bluuebunny/crossref_metadata_2025 huggingface dataset:
EWI-2 regulates α3β1 integrin–dependent cell functions on laminin-5
Authors: Stipp Christopher S. , Kolesnikova Tatiana V. , Hemler Martin E.
Published: December 2003

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (α2β1 integrin ligand). However, on laminin-5 (α3β1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to α3β1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced α3β1–CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance α3β1–CD81 complex formation. These results show how laterally associated EWI-2 might regulate α3β1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.
ASalmonellaprotein causes macrophage cell death by inducing autophagy
Authors: Hernandez Lorraine D. , Pypaert Marc , Flavell Richard A. , Galán Jorge E.
Published: December 2003

Salmonella enterica, the causative agent of food poisoning and typhoid fever, induces programmed cell death in macrophages, a process found to be dependent on a type III protein secretion system, and SipB, a protein with membrane fusion activity that is delivered into host cells by this system. When expressed in cultured cells, SipB caused the formation of and localized to unusual multimembrane structures. These structures resembled autophagosomes and contained both mitochondrial and endoplasmic reticulum markers. A mutant form of SipB devoid of membrane fusion activity localized to mitochondria, but did not induce the formation of membrane structures. Upon Salmonella infection of macrophages, SipB was found in mitochondria, which appeared swollen and devoid of christae. Salmonella-infected macrophages exhibited marked accumulation of autophagic vesicles. We propose that Salmonella, through the action of SipB, kills macrophages by disrupting mitochondria, thereby inducing autophagy and cell death.
Peritoneal macrophages express both P-selectin and PSGL-1
Authors: Tchernychev Boris , Furie Bruce , Furie Barbara C.
Published: December 2003

Macrophages, phagocytic cells involved in an early phase of host defense, are known to express the P-selectin ligand, PSGL-1. Heretofore, P-selectin has only been found on platelets and endothelial cells. Here, we demonstrate that peritoneal macrophages isolated by peritoneal lavage of unchallenged mice express P-selectin on the plasma membrane. The peritoneal macrophages synthesize P-selectin, as indicated by metabolic labeling experiments. P-Selectin is constitutively expressed on the extracellular surface of macrophages but is only partially colocalized with PSGL-1. P-Selectin is rapidly translocated from the macrophage plasma membrane to intracellular vesicles and to lysosomes. Peritoneal macrophages assemble into cell strings under flow conditions based upon macrophage–macrophage interactions mediated by P-selectin and PSGL-1. This is the first description of a leukocyte shown to express both P-selectin and PSGL-1.
Water: the bloodstream of the biosphere
Authors: Ripl Wilhelm
Published: December 2003

Water, the bloodstream of the biosphere, determines the sustainability of living systems. The essential role of water is expanded in a conceptual model of energy dissipation, based on the water balance of whole landscapes. In this model, the underlying role of water phase changes--and their energy-dissipative properties--in the function and the self-organized development of natural systems is explicitly recognized. The energy-dissipating processes regulate the ecological dynamics within the Earth's biosphere, in such a way that the development of natural systems is never allowed to proceed in an undirected or random way. A fundamental characteristic of self-organized development in natural systems is the increasing role of cyclic processes while loss processes are correspondingly reduced. This gives a coincidental increase in system efficiency, which is the basis of growing stability and sustainability. Growing sustainability can be seen as an increase of ecological efficiency, which is applicable at all levels up to whole landscapes. Criteria for necessary changes in society and for the design of the measures that are necessary to restore sustainable landscapes and waters are derived.
Global analysis of river systems: from Earth system controls to Anthropocene syndromes
Authors: Meybeck Michel
Published: December 2003

