bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetics of dark skin in mice
Authors: Fitch Karen R. , McGowan Kelly A. , van Raamsdonk Catherine D. , Fuchs Helmut , Lee Daekee , Puech Anne , Hérault Yann , Threadgill David W. , de Angelis Martin Hrabé , Barsh Gregory S.
Published: January 2003

Chemical mutagenesis in the mouse is a powerful approach for phenotype-driven genetics, but questions remain about the efficiency with which new mutations ascertained by their phenotype can be localized and identified, and that knowledge applied to a specific biological problem. During a global screen for dominant phenotypes in about 30,000 animals, a novel class of pigmentation mutants were identified by dark skin (Dsk). We determined the genetic map location, homozygous phenotype, and histology of 10 newDskand 2 new dark coat (Dcc) mutations, and identified mutations inAgouti(Met1Leu,Dcc4),Sox18(Leu220ter,Dcc1),Keratin 2e(Thr500Pro,Dsk2), andEgfr(Leu863Gln,Dsk5). Cutaneous effects of mostDskmutations are limited to melanocytes, except for theKeratin 2eandEgfrmutations, in which hyperkeratosis and epidermal thickening precede epidermal melanocytosis by 3–6 wk. TheDsk2mutation is likely to impair intermediate filament assembly, leading to cytolysis of suprabasal keratinocytes and secondary hyperkeratosis and melanocytosis. TheDsk5mutation causes increased tyrosine kinase activity and a decrease in steady-state receptor levels in vivo. TheDskmutations represent genes or map locations not implicated previously in pigmentation, and delineate a developmental pathway in which mutations can be classified on the basis of body region, microscopic site, and timing of pigment accumulation.
An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation inCaenorhabditis elegans
Authors: Miller Michael A. , Ruest Paul J. , Kosinski Mary , Hanks Steven K. , Greenstein David
Published: January 2003

During sexual reproduction in most animals, oocytes arrest in meiotic prophase and resume meiosis (meiotic maturation) in response to sperm or somatic cell signals. Despite progress in delineating mitogen-activated protein kinase (MAPK) and CDK/cyclin activation pathways involved in meiotic maturation, it is less clear how these pathways are regulated at the cell surface. TheCaenorhabditis elegansmajor sperm protein (MSP) signals oocytes, which are arrested in meiotic prophase, to resume meiosis and ovulate. We used DNA microarray data and an in situ binding assay to identify the VAB-1 Eph receptor protein–tyrosine kinase as an MSP receptor. We show that VAB-1 and a somatic gonadal sheath cell-dependent pathway, defined by the CEH-18 POU-class homeoprotein, negatively regulate meiotic maturation and MAPK activation. MSP antagonizes these inhibitory signaling circuits, in part by binding VAB-1 on oocytes and sheath cells. Our results define a sperm-sensing control mechanism that inhibits oocyte maturation, MAPK activation, and ovulation when sperm are unavailable for fertilization. MSP-domain proteins are found in diverse animal taxa, where they may regulate contact-dependent Eph receptor signaling pathways.
c-MYC apoptotic function is mediated by NRF-1 target genes
Authors: Morrish Fionnuala , Giedt Christopher , Hockenbery David
Published: January 2003

A detailed understanding of the signaling pathways by which c-Myc elicits apoptosis has proven elusive. In the current study, we have evaluated whether the activation of the mitochondrial apoptotic signaling pathway is linked to c-Myc induction of a subset of genes involved in mitochondrial biogenesis. Cytochrome c and other nuclear-encoded mitochondrial genes are regulated by the transcription factor nuclear respiratory factor-1 (NRF-1). The consensus binding sequence (T/C)GCGCA(C/T)GCGC(A/G) of NRF-1 includes a noncanonical CA(C/T)GCG Myc:MAX binding site. In this study, we establish a link between the induction of NRF-1 target genes and sensitization to apoptosis on serum depletion. We demonstrate, by using Northern analysis, transactivation assays, and in vitro and in vivo promoter binding assays that cytochrome c is a direct target of c-Myc. Like c-Myc, NRF-1 overexpression sensitizes cells to apoptosis on serum depletion. We also demonstrate that selective interference with c-Myc induction of NRF-1 target genes by using a dominant-negative NRF-1 prevented c-Myc-induced apoptosis, without affecting c-Myc-dependent proliferation. These results suggest thatc-mycexpression leads to mitochondrial dysfunction and apoptosis by deregulating genes involved in mitochondrial function.
Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development
Authors: Mill Pleasantine , Mo Rong , Fu Hong , Grachtchouk Marina , Kim Peter C.W. , Dlugosz Andrzej A. , Hui Chi-chung
Published: January 2003

