Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H(+)-dependent F(0)F(1) type, one Na(+)-dependent V type).     

Urease-Encoding Genes in Ammonia-Oxidizing Bacteria

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the β-proteobacterium Nitrosospira sp. strain NpAV and the γ-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several β-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from β-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other β-proteobacteria. Instead, it appears that urease in β-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of γ- and β-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from β-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.     

Worldwide Distribution of Nitrosococcus oceani, a Marine Ammonia-Oxidizing γ-Proteobacterium, Detected by PCR and Sequencing of 16S rRNA and amoA Genes

Diversity of cultured ammonia-oxidizing bacteria in the γ-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of Nitrosococcus. These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.     

The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 reveals a large island involved in pathogenicity

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.     

Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO₂ fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min⁻¹mg protein⁻¹. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP_i. The enzyme was purified 95-fold to a specific activity of 18 μmol min⁻¹ mg protein⁻¹ from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO₂ fixation in this archaeon and possibly other members of the Sulfolobaceae family.     

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K⁺ (K⁺_in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K⁺_in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K⁺_in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs⁺, when applied externally, inhibited SKT1-mediated K⁺_in currents half-maximally with an inhibitor concentration (IC₅₀) of 105 μm. An almost identical high Cs⁺ sensitivity (IC₅₀ = 90 μm) was found for the potato guard-cell K⁺_in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺_in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.     

Biochemical and Genetic Characterization of an Extracellular Protease from Pseudomonas fluorescens CY091

Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl₂ or SrCl₂ but not in media containing ZnCl₂, MgCl₂, or MnCl₂. The requirement of Ca²⁺ (or Sr²⁺) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca²⁺ and Zn²⁺ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.     

Bioinformatic and Expression Analyses of Genes Mediating Zinc Homeostasis in Nostoc punctiforme

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn²⁺ over 14 days showed that ZnCl₂ levels above 22 μM significantly reduced cell viability. After 3 days in 22 μM ZnCl₂, ca. 12% of the Zn²⁺ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 μM relative to 0 μM treatment. Radiolabeled ⁶⁵Zn showed Zn²⁺ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn²⁺ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 μM ZnCl₂ within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.     

Complete Genome Sequence of Francisella tularensis Subspecies holarctica FTNF002-00

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD₂₃ deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997–1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also ∼5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1–1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H⁺ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.     

Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny,physiology and stable isotope fractionation

Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the North Pacific Ocean that have been maintained in laboratory culture for over 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98–99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH₃) to nitrite (NO₂⁻). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (¹⁵ɛ_NH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal ¹⁵ɛ_NH3 ranged from 13‰ to 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by ¹⁵ɛ_NH3 is similar.     

Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd²⁺ and Cu²⁺ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu²⁺ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu²⁺ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu²⁺ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.     

Properties of an Inwardly Rectifying ATP-sensitive K+ Channel in the Basolateral Membrane of Renal Proximal Tubule

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na⁺,K⁺-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K⁺ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K⁺ channels (γ_{slope, in} = 24.5 ± 0.6 pS, γ_{chord, out} = 3.7 ± 0.4 pS). The rectification is mediated in part by internal Mg²⁺. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding–unbinding model. The channel conducts Tl⁺ and K⁺, but there is no significant conductance for Na⁺, Rb⁺, Cs⁺, Li⁺, NH₄⁺, or Cl⁻. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0.2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K⁺ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K⁺ channel is the major ATP-sensitive basolateral K⁺ conductance in the proximal tubule.     