Continental aquatic systems from rivers to the coastal zone are considered within two perspectives: (i) as a major link between the atmosphere, pedosphere, biosphere and oceans within the Earth system with its Holocene dynamics, and (ii) as water and aquatic biota resources progressively used and transformed by humans. Human pressures have now reached a state where the continental aquatic systems can no longer be considered as being controlled by only Earth system processes, thus defining a new era, the Anthropocene. Riverine changes, now observed at the global scale, are described through a first set of syndromes (flood regulation, fragmentation, sediment imbalance, neo–arheism, salinization, chemical contamination, acidification, eutrophication and microbial contamination) with their related causes and symptoms. These syndromes have direct influences on water uses, either positive or negative. They also modify some Earth system key functions such as sediment, water, nutrient and carbon balances, greenhouse gas emissions and aquatic biodiversity. Evolution of river syndromes over the past 2000 years is complex: it depends upon the stages of regional human development and on natural conditions, as illustrated here for the chemical contamination syndrome. River damming, eutrophication and generalized decrease of river flow due to irrigation are some of the other global features of river changes. Future management of river systems should also consider these long–term impacts on the Earth system.
Groundwater: the processes and global significance of aquifer degradation
Authors: Foster S. S. D. , Chilton P. J.
Published: December 2003

The exploitation of groundwater resources for human use dates from the earliest civilizations, but massive resource development has been largely restricted to the past 50 years. Although global in scope, the emphasis of this paper is on groundwater–based economies in a developing nation context, where accelerated resource development has brought major social and economic benefits over the past 20 years. This results from groundwater's significant role in urban water supply and in rural livelihoods, including irrigated agriculture. However, little of the economic benefit of resource development has been reinvested in groundwater management, and concerns about aquifer degradation and resource sustainability began to arise.

A general review, for a broad–based audience, is given of the mechanisms and significance of three semi–independent facets of aquifer degradation. These are (i) depletion of aquifer storage and its effects on groundwater availability, terrestrial and aquatic ecosystems; (ii) groundwater salinization arising from various different processes of induced hydraulic disturbance and soil fractionation; and (iii) vulnerability of aquifers to pollution from land–use and effluent discharge practices related to both urban development and agricultural intensification.

Globally, data with which to assess the status of aquifer degradation are of questionable reliability, inadequate coverage and poor compilation. Recourse has to be made to ‘type examples’ and assumptions about the extension of similar hydrogeological settings likely to be experiencing similar conditions of groundwater demand and subsurface contaminant load. It is concluded that (i) aquifer degradation is much more than a localized problem because the sustainability of the resource base for much of the rapid socio–economic development of the second half of the twentieth century is threatened on quite a widespread geographical basis; and (ii) major (and long overdue) investments in groundwater resource and quality protection are urgently needed. These investments include appropriate institutional provisions, demand–side management, supply–side enhancement and pollution control.
Land cover change and water vapour flows: learning from Australia
Authors: Gordon Line , Dunlop Michael , Foran Barney
Published: December 2003

Australia is faced with large–scale dryland salinization problems, largely as a consequence of the clearing of native vegetation for cropland and grassland. We estimate the change in continental water vapour flow (evapotranspiration) of Australia during the past 200 years. During this period there has been a substantial decrease in woody vegetation and a corresponding increase in croplands and grasslands. The shift in land use has caused aca. 10% decrease in water vapour flows from the continent. This reduction corresponds to an annual freshwater flow of almost 340 km3. The society–induced alteration of freshwater flows is estimated at more than 15 times the volume of run–off freshwater that is diverted and actively managed in the Australian society. These substantial water vapour flow alterations were previously not addressed in water management but are now causing serious impacts on the Australian society and local economies. Global and continental freshwater assessments and policy often neglects the interplay between freshwater flows and landscape dynamics. Freshwater issues on both regional and global levels must be rethought and the interplay between terrestrial ecosystems and freshwater better incorporated in freshwater and ecosystem management.
Dimensions and approaches for Third World city water security
Authors: Lundqvist Jan , Appasamy Paul , Nelliyat Prakash
Published: December 2003