Sonic hedgehog (Shh) signaling plays a critical role in hair follicle development and skin cancer, but how it controls these processes remains unclear. Of the three Gli transcription factors involved in transducing Shh signals in vertebrates, we demonstrate here that Gli2 is the key mediator of Shh responses in skin. Similar toShh−/−mice,Gli2−/−mutants exhibit an arrest in hair follicle development with reduced cell proliferation and Shh-responsive gene expression, but grossly normal epidermal differentiation. By transgenic rescue experiments, we show that epidermal Gli2 function alone is sufficient to restore hair follicle development inGli2−/−skin. Furthermore, only a constitutively active form of Gli2, but not wild-type Gli2, can activate Shh-responsive gene expression and promote cell proliferation inShh−/−skin. These observations indicate that Shh-dependent Gli2 activator function in the epidermis is essential for hair follicle development. Our data also reveal that Gli2 mediates the mitogenic effects of Shh by transcriptional activation ofcyclin D1andcyclin D2in the developing hair follicles. Together, our results suggest that Shh-dependent Gli2 activation plays a critical role in epithelial homeostasis by promoting proliferation through the transcriptional control of cell cycle regulators.
Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA
Authors: Pestova Tatyana V. , Hellen Christopher U.T.
Published: January 2003

Reconstitution of translation elongation from purified components confirmed that ribosomes that assembled on the Cricket paralysis virus intercistronic internal ribosomal entry site (IRES) without the involvement of initiation factors or initiator tRNA were active in elongation and are, therefore, true initiation complexes. The first elongation cycle occurred without peptide bond formation on 80S ribosomes that did not contain tRNA in the P site. It required elongation factors 1A and 2 and A site-cognate aminoacylated tRNA. Cycloheximide arrested ribosomes on the IRES only after two cycles of elongation, when the first deacylated tRNA reached the E-site after translocation from the A-site.
Proapoptotic BID is required for myeloid homeostasis and tumor suppression
Authors: Zinkel Sandra S. , Ong Christy C. , Ferguson David O. , Iwasaki Hiromi , Akashi Koichi , Bronson Roderick T. , Kutok Jeffery L. , Alt Frederick W. , Korsmeyer Stanley J.
Published: January 2003

The proper expansion and contraction of hematopoietic cells requires tight regulation of cell death. BID, a “BH3-only” molecule, amplifies death receptor signals connecting the extrinsic to intrinsic pathways by triggering the mitochondrial pathway of apoptosis.Bid-deficient mice, as they age, spontaneously develop a myeloproliferative disorder, which progresses from myeloid hyperplasia to a fatal, clonal malignancy closely resembling chronic myelomonocytic leukemia (CMML). Thus, an apoptotic defect can result in myeloid leukemogenesis. PremalignantBid−/− myeloid precursor cells are resistant to death receptor-induced apoptosis. Furthermore, a competitive reconstitution assay demonstrates thatBid-deficient long-term repopulating cells give rise to expanded myelomonocytic cells in vivo. Surprisingly, a single BH3-only molecule operating in the extrinsic death receptor pathway proved essential in vivo for physiologic cell death required to maintain myeloid homeostasis. Moreover, progression to CMML indicates that an upstream BH3-only molecule, BID, is required to suppress tumorigenesis.
Tbx1is regulated by tissue-specific forkhead proteins through a common Sonic hedgehog-responsive enhancer
Authors: Yamagishi Hiroyuki , Maeda Jun , Hu Tonghuan , McAnally John , Conway Simon J. , Kume Tsutomu , Meyers Erik N. , Yamagishi Chihiro , Srivastava Deepak
Published: January 2003

