suggesting heterogenous control of the regulon. One prg locus, prgH, was demonstrated to contribute to mouse virulence by both the oral and the intraperitoneal routes. prgH was located at 59 min on the Salmonella chromosome, a region where other genes essential to invasion of epithelial cells are clustered. The prgH locus was highly linked to one invasion locus, hil (C.A. Lee, B.D. Jones, and S. Falkow, Proc. Natl. Acad. Sci. USA 89:1847-1851, 1992), although transcription of prgH was opposite that of the Tn5B50-encoded promoters that result in a hyperinvasive or hil phenotype. Both PrgH and PhoPc mutant S. typhimurium were found to be defective in induction of endocytosis by Madin-Darby canine kidney (MDCK) epithelial cells. The invasion defect of PrgH but not that of PhoPc mutant bacteria was complemented by plasmids containing prgH (hil) DNA. Therefore, two virulence properties of Salmonella species, induction of endocytosis by epithelial cells and survival within macrophages, are oppositely modulated by the PhoP/PhoQ virulence regulators.
Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae
Authors: Lim C K , Cooksey D A
Published: November 2016

Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD). cop homologs were cloned from the chromosome of P. syringae pv. tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P. syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance. Two chromosomal cop homolog regions were cloned. Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS. Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P. syringae. However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC. In addition, homolog 2 conferred a high frequency of mutation to full copper resistance. In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed. pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs. Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.
The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system
Authors: Rathsam C , Giffard P M , Jacques N A
Published: November 2016

The ftf gene, coding for the cell-bound beta-D-fructosyltransferase (FTF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FTF of Streptococcus mutans and the levansucrases (SacBs) of Bacillus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivarius ftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host.
Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100
Authors: Ng W L , DasSarma S
Published: November 2016

We have identified the replication origin of pNRC100, a 200-kb plasmid of Halobacterium halobium, by assaying for replication ability of miniplasmids containing cloned fragments of pNRC100 and the mevinolin resistance selectable marker of Haloferax volcanii. First, we showed the replication ability of plasmid pNGHCMEV1, which contains the 19-kb HindIII-C fragment of pNRC100, by recovery of plasmid DNA from mevinolin-resistant transformants of H. halobium. The minimal replication origin of approximately 3.9 kb was defined by subcloning successively smaller regions of pNGHCMEV1 and assaying for plasmid replication in either H. halobium or H. volcanii. The same replication origin was also recovered after transformation of H. volcanii with a library of partial Sau3AI fragments of pNRC100. The nucleotide sequence of the minimal replication origin was determined and found to contain a long open reading frame, named repH, transcribed away from a highly A+T-rich region. The transcription start site was identified by primer extension analysis to be 17 to 18 nucleotides 5' to a putative repH start codon. The predicted product of the repH gene, an acidic protein with a molecular weight of 113,442, showed 24 to 27% identity with predicted gene products of H. volcanii plasmid pHV2 and H. halobium plasmid p phi HL, suggesting that each is involved in plasmid replication. One pNRC100 minireplicon, pNG11 delta 12, was analyzed by linker scanning mutagenesis, which showed the requirement of repH for replication. Restoration of the repH reading frame of one replication-defective pNG11 delta 12 derivative by introduction of a second small insertion resulted in reversion to replication proficiency. The replication ability of pNG11delta12 was lost when the entire A+T-rich region, about 550 bp long, was deleted but not when small insertions or deletions were introduced into this region. The presence of only 52 bp of the A+T-rich segment was sufficient to permit replication. The pNG11delta12 minireplicon was lost at high frequency from cells grown without mevinolin selection, suggesting that the plasmid partitioning locus of pNRC100 is absent in the minimal replication origin region. We discuss the possible roles of the repH gene and the A+T-rich region in replication of pNRC100.
Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa
Authors: Poole K , Neshat S , Krebes K , Heinrichs D E
Published: November 2016

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.
Characterization of cytR mutations that influence oligomerization of mutant repressor subunits
Authors: Barbier C S , Short S A
Published: November 2016

In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.
DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro
Authors: Scarlato V , Aricò B , Rappuoli R
Published: November 2016

The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription.
Dynamic structure of Agrobacterium tumefaciens Ti plasmids
Authors: Fortin C , Marquis C , Nester E W , Dion P
Published: November 2016