A rapid expansion of urban systems, particularly in less–developed countries, pose considerable challenges. Urbanization also provides opportunities for socio–economic progress. Relative contribution from the urban sector to national economic growth is very high. The fate and the role of the socio–economic system in local, regional and national development hinges on many circumstances. Apart from delicate social issues, deficiencies in water provision, internal distribution and a hazardous water and environmental quality represent basic and tangible daily problems. Urban water security requires fresh thinking at two levels. Some kind of basin authority (corresponding to a county council, i.e. a formal administrative and regulatory body for the geographical area within a river basin) in combination with a national water policy is required, notably in countries that contemplate, or are in the process of implementing, regional and sometimes inter–basin schemes to augment supply to growing conglomerations. Similarly, the generation of large volumes of waste water and the associated threat to downstream areas cannot be effectively tackled through conventional urban planning. Within the urban area, and particularly in non–regulated parts, there is an urgent need for institutional arrangements that facilitate operations for providers who have the capacity and ability to function under the prevailing circumstances. Introduction of effective production and treatment technologies are other necessary and urgent prerequisites to reach urban water security in Third World cities.
The sharing of water between society and ecosystems: from conflict to catchment–based co–management
Authors: Wallace J. S. , Acreman M. C. , Sullivan C. A.
Published: December 2003

Human uses of freshwater resources are increasing rapidly as the world population rises. As this happens, less water is left to support aquatic and associated ecosystems. To minimize future human water shortages and undesirable environmental impacts, more equitable sharing of water resources between society and nature is required. This will require physical quantities and social values to be placed on both human and aquatic ecosystem requirements. Current water valuation systems are dominated by economic values and this paper illustrates new quantification and valuation methods that take more account of human well–being and environmental impacts.

The key to the effective implementation of these more equitable water allocation methods is the use of catchment–based integrated water resources management. This holistic framework makes it possible for human and ecosystem water requirements and the interactions between them to be better understood. This knowledge provides the foundation for incorporating relevant social factors so that water policies and laws can be developed to make best use of limited water resources. Catchment–based co–management can therefore help to ensure more effective sharing of water between people and nature.
Freshwater for resilience: a shift in thinking
Authors: Folke Carl
Published: December 2003

Humanity shapes freshwater flows and biosphere dynamics from a local to a global scale. Successful management of target resources in the short term tends to alienate the social and economic development process from its ultimate dependence on the life-supporting environment. Freshwater becomes transformed into a resource for optimal management in development, neglecting the multiple functions of freshwater in dynamic landscapes and its fundamental role as the bloodstream of the biosphere. The current tension of these differences in worldview is exemplified through the recent development of modern aquaculture contrasted with examples of catchment-based stewardship of freshwater flows in dynamic landscapes. In particular, the social and institutional dimension of catchment management is highlighted and features of social-ecological systems for resilience building are presented. It is concluded that this broader view of freshwater provides the foundation for hydrosolidarity.
Freshwater as shared between society and ecosystems: from divided approaches to integrated challenges
Authors: Falkenmark Malin
Published: December 2003

The paper has its focus on water's key functions behind ecosystem dynamics and the water–related balancing involved in a catchment–based ecosystem approach. A conceptual framework is being developed to address fundamental trade–offs between humans and ecosystems. This is done by paying attention to society's unavoidable landscape modifications and their unavoidable ecological effects mediated by water processes. Because the coevolution of societal and environmental processes indicates resonance rather than a cause–effect relationship, humanity will have to learn to live with change while securing ecosystem resilience. In view of the partial incompatibility of the social imperative of the millennium goals and its environmental sustainability goal, human activities and ecosystems have to be orchestrated for compatibility. To this end a catchment–based approach has to be taken by integrating water, land use and ecosystems. It is being suggested that ecosystem protection has to be thought of in two scales: site–specific biotic landscape components to be protected for their social value, and a catchment–based ecosystem approach to secure sustainable supply of crucial ecosystem goods and services on which social and economic development depends.
Water for food and nature in drought–prone tropics: vapour shift in rain–fed agriculture
Authors: Rockström Johan
Published: December 2003

This paper quantifies the eco–hydrological challenge up until 2050 of producing food in balance with goods and services generated by water–dependent ecosystems in nature. Particular focus is given to the savannah zone, covering 40% of the land area in the world, where water scarcity constitutes a serious constraint to sustainable development. The analysis indicates an urgent need for a new green revolution, which focuses on upgrading rain–fed agriculture.