Haploinsufficiency of Tbx1 is likely a major determinant of cardiac and craniofacial birth defects associated with DiGeorge syndrome. Although mice deficient in Tbx1 exhibit pharyngeal and aortic arch defects, the developmental program and mechanisms through which Tbx1 functions are relatively unknown. We identified a singlecis-element upstream ofTbx1that recognized winged helix/forkhead box (Fox)-containing transcription factors and was essential for regulation ofTbx1transcription in the pharyngeal endoderm and head mesenchyme. TheTbx1regulatory region was responsive to signaling by Sonic hedgehog (Shh) in vivo. We show that Shh is necessary for aortic arch development, similar to Tbx1, and is also required for expression ofFoxa2andFoxc2in the pharyngeal endoderm and head mesenchyme, respectively. Foxa2, Foxc1, or Foxc2 could bind and activate transcription through the criticalcis-element upstream ofTbx1, and Foxc proteins were required, within their expression domains, forTbx1transcription in vivo. We propose thatTbx1is a direct transcriptional target of Fox proteins and that Fox proteins may serve an intermediary role in Shh regulation ofTbx1.
Senescence, aging, and malignant transformation mediated by p53 in mice lacking the Brca1 full-length isoform
Authors: Cao Liu , Li Wenmei , Kim Sangsoo , Brodie Steven G. , Deng Chu-Xia
Published: January 2003

Senescence may function as a two-edged sword that brings unexpected consequences to organisms. Here we provide evidence to support this theory by showing that the absence of the Brca1 full-length isoform causes senescence in mutant embryos and cultured cells as well as aging and tumorigenesis in adult mice. Haploid loss of p53 overcame embryonic senescence but failed to prevent the adult mutant mice from prematurely aging, which included decreased life span, reduced body fat deposition, osteoporosis, skin atrophy, and decreased wound healing. We further demonstrate that mutant cells that escaped senescence had undergone clonal selection for faster proliferation and extensive genetic/molecular alterations, including overexpression of cyclin D1 and cyclin A and loss of p53. These observations provide the first in vivo evidence that links cell senescence to aging due to impaired function of Brca1 at the expense of tumorigenesis.
TheArabidopsis SRR1gene mediates phyB signaling and is required for normal circadian clock function
Authors: Staiger Dorothee , Allenbach Laure , Salathia Neeraj , Fiechter Vincent , Davis Seth J. , Millar Andrew J. , Chory Joanne , Fankhauser Christian
Published: January 2003

Plants possess several photoreceptors to sense the light environment. InArabidopsiscryptochromes and phytochromes play roles in photomorphogenesis and in the light input pathways that synchronize the circadian clock with the external world. We have identifiedSRR1(sensitivity tored lightreduced), a gene that plays an important role in phytochrome B (phyB)-mediated light signaling. The recessivesrr1null allele andphyBmutants display a number of similar phenotypes indicating thatSRR1is required for normal phyB signaling. Genetic analysis suggests thatSRR1works both in the phyB pathway but also independently of phyB.srr1mutants are affected in multiple outputs of the circadian clock in continuous light conditions, including leaf movement and expression of the clock components,CCA1andTOC1. Clock-regulated gene expression is also impaired during day–night cycles and in constant darkness. The circadian phenotypes ofsrr1mutants in all three conditions suggest that SRR1 activity is required for normal oscillator function. TheSRR1gene was identified and shown to code for a protein conserved in numerous eukaryotes including mammals and flies, implicating a conserved role for this protein in both the animal and plant kingdoms.
Listening and learning
Authors: Rockman Ilene F.
Published: January 2003

Looks at papers presented at the LOEX‐of‐the‐West Conference “Expanded conversations: collaborating for student learning” held in 2002 in Eugene, Oregon, USA, which cover innovative research projects in the areas of information literacy. Concludes that by listening to campus colleagues and students unexpected benefits can be gained.
Building reference strengths through peer training
Authors: Stec Dankert Holly , Dempsey Paula R.
Published: January 2003

What began as peer training instruction sessions for DePaul University librarians has grown into an electronic knowledge base available 24 × 7 for ongoing staff development. Using Blackboard course management software, DePaul University Libraries developed peer to peer instruction to keep communication open among staff members at all eight campus libraries while sharing subject expertise among librarians and sharpening skills needed in group and one‐on‐one instruction. In order to facilitate continued communication between librarians at different campuses at DePaul University, an annual program of structured peer training sessions was developed and implemented within the last two years to provide ongoing professional development.
Involvement of a Protein Tyrosine Kinase in Production of the Polymeric Bioemulsifier Emulsan from the Oil-Degrading StrainAcinetobacter lwoffiiRAG-1
Authors: Nakar David , Gutnick David L.
Published: January 2003