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.
Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells
Authors: Hsia R C , Small P L , Bavoil P M
Published: November 2016

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.
Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences
Authors: Belogurov A A , Delver E P , Rodzevich O V
Published: November 2016

The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously. The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli. Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E. coli and only slightly affects the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system. Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively. The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins. We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids. It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating.
Delayed expression of in vivo restriction activity following conjugal transfer of Escherichia coli hsdK (restriction-modification) genes
Authors: Prakash-Cheng A , Ryu J
Published: November 2016

Following conjugal transfer of the hsdK genes (hsdRK, hsdMK, and hsdSK) of Escherichia coli K-12, restriction activity was first detected only after approximately 15 generations, whereas modification activity was observed immediately. This sequential expression explains the establishment of hsdK genes in a nonmodified host and suggests regulation of restriction activity after conjugal transfer.
Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli
Authors: Sharma V , Meganathan R , Hudspeth M E
Published: November 2016

The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation. The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein. A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found. However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes. Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene.
The tet(K) gene from Staphylococcus aureus mediates the transport of potassium in Escherichia coli
Authors: Guay G G , Tuckman M , McNicholas P , Rothstein D M
Published: November 2016

The tet(K) gene, encoding the tetracycline efflux protein from Staphylococcus aureus, mediates the transport of potassium in an Escherichia coli mutant defective in potassium uptake. Deletion mapping indicates that the first third of the tet(K) gene is sufficient to mediate potassium transport.
Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12
Authors: Plumbridge J A , Cochet O , Souza J M , Altamirano M M , Calcagno M L , Badet B
Published: November 2016

The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene glmS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine. This regulation occurs at the level of glmS transcription. It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism of N-acetylglucosamine) also repress the expression of glmS in the absence of exogenous amino sugars.
Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa
Authors: Turner L R , Lara J C , Nunn D N , Lory S
Published: November 2016

The process of extracellular secretion in Pseudomonas aeruginosa requires specialized machinery which is widely distributed among bacteria that actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the biogenesis of type IV pili. Both XcpR and PilB are characterized by the presence of a conserved ATP-binding motif (Walker sequence). The codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these substitutions were examined. Bacteria expressing mutant XcpR or PilB were unable to secrete exotoxin A or assemble pili, respectively. In addition, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from the cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and assembly of pili, respectively. Moreover, the observed dominant negative phenotype of mutant XcpR suggests that this protein functions as a multimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.
OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis
Authors: Misra R
Published: November 2016

This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane. The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene. A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+). Assembly mutants were isolated in two steps. First, amber mutations were obtained that mapped in ompF-Dex. This resulted in a Dex- phenotype. Starting with these Dex- strains, Dex+ revertants were isolated. Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized. Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein. All three mutant OmpF proteins showed an assembly defect. This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel. Suppressor mutations were sought that corrected the assembly defect of OmpF. These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome. The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele. In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner. The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species. At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product. The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.
Staphylococcus aureus has clustered tRNA genes
Authors: Green C J , Vold B S
Published: November 2016

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.
Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002
Authors: Chen X , Widger W R
Published: November 2016

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.
Pseudomonas aeruginosa lipopolysaccharide: evidence that the O side chains and common antigens are on the same molecule
Authors: Hatano K , Goldberg J B , Pier G B
Published: November 2016

We investigated whether Pseudomonas aeruginosa produces two distinct lipopolysaccharides (LPS) containing either serologically variable O side chains or a neutral polysaccharide common antigen, designated A bands, that reacts with monoclonal antibody (MAb) E87. Immunoprecipitation of LPS and free O side chains with O-side-chain-specific antibodies or MAb E87 resulted in coprecipitation of both polysaccharides when antibody of either specificity was employed. Chromatography of LPS and free O side chains in a disaggregating deoxycholate buffer indicated the two polysaccharide antigens cochromatograph when eluates were analyzed by sensitive and specific enzyme-linked immunosorbent assay inhibitions. The LPS from a mutant of strain PAO1 that lacks polymerized O side chains but retains the common antigen eluted in fractions containing smaller LPS molecules, indicating the necessity of polymerized O side chains for elution in early fractions containing large LPS monomers. A phosphomannomutase mutant of P. aeruginosa PAO1 makes a rough LPS lacking both O side chains and common antigen but still produces a small (< 6-kDa) common antigen component detectable in cell lysates. Introduction of the cloned pmm gene into this strain restored production of a smooth LPS expressing large MAb E87-reactive common antigen. Destruction with NaOH of O side chains on recombinant LPS molecules eluting early from the molecular sieve column resulted in a shift of the MAb E87-reactive antigen to the late-eluting fractions. These results indicate that on most P. aeruginosa LPS molecules, O side chains and neutral polysaccharide common antigens are both present.
Roles of the GcvA and PurR proteins in negative regulation of the Escherichia coli glycine cleavage enzyme system
Authors: Wilson R L , Stauffer L T , Stauffer G V
Published: November 2016