Water requirements to produce adequate diets for humans are shown to be relatively generic irrespective of hydro–climate, amounting to a global average of 1300 m
3
cap
−1
yr
−1
. Present food production requires an estimated 6800 km
3
yr
−1
of consumptive green water (5000 km
3
yr
−1
in rain–fed agriculture and 1800 km
3
yr
−1
from irrigated crops). Without considering water productivity gains, an additional 5800 km
3
yr
−1
of water is needed to feed a growing population in 2050 and eradicate malnutrition. It is shown that the bulk of this water will be used in rain–fed agriculture.

A dynamic analysis of water productivity and management options indicates that large ‘crop per drop’ improvements can be achieved at the farm level. Vapour shift in favour of productive green water flow as crop transpiration could result in relative water savings of 500 km
3
yr
−1
in semi–arid rain–fed agriculture.
Integrated management of land and water resources based on a collective approach to fragmented international conventions
Authors: Duda Alfred M.
Published: December 2003

Interlinked crises of land degradation, food security, ecosystem decline, water quality and water flow depletion stand in the way of poverty reduction and sustainable development. These crises are made worse by increased fluctuations in climatic regimes. Single–purpose international conventions address these crises in a piecemeal, sectoral fashion and may not meet their objectives without greater attention to policy, legal, and institutional reforms related to: (i) balancing competing uses of land and water resources within hydrologic units; (ii) adopting integrated approaches to management; and (iii) establishing effective governance institutions for adaptive management within transboundary basins. This paper describes this global challenge and argues that peace, stability and security are all at stake when integrated approaches are not used. The paper presents encouraging results from a decade of transboundary water projects supported by the Global Environment Facility in developing countries that test practical applications of processes for facilitating reforms related to land and water that are underpinned by science–based approaches. Case studies of using these participative processes are described that collectively assist in the transition to integrated management. A new imperative for incorporatiing interlinkages among food, water, and environment security at the basin level is identified.
Introduction
Authors: Falkenmark Malin , Folke Carl
Published: December 2003

Introduction
Two MerR homologues that affect copper induction of the Bacillus subtilis
copZA operon
Authors: Gaballa Ahmed , Cao Min , Helmann John D.
Published: December 2003

Copper ions induce expression of the Bacillus subtilis
copZA operon encoding a metallochaperone, CopZ, and a CPx-type ATPase efflux protein, CopA. The copZA promoter region contains an inverted repeat sequence similar to that recognized by the mercury-sensing MerR protein. To investigate the possible involvement of MerR homologues in copZA regulation, null mutations were engineered affecting each of four putative MerR-type regulators: yyaN, yraB, yfmP and yhdQ. Two of these genes affected copper regulation. Mutation of yhdQ (hereafter renamed cueR) dramatically reduced copper induction of copZA, and purified CueR bound with high affinity to the copZA promoter region. These results suggest that CueR is a direct regulator of copZA transcription that mediates copper induction. Surprisingly, a yfmP mutation also reduced copper induction of copZA. Sequence analysis suggested that yfmP was cotranscribed with yfmO, encoding a putative multidrug efflux protein. The yfmPO operon is autoregulated: a yfmP mutation derepressed the yfmP promoter and purified YfmP bound the yfmP promoter region, but not the copZA promoter region. Since the yfmP mutant strain was predicted to express elevated levels of the YfmO efflux pump, it was hypothesized that copper efflux might be responsible for the reduced copZA induction. Consistent with this model, in a yfmP
yfmO double mutant copper induction of copZA was normal. The results demonstrate the direct regulation of the B. subtilis copper efflux system by CueR, and indirect regulation by a putative multidrug efflux system.
Compaction of the Escherichia coli nucleoid caused by Cyt1Aa
Authors: Manasherob Robert , Zaritsky Arieh , Metzler Yifah , Ben-Dov Eitan , Itsko Mark , Fishov Itzhak
Published: December 2003