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degradingAcinetobacter lwoffiiRAG-1 are clustered within a 27-kbp region termed theweecluster. This report demonstrates the involvement of two genes of theweecluster of RAG-1,wzbandwzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substratep-nitrophenyl phosphate (PNPP) Wzb exhibited aVmaxof 12 μmol of PNPP min−1mg−1and aKmof 8 mM PNPP at 30°C. The emulsifying activity of mutants lacking eitherwzborwzcwas 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.
The Iron-Binding Protein Dps Confers Hydrogen Peroxide Stress Resistance to
Campylobacter jejuni
Authors: Ishikawa Takahiko , Mizunoe Yoshimitsu , Kawabata Shun-ichiro , Takade Akemi , Harada Mine , Wai Sun Nyunt , Yoshida Shin-ichi
Published: January 2003

We identified and characterized the iron-binding protein Dps from
Campylobacter jejuni
. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (
dps
) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between
C. jejuni
Dps and other Dps family proteins. In
C. jejuni
Dps, there are iron-binding motifs, as reported in other Dps family proteins.
C. jejuni
Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic
dps
-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H
2
O
2
or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in
C. jejuni
plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.
Comparative Analyses of the Complete Genome Sequences of Pierce's Disease and Citrus Variegated Chlorosis Strains of
Xylella fastidiosa
Authors: Van Sluys M. A. , de Oliveira M. C. , Monteiro-Vitorello C. B. , Miyaki C. Y. , Furlan L. R. , Camargo L. E. A. , da Silva A. C. R. , Moon D. H. , Takita M. A. , Lemos E. G. M. , Machado M. A. , Ferro M. I. T. , da Silva F. R. , Goldman M. H. S. , Goldman G. H. , Lemos M. V. F. , El-Dorry H. , Tsai S. M. , Carrer H. , Carraro D. M. , de Oliveira R. C. , Nunes L. R. , Siqueira W. J. , Coutinho L. L. , Kimura E. T. , Ferro E. S. , Harakava R. , Kuramae E. E. , Marino C. L. , Giglioti E. , Abreu I. L. , Alves L. M. C. , do Amaral A. M. , Baia G. S. , Blanco S. R. , Brito M. S. , Cannavan F. S. , Celestino A. V. , da Cunha A. F. , Fenille R. C. , Ferro J. A. , Formighieri E. F. , Kishi L. T. , Leoni S. G. , Oliveira A. R. , Rosa V. E. , Sassaki F. T. , Sena J. A. D. , de Souza A. A. , Truffi D. , Tsukumo F. , Yanai G. M. , Zaros L. G. , Civerolo E. L. , Simpson A. J. G. , Almeida N. F. , Setubal J. C. , Kitajima J. P.
Published: January 2003

Xylella fastidiosa
is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee.
X. fastidiosa
has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of
X. fastidiosa
isolates that were expected to be genetically divergent. Here we report the genome sequence of
X. fastidiosa
(Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced
X. fastidiosa
strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD
X. fastidiosa
Temecula genes are shared with the CVC
X. fastidiosa
strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other
X. fastidiosa
strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.
Rhamnolipid Surfactant Production Affects Biofilm Architecture in
Pseudomonas aeruginosa
PAO1
Authors: Davey Mary E. , Caiazza Nicky C. , O'Toole George A.
Published: January 2003

In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen
Pseudomonas aeruginosa
in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.
pIII
CTX
, a Predicted CTXφ Minor Coat Protein, Can Expand the Host Range of Coliphage fd To Include
Vibrio cholerae
Authors: Heilpern Andrew J. , Waldor Matthew K.
Published: January 2003

CTXφ is a filamentous bacteriophage that encodes cholera toxin. CTXφ infection of its host bacterium,
Vibrio cholerae
, requires the toxin-coregulated pilus (TCP) and the products of the
V. cholerae tolQRA
genes. Here, we have explored the role of OrfU, a predicted CTXφ minor coat protein, in CTXφ infection. Prior to the discovery that it was part of a prophage,
orfU
was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXφ genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXφ infection as well as playing a role in CTXφ assembly and release. Deletion of
orfU
from CTXφ dramatically reduced the number of CTXφ virions detected in supernatants from CTXφ-bearing cells. This defect was complemented by expression of
orfU
in
trans
, thereby confirming a role for this gene in CTXφ assembly and/or release. To evaluate the requirement for OrfU in CTXφ infection, we introduced fragments of
orfU
into
gIII
in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect
V. cholerae
, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect
V. cholerae
in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXφ pIII, we propose to rename OrfU as pIII
CTX
. Our data also provide new evidence for a conserved pathway for filamentous phage infection.
The VirB4 Family of Proposed Traffic Nucleoside Triphosphatases: Common Motifs in Plasmid RP4 TrbE Are Essential for Conjugation and Phage Adsorption
Authors: Rabel Christian , Grahn A. Marika , Lurz Rudi , Lanka Erich
Published: January 2003