When Escherichia coli was grown in medium containing both inosine and glycine, the PurR repressor protein was shown to be responsible for a twofold reduction from the fully induced glycine cleavage enzyme levels. This twofold repression was also seen by measuring beta-galactosidase levels in cells carrying a lambda gcvT-lacZ gene fusion. In this fusion, the synthesis of beta-galactosidase is under the control of the gcv regulatory region. A DNA fragment carrying the gcv control region was shown by gel mobility shift assay and DNase I footprinting to bind purified PurR protein, suggesting a direct involvement of the repressor in gcv regulation. A separate mechanism of purine-mediated regulation of gcv was shown to be independent of the purR gene product and resulted in an approximately 10-fold reduction of beta-galactosidase levels when cells were grown in medium containing inosine but lacking the inducer glycine. This additional repression was dependent upon a functional gcvA gene, a positive activator for the glycine cleavage enzyme system. A dual role for the GcvA protein as both an activator in the presence of glycine and a repressor in the presence of inosine is suggested.
Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation
Authors: Gerin P A , Dufrene Y , Bellon-Fontaine M N , Asther M , Rouxhet P G
Published: November 2016

The conidiospores of the white rot basidiomycete Phanerochaete chrysosporium tend to aggregate during swelling and germination in agitated liquid medium; as time passes, the initial aggregates tend to associate together and to capture conidiospores that remain isolated. The surface chemical compositions of the conidiospores and of developed hyphae were analyzed by X-ray photoelectron spectroscopy. The data were interpreted by modelling the surface in terms of proteins, polysaccharides and hydrocarbonlike compounds. The surface molecular composition of the dormant conidiospores was estimated to be about 45% proteins, 20% carbohydrates, and 35% hydrocarbonlike compounds. There was an increase in the polysaccharide content during germination. Later, when the hyphae were developed, the polysaccharide content became still higher, and the protein content dropped. The initial step of aggregation is attributed to polysaccharide bridging; its occurrence cannot be explained by a change of the overall hydrophobicity or electrical properties of the conidiospores.
Characterization of genes for synthesis and catabolism of a new rhizopine induced in nodules by Rhizobium meliloti Rm220-3: extension of the rhizopine concept
Authors: Saint C P , Wexler M , Murphy P J , Tempé J , Tate M E , Murphy P J
Published: November 2016

Rhizopines are selective growth substrates synthesized in nodules only by strains of rhizobia capable of their catabolism. We report the isolation and study of genes for the synthesis and catabolism of a new rhizopine, scyllo-inosamine (sIa), from alfalfa nodules induced by Rhizobium meliloti Rm220-3. This compound is similar in structure to the previously described rhizopine 3-O-methyl-scyllo-inosamine from R. meliloti L5-30 (P.J. Murphy, N. Heycke, Z. Banfalvi, M.E. Tate, F.J. de Bruijn, A. Kondorosi, J. Tempé, and J. Schell, Proc. Natl. Acad. Sci. USA 84:493-497, 1987). The synthesis (mos) and catabolism (moc) genes for the Rm220-3 rhizopine are closely linked and located on the nod-nif Sym plasmid. The mos genes are directly controlled by the NifA/NtrA regulatory system. A comparison of the sequence of the 5' regions of the two mos loci shows very extensive conservation of sequence as well as strong homology to the nifH coding region. Restriction mapping and hybridization to DNA from the four open reading frames (ORFs) of the L5-30 mos locus indicate the absence of mosA and presence of the other three ORFs (ORF1 and mosB and -C) in Rm220-3. We suggest that the L5-30 mosA gene product is involved in the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine. Restriction fragment length polymorphism analysis of the moc regions of both strains shows that they are very similar. Regulation studies indicate that the moc region is not controlled by the common regulatory gene nifA, ntrA, and ntrC. We discuss the striking similarities in gene structure, location, and regulation between these two rhizopine loci in relation to the rhizopine concept.
Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function
Authors: Murray B E , Singh K V , Ross R P , Heath J D , Dunny G M , Weinstock G M
Published: November 2016