Compaction of theEscherichia colinucleoid in the cell's centre was associated with the loss of colony-forming ability; these effects were caused by induction of Cyt1Aa, the cytotoxic 27 kDa protein fromBacillus thuringiensissubsp.israelensis. Cyt1Aa-affected compaction of the nucleoids was delayed but eventually more intense than compaction caused by chloramphenicol. The possibility that small, compact nucleoids in Cyt1Aa-expressing cells resulted in DNA replication run-out and segregation following cell division was ruled out by measuring relative nucleoid length. Treatments with membrane-perforating substances other than Cyt1Aa did not cause such compaction of the nucleoids, but rather the nucleoids overexpanded to occupy nearly all of the cell volume. These findings support the suggestion that, in addition to its perforating ability, Cyt1Aa causes specific disruption of nucleoid associations with the cytoplasmic membrane.In situimmunofluorescence labelling with Alexa did not demonstrate a great amount of Cyt1Aa associated with the membrane. Clear separation between Alexa-labelled Cyt1Aa and 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA indicates that the nucleoid does not bind Cyt1Aa. Around 2 h after induction, nucleoids in Cyt1Aa-expressing cells started to decompact and expanded to fill the whole cell volume, most likely due to partial cell lysis without massive peptidoglycan destruction.
Mitochondrial-type iron–sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum
Authors: LaGier Michael J. , Tachezy Jan , Stejskal Frantisek , Kutisova Katerina , Keithly Janet S.
Published: December 2003

Several reports have indicated that the iron–sulfur cluster [Fe–S] assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterized and most highly conserved [Fe–S] assembly proteins are a pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe–S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CpIscS and CpIscU) and phylogenetic studies (CpIscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CpIscS and CpIscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe–S] assembly.
Identification of functionally important regions of a haemoglobin receptor from Neisseria meningitidis
Authors: Perkins-Balding D. , Baer M. T. , Stojiljkovic I.
Published: December 2003

The HmbR outer-membrane receptor enablesNeisseria meningitidisto use haemoglobin (Hb) as a source of iron. This protein functions by binding Hb, removing haem from it, and releasing the haem into the periplasm. Functionally important HmbR receptor domains were discerned using a series of HmbR deletions and site-directed mutations. Mutations exhibiting similar defective phenotypes inN. meningitidisfell into two groups. The first group of mutations affected Hb binding and were located in putative extracellular loops (L) L2 (amino acid residues (aa) 192–230) and L3 (aa 254–284). The second group of mutations resulted in a failure to utilize Hb but proficiency in Hb binding was retained. These mutations localized to the putative extracellular loops L6 (aa 420–462) and L7 (aa 486–516). A highly conserved protein motif found in all haem/Hb receptors, within putative extracellular loop L7 of HmbR, is essential for Hb utilization but not required for Hb binding. This finding suggests a mechanistic involvement of this motif in haem removal from Hb. In addition, an amino-terminal deletion in the putative cork-like domain of HmbR affected Hb usage but not Hb binding. This result supports a role of the cork domain in utilization steps that are subsequent to Hb binding.
Gradual evolution in bacteria: evidence from Bacillus systematics
Authors: Feldgarden Michael , Byrd Noah , Cohan Frederick M.
Published: December 2003

The bacterial genome projects have suggested a central role for horizontal transfer in bacterial adaptation, but it is difficult to rule out an adaptive role for ordinary genetic change in existing genes. The bacterial systematics literature can readily address the importance of gene acquisition in adaptive evolution, since phenotypic characterization typically assesses presence versus absence of metabolic capabilities, and metabolic gains and losses are most likely due to horizontal transfer and/or gene loss. Bacterial systematists have not geared their studies toward quantitative differences in metabolic capabilities, which are more likely to involve adjustments of existing genes. Here, quantitative variation in metabolism within and between three closely relatedBacillustaxa has been assayed. While these taxa show no qualitative (i.e. presence versus absence) differences in resource utilization, they are quantitatively different in utilization of 8 % of 95 resources tested. Moreover, 93 % of the resources tested showed significant quantitative variation among strains within a single taxon. These results suggest that ordinary genetic changes in existing genes may play an important role in adaptation. If these results are typical, future genomically based assays of quantitative variation in phenotype (e.g. microarray analysis of mRNA concentrations) may identify hundreds of genes whose expression has been modified. A protocol is presented for identifying those modifications of gene expression and those gene acquisitions that are most likely to have played a role in adaptive evolution.
The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects
Authors: Gordon David M. , Cowling Ann
Published: December 2003