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in
trbE
resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in
Escherichia coli
. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.
Traffic at the tmRNA Gene
Authors: Williams Kelly P.
Published: January 2003

A partial screen for genetic elements integrated into completely sequenced bacterial genomes shows more significant bias in specificity for the tmRNA gene (
ssrA
) than for any type of tRNA gene. Horizontal gene transfer, a major avenue of bacterial evolution, was assessed by focusing on elements using this single attachment locus. Diverse elements use
ssrA
; among enterobacteria alone, at least four different integrase subfamilies have independently evolved specificity for
ssrA
, and almost every strain analyzed presents a unique set of integrated elements. Even elements using essentially the same integrase can be very diverse, as is a group with an
ssrA
-specific integrase of the P4 subfamily. This same integrase appears to promote damage routinely at attachment sites, which may be adaptive. Elements in arrays can recombine; one such event mediated by invertible DNA segments within neighboring elements likely explains the monophasic nature of
Salmonella enterica
serovar Typhi. One of a limited set of conserved sequences occurs at the attachment site of each enterobacterial element, apparently serving as a transcriptional terminator for
ssrA
. Elements were usually found integrated into tRNA-like sequence at the 3′ end of
ssrA
, at subsites corresponding to those used in tRNA genes; an exception was found at the non-tRNA-like 3′ end produced by
ssrA
gene permutation in cyanobacteria, suggesting that, during the evolution of new site specificity by integrases, tropism toward a conserved 3′ end of an RNA gene may be as strong as toward a tRNA-like sequence. The proximity of
ssrA
and
smpB
, which act in concert, was also surveyed.
Microarray Analysis of Global Gene Expression in Mucoid
Pseudomonas aeruginosa
Authors: Firoved Aaron M. , Deretic Vojo
Published: January 2003

Pseudomonas aeruginosa
is the dominant pathogen causing chronic respiratory infections in cystic fibrosis (CF). After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of
P. aeruginosa
to the mucoid, exopolysaccharide alginate-overproducing phenotype. The emergence of mucoid
P. aeruginosa
in CF is associated with respiratory decline and poor prognosis. The switch to mucoidy in most CF isolates is caused by mutations in the
mucA
gene encoding an anti-sigma factor. The mutations in
mucA
result in the activation of the alternative sigma factor AlgU, the
P. aeruginosa
ortholog of
Escherichia coli
extreme stress sigma factor σ
E
. Because of the global nature of the regulators of mucoidy, we have hypothesized that other genes, in addition to those specific for alginate production, must be induced upon conversion to mucoidy, and their production may contribute to the pathogenesis in CF. Here we applied microarray analysis to identify on the whole-genome scale those genes that are coinduced with the AlgU sigmulon upon conversion to mucoidy. Gene expression profiles of AlgU-dependent conversion to mucoidy revealed coinduction of a specific subset of known virulence determinants (the major protease elastase gene, alkaline metalloproteinase gene
aprA
, and the protease secretion factor genes
aprE
and
aprF
) or toxic factors (cyanide synthase) that may have implications for disease in CF. Analysis of promoter regions of the most highly induced genes (>40-fold,
P
≤ 10
−4
) revealed a previously unrecognized, putative AlgU promoter upstream of the osmotically inducible gene
osmE
. This newly identified AlgU-dependent promoter of
osmE
was confirmed by mapping the mRNA 5′ end by primer extension. The recognition of genes induced in mucoid
P. aeruginosa
, other than those associated with alginate biosynthesis, reported here revealed the identity of previously unappreciated factors potentially contributing to the morbidity and mortality caused by mucoid
P. aeruginosa
in CF.
Attachment Organelle Formation Represented by Localization of Cytadherence Proteins and Formation of the Electron-Dense Core in Wild-Type and Mutant Strains of
Mycoplasma pneumoniae
Authors: Seto Shintaro , Miyata Makoto
Published: January 2003