Source and Magnitude of Ammonium Generation in Maize Roots

Studies with ¹⁵N indicate that appreciable generation of NH₄⁺ from endogenous sources accompanies the uptake and assimilation of exogenous NH₄⁺ by roots. To identify the source of NH₄⁺ generation, maize (Zea mays L.) seedlings were grown on ¹⁴NH₄⁺ and then exposed for 3 d to highly labeled ¹⁵NH₄⁺. More of the entering ¹⁵NH₄⁺ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the ¹⁴NH₄⁺ content of roots declined rapidly to less than 1 μmol per plant, efflux of ¹⁴NH₄⁺ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative ¹⁴NH₄⁺ efflux during the 3-d period accounted for 25% of the total ¹⁴N initially present in the root. Although soluble organic ¹⁴N in roots declined during the 3-d period, insoluble ¹⁴N remained relatively constant. In shoots both soluble organic ¹⁴N and ¹⁴NH₄⁺ declined, but a comparable increase in insoluble ¹⁴N was noted. Thus, total ¹⁴N in shoots remained constant, reflecting little or no net redistribution of ¹⁴N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH₄⁺ generation in maize roots.     

Nitrate-ammonium synergism in rice. A subcellular flux analysis

Many reports have shown that plant growth and yield is superior on mixtures of NO₃⁻ and NH₄⁺ compared with provision of either N source alone. Despite its clear practical importance, the nature of this N-source synergism at the cellular level is poorly understood. In the present study we have used the technique of compartmental analysis by efflux and the radiotracer ¹³N to measure cellular turnover kinetics, patterns of flux partitioning, and cytosolic pool sizes of both NO₃⁻ and NH₄⁺ in seedling roots of rice (Oryza sativa L. cv IR72), supplied simultaneously with the two N sources. We show that plasma membrane fluxes for NH₄⁺, cytosolic NH₄⁺ accumulation, and NH₄⁺ metabolism are enhanced by the presence of NO₃⁻, whereas NO₃⁻ fluxes, accumulation, and metabolism are strongly repressed by NH₄⁺. However, net N acquisition and N translocation to the shoot with dual N-source provision are substantially larger than when NO₃⁻ or NH₄⁺ is provided alone at identical N concentrations.     

Capacitative Ca2+ Entry Is Closely Linked to the Filling State of Internal Ca2+ Stores: A Study Using Simultaneous Measurements of ICRAC and Intraluminal [Ca2+]

I_CRAC (the best characterized Ca²⁺ current activated by store depletion) was monitored concurrently for the first time with [Ca²⁺] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca²⁺] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca²⁺ chelator N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca²⁺] within the ER can be lowered and restored to its original level within 10–15 s without modifications of Ca²⁺ pumps or release channels. Using these new tools, we demonstrate here that Ca²⁺ release–activated Ca²⁺ current (I_CRAC) is activated (a) solely by reduction of free [Ca²⁺] within the ER and (b) by any measurable decrease in [Ca²⁺]_ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.     

The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

Unlike other characterized phages, the lytic coliphage N4 must inject the 360-kDa virion RNA polymerase (vRNAP), in addition to its 72-kbp genome, into the host for successful infection. The process of adsorption to the host sets up and elicits the necessary conformational changes in the virion to allow genome and vRNAP injection. Infection of suppressor and nonsuppressor strains, Escherichia coli W3350 supF and E. coli W3350, with a mutant N4 isolate (N4am229) harboring an amber mutation in Orf65 yielded virions containing (N4gp65⁺) and lacking (N4gp65⁻) gp65, respectively. N4gp65⁺ but not N4gp65⁻ phage was able to adsorb to the host. Recombinant gp65 with a hexahistidine tag at the N terminus or hexahistidine and c-myc tags at the C terminus was able to complement N4gp65⁻ virions in vivo and in vitro. Immunogold detection of gp65 in vivo complemented virions revealed its localization at the N4 tail. Finally, we show both in vitro and in vivo that gp65 interacts with the previously determined N4 outer membrane receptor, NfrA.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Influence of Starvation on Potential Ammonia-Oxidizing Activity and amoA mRNA Levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO_x biosensor. The apparent half-saturation constant [K_m(app)] value of the NH₃ oxidation of N. briensis was 3 μM NH₃ for cultures grown both in continuous and batch cultures as determined by a NO_x biosensor. Cells grown on the wall of the vessel had a lower K_m(app) value of 1.8 μM NH₃. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 μM N h⁻¹, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH₄⁺, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH₄⁺ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH₄⁺ availability.