Escherichia coliwas isolated from more than 2300 non-domesticated vertebrate hosts living in Australia.E. coliwas most prevalent in mammals, less prevalent in birds and uncommon in fish, frogs and reptiles. Mammals were unlikely to harbourE. coliif they lived in regions with a desert climate and less likely to haveE. coliif they lived in the tropics than if they lived in semi-arid or temperate regions. In mammals, the likelihood of isolatingE. colifrom an individual depended on the diet of the host andE. coliwas less prevalent in carnivores than in herbivores or omnivores. In both birds and mammals, the probability of isolatingE. coliincreased with the body mass of the host. Hosts living in close proximity to human habitation were more likely to harbourE. colithan hosts living away from people. The relative abundance ofE. coligroups A, B1, B2 and D strains in mammals depended on climate, host diet and body mass. Group A strains were uncommon, but were isolated from both ectothermic and endothermic vertebrates. Group B1 strains could also be isolated from any vertebrate group, but were predominant in ectothermic vertebrates, birds and carnivorous mammals. Group B2 strains were unlikely to be isolated from ectotherms and were most abundant in omnivorous and herbivorous mammals. Group D strains were rare in ectotherms and uncommon in endotherms, but were equally abundant in birds and mammals. The results of this study suggest that, at the species level, the ecological niche ofE. coliis mammals with hindgut modifications to enable microbial fermentation, or in the absence of a modified hindgut,E. colican only establish a population in ‘large-bodied’ hosts. The non-random distribution ofE. coligenotypes among the different host groups indicates that strains of the fourE. coligroups may differ in their ecological niches and life-history characteristics.
Extracting phylogenetic information from whole-genome sequencing projects: the lactic acid bacteria as a test case
Authors: Coenye Tom , Vandamme Peter
Published: December 2003

The availability of an ever increasing number of complete genome sequences of diverse prokaryotic taxa has led to the introduction of novel approaches to infer phylogenetic relationships among bacteria. In the present study the sequences of the 16S rRNA gene and nine housekeeping genes were compared with the fraction of shared putative orthologous protein-encoding genes, conservation of gene order, dinucleotide relative abundance and codon usage among 11 genomes of species belonging to the lactic acid bacteria. In general there is a good correlation between the results obtained with various approaches, although it is clear that there is a stronger phylogenetic signal in some datasets than in others, and that different parameters have different taxonomic resolutions. It appears that trees based on different kinds of information derived from whole-genome sequencing projects do not provide much additional information about the phylogenetic relationships among bacterial taxa compared to more traditional alignment-based methods. Nevertheless, it is expected that the study of these novel forms of information will have its value in taxonomy, to determine which genes are shared, when genes or sets of genes were lost in evolutionary history, to detect the presence of horizontally transferred genes and/or confirm or enhance the phylogenetic signal derived from traditional methods. Although these conclusions are based on a relatively small dataset, they are largely in agreement with other studies and it is anticipated that similar trends will be observed when comparing other genomes.
PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress
Authors: Chandu Dilip , Nandi Dipankar
Published: December 2003

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity inEscherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and twopepNmutants, 9218 and DH5αΔpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in bothpepNmutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in bothpepNmutants. This decreased ability to cleave these substrates ispepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates inE. colimutants lacking other peptidases,pepA,pepBorpepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity inE. coli. Furtherin vitrostudies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference ofE. coliPepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress inE. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.
Pathways for phosphatidylcholine biosynthesis in bacteria
Authors: Martínez-Morales Fernando , Schobert Max , López-Lara Isabel M. , Geiger Otto
Published: December 2003

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipidN-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated thatSinorhizobium meliloti,Agrobacterium tumefaciens,Rhizobium leguminosarum,Bradyrhizobium japonicum,Mesorhizobium lotiandLegionella pneumophilahave both PMT and PCS activities. In addition,Rhodobacter sphaeroideshas PMT activity andBrucella melitensis,Pseudomonas aeruginosaandBorrelia burgdorferihave PCS activities. Genes fromM. lotiandL. pneumophilaencoding a Pmt or a Pcs activity and the genes fromP. aeruginosaandBorrelia burgdorferiresponsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such asBrucella melitensis,P. aeruginosaandBorrelia burgdorferiseem to be exceptional as they possess only the PCS pathway for PC formation.
Variation of the natural transformation frequency of Campylobacter jejuni in liquid shake culture
Authors: Wilson David L. , Bell Julia A. , Young Vincent B. , Wilder Stacey R. , Mansfield Linda S. , Linz John E.
Published: December 2003