Cytadherence proteins of
Mycoplasma pneumoniae
are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division. The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy. In the class I-2 mutant, which has a frameshift mutation in the
hmw2
gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing. However, foci for P90, P40, P30, and P65 were not observed in this mutant. In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused. In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell. These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65. The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core. Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain. HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3. These differences were more pronounced in cytadherence mutants. These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65.
Characterization of the CipA Scaffolding Protein and In Vivo Production of a Minicellulosome in
Clostridium acetobutylicum
Authors: Sabathé Fabrice , Soucaille Philippe
Published: January 2003

The
cipA
gene encoding the
Clostridium acetobutylicum
scaffolding protein CipA was cloned and expressed in
Escherichia coli
. CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains. The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain. Affinity-purified CipA was used in equilibrium-binding experiments to characterize the interaction of CipA with crystalline cellulose. A

K
d

of 0.038 μM and a [C]
max
of 0.43 μmol of CipA bound per g of Avicel were determined. A mini-CipA polypeptide consisting of a CBD3a and two cohesin domains was overexpressed in
C. acetobutylicum
, yielding the in vivo formation of a minicellulosome. This is to our knowledge the first demonstration of the in vivo assembly of a recombinant minicellulosome.
Plasmid DNA Supercoiling and Gyrase Activity in
Escherichia coli
Wild-Type and
rpoS
Stationary-Phase Cells
Authors: Reyes-Domínguez Yazmid , Contreras-Ferrat Gabriel , Ramírez-Santos Jesús , Membrillo-Hernández Jorge , Gómez-Eichelmann M. Carmen
Published: January 2003

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase
rpoS
cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that
rpoS
plays a role in the regulation of plasmid topology during the stationary phase.
The NorM Efflux Pump of
Neisseria gonorrhoeae
and
Neisseria meningitidis
Recognizes Antimicrobial Cationic Compounds
Authors: Rouquette-Loughlin Corinne , Dunham Steven A. , Kuhn Michael , Balthazar Jacqueline T. , Shafer William M.
Published: January 2003

In
Neisseria gonorrhoeae
and
Neisseria meningitidis
, we identified a gene that would encode a protein highly similar to NorM of
Vibrio parahaemolyticus
(Y. Morita et al., Antimicrob. Agents Chemother. 42:1778-1782, 1998). A nonpolar insertional mutation in either the gonococcal or meningococcal
norM
gene resulted in increased bacterial sensitivity to compounds harboring a quaternary ammonium on an aromatic ring (e.g., ethidium bromide, acriflavine hydrochloride, 2-
N
-methylellipticinium, and berberine). The presence of point mutations within the −35 region of a putative
norM
promoter or a likely ribosome binding site resulted in an increased resistance of gonococci and meningococci to the same compounds, as well as to norfloxacin and ciprofloxacin. Structure-activity relationship studies with putative NorM substrates have found that a cationic moiety is essential for NorM recognition.
The High-Pathogenicity Island Is Absent in Human Pathogens of
Salmonella enterica
Subspecies I but Present in Isolates of Subspecies III and VI
Authors: Oelschlaeger T. A. , Zhang D. , Schubert S. , Carniel E. , Rabsch W. , Karch H. , Hacker J.
Published: January 2003

In this study we tested 74
Salmonella
strains of all eight
Salmonella
groups and were able to demonstrate the presence of two high-pathogenicity island types in strains of
Salmonella
groups IIIa, IIIb, and VI. Most high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were positive for the high-molecular-weight proteins HMWP1 and HMWP2.
Genetic and Biochemical Studies of Phosphatase Activity of PhoR
Authors: Carmany Daniel O. , Hollingsworth Kristine , McCleary William R.
Published: January 2003

In
Escherichia coli
, PhoR is the histidine kinase of the phosphate regulon. It has been postulated that PhoR may function as a phospho-PhoB phosphatase. Experiments with four precise
phoR
deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain. This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.
Expression of Primary Sigma Factor (PSF) and PSF-Like Sigma Factors in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Tuominen Ilona , Tyystjärvi Esa , Tyystjärvi Taina
Published: January 2003

Large amounts of
sigA
mRNA, encoding the primary σ factor (PSF) in
Synechocystis
sp. strain PCC 6803, accumulated under standard growth conditions, while stress conditions like heat or high salinity led to a rapid decrease in
sigA
mRNA content. The
sigB
,
sigC
,
sigD
, and
sigE
genes, encoding PSF-like σ factors, were under strict physiological control.
Chromosomal Arm Replacement in
Streptomyces griseus
Authors: Uchida Tetsuya , Miyawaki Mariko , Kinashi Haruyasu
Published: January 2003