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.
Characterization of twenty-six new heat shock genes of Escherichia coli
Authors: Chuang S E , Blattner F R
Published: November 2016

Most organisms respond to heat by substantial alteration of the pattern of gene expression. This has been particularly well studied with Escherichia coli although the response has by no means been completely characterized. Here we report the characterization of 26 new heat shock genes of E. coli, termed hsl, discovered by global transcription analysis with an overlapping lambda clone bank. We have measured the molecular weights of the corresponding heat shock proteins and mapped each of them to within a few kilobases on the E. coli genome. In vitro, 16 of them can be activated by the E sigma 32 RNA polymerase, which specifically transcribes heat shock genes. In vivo expression kinetics of seven of eight examined new proteins were found to be similar to those of the four most studied heat shock proteins, DnaK, DnaJ, GroEL (MopA), and GroES (MopB). In the course of this work, we confirmed that the catalytic subunit of the ATP-dependent Clp protease (also known as Ti protease), ClpP, is derived from a larger precursor protein. Possible assignments of some of the hsl genes to known proteins are discussed.
Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function
Authors: Ruhfel R E , Manias D A , Dunny G M
Published: November 2016

In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ability of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E. faecalis cell with the ability to specifically remove (apparently by irreversible binding) cCF10 activity from culture medium. The pCF10 DNA encoding this ability was localized to a 3.4-kb segment within a region involved in negative control of expression of conjugal transfer functions. This segment also encoded ability to bind the pheromone inhibitor peptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an adjacent region of pCF10 and could encode a protein with significant similarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducible hemolysin plasmid pAD1, according to F.Y. An and D.B. Clewell (Abstr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene product showed significant relatedness to binding proteins encoded by oligopeptide permease (opp) operons in gram-positive and gram-negative bacteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An, and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.
Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter
Authors: Mathee K , Howe M M
Published: November 2016

Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late. Middle transcription requires the early gene product Mor for its activation. Mor protein overproduction was accomplished by fusing the mor gene to an efficient phage T7 promoter and translation initiation region. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) from the Mor-overproducing strain was able to activate transcription from both the tac promoter (Ptac) and the Mu middle promoter (Pm) in vitro. Transcription initiation from Pm was Mor dependent, and the RNA 5' end was identical to that of in vivo RNA. Addition of anti-sigma 70 antibody to transcription reactions containing Ptac and Pm resulted in inhibition of transcription from both promoters; addition of purified sigma 70 restored transcription. These results indicate that Mor-dependent activation requires sigma 70 and therefore imply that Mor is not an alternate sigma factor. This conclusion was further substantiated by a reconstitution experiment with purified proteins in which all three components, Mor, sigma 70, and core RNA polymerase, were required for Pm-dependent transcription in vitro. The sigma 70 dependence of Mor-specific transcription and the amino acid sequence similarity between Mor and C (an activator for Mu late transcription) both support the hypothesis that Mor functions mechanistically as an activator protein.
Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus
Authors: Branny P , De La Torre F , Garel J R
Published: November 2016

A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli. The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E. coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties. The recombinant protein had the same structural and functional properties as did the original enzyme. The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L. bulgaricus. This gene organization, the same as that in S. citri (C. Chevalier, C. Saillard, and J. M. Bové, J. Bacteriol. 172:2693-2703, 1990) and B. stearothermophilus (D. Walker, W. N. Chia, and H. Muirhead, J. Mol. Biol. 228:265-276, 1992; H. Sakai and T. Ohta, Eur. J. Biochem. 311:851-859, 1993) but different from that in E. coli (H. W. Hellinga and P. R. Evans, Eur. J. Biochem. 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis. The possibility that these genes could be transcribed at the same time suggested that in L. bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity.
Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12
Authors: Iida A , Teshiba S , Mizobuchi K
Published: November 2016

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.
Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides
Authors: Jayaratne P , Keenleyside W J , MacLachlan P R , Dodgson C , Whitfield C
Published: November 2016

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.