Natural transformation, a mechanism that generates genetic diversity in Campylobacter jejuni, was studied in a novel liquid shake culturing system that allowed an approximately 10 000-fold increase in cell density. C. jejuni transformation frequency was analysed in this system under 10 %, 5·0 % and 0·7 % CO2 atmospheres. At 5·0 % and 10 % CO2 concentrations, when purified isogenic chromosomal DNA was used to assess competence, transformation frequency ranged from 10−3 to 10−4 at low cell concentrations and declined as cell density increased. Transformation frequency under a 0·7 % CO2 atmosphere was more stable, maintaining 10−3 levels at high cell densities, and was 10- to 100-fold higher than that under a 10 % CO2 atmosphere. Three of four C. jejuni strains tested under a 5·0 % CO2 atmosphere were naturally competent for isogenic DNA; competent strains demonstrated a lack of barriers to intraspecies genetic exchange by taking up and incorporating chromosomal DNA from multiple C. jejuni donors. C. jejuni showed a preference for its own DNA at the species level, and co-cultivation demonstrated that DNA transfer via natural transformation occurred between isogenic populations during short periods of exposure in liquid medium when cell density and presumably DNA concentrations were low. Transformation frequency during co-cultivation of isogenic populations was also influenced by CO2 concentration. Under a 0·7 % CO2 atmosphere, co-cultivation transformation frequency increased approximately 500-fold in a linear fashion with regard to cell density, and was 1000- to 10 000-fold higher during late-exponential-phase growth when compared to cultures grown under a 10 % CO2 atmosphere.
Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system
Authors: Tomaras Andrew P. , Dorsey Caleb W. , Edelmann Richard E. , Actis Luis A.
Published: December 2003

Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid–air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.
Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans
Authors: Mazoch Jiří , Kuňák Michal , Kučera Igor , van Spanning Rob J. M.
Published: December 2003

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, β-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of β-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic β-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N2O. Additions of nitrate or nitroprusside lowered β-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.
Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens
Authors: Baysse Christine , Matthijs Sandra , Schobert Max , Layer Gunhild , Jahn Dieter , Cornelis Pierre
Published: December 2003

The cytoplasmic membrane protein CcmC is, together with other Ccm proteins, a component for the maturation of c-type cytochromes in Gram-negative bacteria. A Pseudomonas fluorescens ATCC 17400 ccmC mutant is cytochrome c-deficient and shows considerably reduced production of the two siderophores pyoverdine and quinolobactin, paralleled by a general inability to utilize various iron sources, with the exception of haem. The ccmC mutant accumulates in a 5-aminolevulinic acid-dependent synthesis a reddish, fluorescent pigment identified as protoporphyrin IX. As a consequence a visA phenotype similar to that of a ferrochelatase-deficient hemH mutant characterized by drastically reduced growth upon light exposure was observed for the ccmC mutant. The defect of iron–protoporphyrin formation was further demonstrated by the failure of ccmC cell-free proteinase K-treated extracts to stimulate the growth of a haem auxotrophic hemH indicator strain, compared to similarly prepared wild-type extracts. In addition, the ccmC mutant did not sustain hemH growth in cross-feeding experiments while the wild-type did. Significantly reduced resistance to oxidative stress mediated by haem-containing catalases was observed for the ccmC mutant. A double hemH ccmC mutant could not be obtained in the presence of external haem without the hemH gene in trans, indicating that the combination of the two mutations is lethal. It was concluded that CcmC, apart from its known function in cytochrome c biogenesis, plays a role in haem biosynthesis. A function in the regulatory co-ordination of iron acquisition via siderophores, iron insertion into porphyrin via ferrochelatase and iron–protoporphyrin export for cytochrome c formation is predicted.
Use of a two-hybrid assay to study the assembly of a complex multicomponent protein machinery: bacterial septosome differentiation
Authors: Di Lallo G. , Fagioli M. , Barionovi D. , Ghelardini P. , Paolozzi L.
Published: December 2003