UV irradiation of
Streptomyces griseus
2247 yielded a new chromosomal deletion mutant, MM9. Restriction and sequencing analysis revealed that homologous recombination between two similar lipoprotein-like open reading frames, which are located 450 and 250 kb from the left and right ends, respectively, caused chromosomal arm replacement. As a result, new 450-kb terminal inverted repeats (TIRs) were formed in place of the original 24-kb TIRs. Frequent homologous recombinations in
Streptomyces
strains suggest that telomere deletions can usually be repaired by recombinational DNA repair functioning between the intact and deleted TIR sequences on the same chromosome.
Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of
Ruminococcus flavefaciens
Authors: Rincon Marco T. , Ding Shi-You , McCrae Sheila I. , Martin Jennifer C. , Aurilia Vincenzo , Lamed Raphael , Shoham Yuval , Bayer Edward A. , Flint Harry J.
Published: January 2003

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe
Ruminococcus flavefaciens
17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in
E. coli
; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in
R. flavefaciens
plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative
R. flavefaciens
bands, including an ∼110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in
R. flavefaciens
.
ISCce
1
and ISCce
2
, Two Novel Insertion Sequences in
Clostridium cellulolyticum
Authors: Maamar Hédia , de Philip Pascale , Bélaich Jean-Pierre , Tardif Chantal
Published: January 2003

Two new insertion sequences, ISCce
1
and ISCce
2
, were found to be inserted into the
cipC
gene of spontaneous mutants of
Clostridium cellulolyticum
. In these insertional mutants, the
cipC
gene was disrupted either by ISCce
1
alone or by both ISCce
1
and ISCce
2
. ISCce
1
is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS
481
and IS
3
families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce
1
. ISCce
2
is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS
256
family. Upon transposition, both ISCce
1
and ISCce
2
generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce
1
is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce
2
was found to be mostly inserted into ISCce
1
. In addition, as neither of the elements was detected in seven other
Clostridium
species, we concluded that they may be specific to the
C. cellulolyticum
strain used.
Characterization of
Helicobacter pylori
Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase
Authors: Mehta Nalini , Olson Jonathan W. , Maier Robert J.
Published: January 2003

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in
Helicobacter pylori
. Here, the two proteins were purified and characterized. HypA bound two Ni
2+
ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants
K
1
and
K
2
for Ni
2+
were 58 and 1.3 μM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of
hypA
(in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 μM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of
hypA
) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.
ATP-Dependent Interactions between
Escherichia coli
Min Proteins and the Phospholipid Membrane In Vitro
Authors: Lackner Laura L. , Raskin David M. , de Boer Piet A. J.
Published: January 2003

Proper placement of the division apparatus in
Escherichia coli
requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg
2+
, whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.
1-Methylguanosine-Deficient tRNA of
Salmonella enterica
Serovar Typhimurium Affects Thiamine Metabolism
Authors: Björk Glenn R. , Nilsson Kristina
Published: January 2003

In
Salmonella enterica
serovar Typhimurium a mutation in the
purF
gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a
purF
mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from
S. enterica
specific for leucine, proline, and arginine contain 1-methylguanosine (m
1
G37) adjacent to and 3′ of the anticodon (position 37). The formation of m
1
G37 is catalyzed by the enzyme tRNA(m
1
G37)methyltransferase, which is encoded by the
trmD
gene. Mutations in this gene, which result in an m
1
G37 deficiency in the tRNA, in a
purF
mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m
1
G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m
1
G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.
Agrobacterium tumefaciens
Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion
Authors: Ding Zhiyong , Christie Peter J.
Published: January 2003

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen
Agrobacterium tumefaciens
. In contrast to wild-type strain A348, a
tatC
null mutant failed to export the green fluorescent protein fused to the trimethylamine
N
-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The
tatC
mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in
vir
induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the
tatC
mutant cells were motile in rich medium, and <0.01% were motile in
vir
induction medium. Nonmotile
tatC
mutant cells lacked detectable flagella, whereas motile
tatC
mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile
tatC
cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The
tatC
mutant was highly attenuated for virulence, only occasionally (∼15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile
tatC
mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the
tatC
mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of
A. tumefaciens
is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.