The ability of each of the nine Escherichia coli division proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, FtsN) to interact with itself and with each of the remaining eight proteins was studied in 43 possible combinations of protein pairs by the two-hybrid system previously developed by the authors' group. Once the presumed interactions between the division proteins were determined, a model showing their temporal sequence of assembly was developed. This model agrees with that developed by other authors, based on the co-localization sequence in the septum of the division proteins fused with GFP. In addition, this paper shows that the authors' assay, which has already proved to be very versatile in the study of prokaryotic and eukaryotic protein interaction, is also a powerful instrument for an in vivo study of the interaction and assembly of proteins, as in the case of septum division formation.
Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum
Authors: Inoue Kaoru , Sobhany Mack , Transue Thomas R. , Oguma Keiji , Pedersen Lars C. , Negishi Masahiko
Published: December 2003

Botulism food poisoning is caused primarily by ingestion of the Clostridium botulinum neurotoxin (BoNT). The 1300 amino acid BoNT forms a progenitor toxin (PTX) that, when associated with a number of other proteins, increases its oral toxicity by protecting it from the low pH of the stomach and from intestinal proteases. One of these associated proteins, HA1, has also been suggested to be involved with internalization of the toxin into the bloodstream by binding to oligosaccharides lining the intestine. Here is reported the crystal structure of HA1 from type C Clostridium botulinum at a resolution of 1·7 Å. The protein consists of two β-trefoil domains and bears structural similarities to the lectin B-chain from the deadly plant toxin ricin. Based on structural comparison to the ricin B-chain lactose-binding sites, residues of type A HA1 were selected and mutated. The D263A and N285A mutants lost the ability to bind carbohydrates containing galactose moieties, implicating these residues in carbohydrate binding.
Facing extremes: archaeal surface-layer (glyco)proteins
Authors: Eichler Jerry
Published: December 2003

Archaea are best known in their capacities as extremophiles, i.e. micro-organisms able to thrive in some of the most drastic environments on Earth. The protein-based surface layer that envelopes many archaeal strains must thus correctly assemble and maintain its structural integrity in the face of the physical challenges associated with, for instance, life in high salinity, at elevated temperatures or in acidic surroundings. Study of archaeal surface-layer (glyco)proteins has thus offered insight into the strategies employed by these proteins to survive direct contact with extreme environments, yet has also served to elucidate other aspects of archaeal protein biosynthesis, including glycosylation, lipid modification and protein export. In this mini-review, recent advances in the study of archaeal surface-layer (glyco)proteins are discussed.
A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus
Authors: Battermann Anja , Disse-Krömker Claudia , Dreiseikelmann Brigitte
Published: December 2003

Plasmid analysis of isolates from a smallParacoccuspopulation revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a completerrnoperon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNAIleand tRNAAlawithin the spacer between the 16S and 23S rRNA genes, and a final tRNAfMetat the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that therrnpromoter region is functional. Therrnoperon encoded by plasmid pOL18/23 is the first completerrnoperon sequenced from a strain of the genusParacoccus, and only the second example of anrrnoperon on a small plasmid.
The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli
Authors: Jovanovic Milija , Lilic Mirjana , Savic Dragutin J. , Jovanovic Goran
Published: December 2003

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor inEscherichia colicells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. ThehslJgene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium.cysBmutants showed upregulation of thehslJ : : lacZgene fusion and exhibited increased novobiocin resistance. In this study thehslJtranscription start point and the corresponding putativeσ70promoter were determined. ThehslJpromoter region was defined by employing differenthslJ–lacZoperon fusions, and transcription of thehslJgene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control. These two regulations compete to some extent but they are not mutually exclusive. CysB acts as a direct repressor ofhslJtranscription and binds thehslJpromoter region that carries the putative CysB repressor site. This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligandN-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations. Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression ofhslJtranscription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy.