The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn²⁺ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn²⁺ oxidation and/or secretion of the Mn²⁺-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn²⁺ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn²⁺-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn²⁺ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.In a number of studies during the last three decades it has been shown that various microbial species are able to stimulate the oxidation of Mn²⁺ through direct catalysis. These organisms produce proteinaceous macromolecules which catalyze the oxidation reaction. Manganese oxidations by a soil Arthrobacter species (24), Oceanospirillum and Vibrio strains (2, 3), Pseudomonas putida MnB1 (22, 30), Leptothrix discophora SS-1 (1, 11), and marine Bacillus strain SG-1 (23) are examples in which enzymes are most likely involved in the process. P. putida MnB1 produces a soluble protein which catalytically oxidizes Mn²⁺ in cell extracts (22). Manganese-oxidizing proteins from L. discophora SS-1 (1, 11) and from the spore coats of Bacillus strain SG-1 (43) have been identified on polyacrylamide gels. The oxidizing proteins have not been quantitatively purified or analyzed so far. In Bacillus strain SG-1, an operon containing seven genes appears to be involved in Mn²⁺ oxidation (46). One of these genes encodes a 137-kDa protein related to the family of multicopper oxidases (47). In a previous study we reported the isolation of a structural gene and its promoter postulated to be involved in Mn²⁺ oxidation in L. discophora (19). The encoded protein also contains the copper-binding signatures of multicopper oxidases. The oxidase-related proteins may represent Mn²⁺-oxidizing enzymes (44), but evidence supporting this hypothesis is still lacking.In this paper we describe a genetic analysis of Mn²⁺ oxidation in a freshwater Pseudomonas strain, strain GB-1. In a previous study (32) this strain was preliminarily identified as a Pseudomonas fluorescens strain, but more recent data (see Materials and Methods) indicate that it should be identified as a P. putida strain. When supplied with Mn²⁺ ions, the cells deposit manganese oxide around the outer membrane in the early stationary growth phase (32). They form brown colonies on Mn²⁺-containing agar. Experiments performed with cell extracts indicated that Mn²⁺ oxidation is catalyzed by a protein. The Mn²⁺-oxidizing factor was partially purified, and electrophoresis on an acrylamide gradient gel revealed oxidizing proteins with apparent molecular weights of ca. 250,000 and 180,000 (32). An additional oxidizing factor with a lower molecular weight (ca. 130,000) was identified in another study by using different isolation and electrophoretic procedures (16). It has been suggested that the Mn²⁺-oxidizing protein isolated is part of a larger complex which disintegrates into smaller fragments that retain activity (32). The protein is supposed to be located in the outer membrane of the bacteria. It has not been chemically characterized, and nothing is known about its cellular function or about the possible involvement of other cellular components, such as electron carriers, in Mn²⁺ oxidation.We used transposon mutagenesis to identify genes relevant to the Mn²⁺-oxidizing process in P. putida GB-1. One of these genes appeared to be part of the cytochrome c maturation operon. Transposon insertion in this gene not only abolished Mn²⁺ oxidation but also led to secretion of siderophores and porphyrins.An accompanying report on the involvement of the cytochrome c maturation operon in Mn²⁺ oxidation in P. putida MnB1 (14) supports our findings.     

cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.     

cumA Multicopper Oxidase Genes from Diverse Mn(II)-Oxidizing and Non-Mn(II)-Oxidizing Pseudomonas Strains

A multicopper oxidase gene, cumA, required for Mn(II) oxidation was recently identified in Pseudomonas putida strain GB-1. In the present study, degenerate primers based on the putative copper-binding regions of the cumA gene product were used to PCR amplify cumA gene sequences from a variety of Pseudomonas strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains. The presence of highly conserved cumA gene sequences in several apparently non-Mn(II)-oxidizing Pseudomonas strains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms. Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters. Overall, our results indicate that both the cumA gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse Pseudomonas strains.     

The Adhesion-Associated sca Operon in Streptococcus gordonii Encodes an Inducible High-Affinity ABC Transporter for Mn2+ Uptake

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of ⁵⁴Mn²⁺ uptake in media containing <0.5 μM Mn²⁺. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn²⁺ (<0.5 μM) and by the addition of ≥20 μM Zn²⁺. Sca permease-mediated uptake of ⁵⁴Mn²⁺ was inhibited by Zn²⁺ but not by Ca²⁺, Mg²⁺, Fe²⁺, or Cu²⁺. Reduced uptake of ⁵⁴Mn²⁺ by sca mutants and by wild-type cells in the presence of Zn²⁺ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn²⁺ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in sca mutants. The addition of 0.1 mM Mn²⁺ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn²⁺ and is essential for growth of streptococci under Mn²⁺-limiting conditions.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

Identification of a Two-Component Regulatory Pathway Essential for Mn(II) Oxidation in Pseudomonas putida GB-1

Bacterial manganese(II) oxidation has a profound impact on the biogeochemical cycling of Mn and the availability of the trace metals adsorbed to the surfaces of solid Mn(III, IV) oxides. The Mn(II) oxidase enzyme was tentatively identified in Pseudomonas putida GB-1 via transposon mutagenesis: the mutant strain GB-1-007, which fails to oxidize Mn(II), harbors a transposon insertion in the gene cumA. cumA encodes a putative multicopper oxidase (MCO), a class of enzymes implicated in Mn(II) oxidation in other bacterial species. However, we show here that an in-frame deletion of cumA did not affect Mn(II) oxidation. Through complementation analysis of the oxidation defect in GB-1-007 with a cosmid library and subsequent sequencing of candidate genes we show the causative mutation to be a frameshift within the mnxS1 gene that encodes a putative sensor histidine kinase. The frameshift mutation results in a truncated protein lacking the kinase domain. Multicopy expression of mnxS1 restored Mn(II) oxidation to GB-1-007 and in-frame deletion of mnxS1 resulted in a loss of oxidation in the wild-type strain. These results clearly demonstrated that the oxidation defect of GB-1-007 is due to disruption of mnxS1, not cumA::Tn5, and that CumA is not the Mn(II) oxidase. mnxS1 is located upstream of a second sensor histidine kinase gene, mnxS2, and a response regulator gene, mnxR. In-frame deletions of each of these genes also led to the loss of Mn(II) oxidation. Therefore, we conclude that the MnxS1/MnxS2/MnxR two-component regulatory pathway is essential for Mn(II) oxidation in P. putida GB-1.In living cells, manganese (Mn) is an essential trace element, required for enzymes such as superoxide dismutase and in photosystem II (7). In the environment, Mn cycles between a soluble reduced form [Mn(II)] and an insoluble oxidized form [Mn(III, IV)] that can adsorb other trace metals from the environment and serve as potent oxidizing agents. Thus, redox cycling of Mn has a profound effect on the bioavailability and geochemical cycling of many essential or toxic elements (40). Microorganisms, particularly bacteria, are capable of catalyzing the oxidation of Mn(II), thereby increasing the rate of formation of Mn(III, IV) by several orders of magnitude (39). Since Mn(III, IV) oxides are able to bind trace metals, the bacteria that catalyze their formation are good candidates for bioremediation of heavy metal contaminated sites (26, 39).Although bacterial Mn(II) oxidation is widespread, little is known about the physiological function of oxidation (40). The oxidation of Mn(II) to Mn(III) or Mn(IV) is thermodynamically favorable; thus, bacteria may derive energy from this reaction, although this has never been unequivocally proven (40). In addition, Mn(II) oxidation could protect cells from reactive oxygen species (4) or UV irradiation (11). Since oxidation occurs on the cell surface, the bacteria become coated with the solid Mn(IV) oxides, which may also provide protection from toxic heavy metals, predation, or phage infection (40). As a strong oxidant, Mn(IV) oxides could allow the bacteria to degrade refractory organic matter to low-molecular-weight compounds that could then be used to support bacterial growth (38). Conversely, Mn(II) oxidation may be a side reaction or the result of nonspecific interactions with cellular products (15). Identifying signals or conditions that regulate oxidation could provide some insight into the role of Mn(II) oxidation in the cell. Aside from a requirement for oxygen (28) and iron (27, 30), as well as the observation that oxidation occurs in stationary phase (23), very little is known about this regulation.The enzymes responsible for Mn(II) oxidation have been tentatively identified from some species of bacteria and in several cases the enzyme is a putative multicopper oxidase (MCO). MCOs are a family of enzymes that use four Cu ion cofactors to catalyze oxidation of diverse substrates such as metals and organic compounds (33). This family of enzymes is found in plants and fungi (laccase) and humans (ceruloplasmin), as well as in bacteria (35). Some fungi have been shown to use a laccase enzyme to oxidize Mn(II) (20). In both Leptothrix discophora SS-1 and Pedomicrobium sp. strain ACM 3067, the Mn(II)-oxidizing MCO was identified genetically (mofA [10] and moxA [31], respectively). A third MCO—MnxG—was identified both biochemically and genetically as the Mn(II) oxidase in Bacillus sp. strain SG-1 and related strains (14, 43). Recent work with the Mn(II)-oxidizing alphaproteobacterium Aurantimonas manganoxydans SI85-9A1 and Erythrobacter sp. strain SD21 has identified a second class of enzyme involved in Mn(II) oxidation: the heme-binding peroxidase named MopA (3). This class of enzyme had previously been shown to be used by fungi to oxidize Mn(II) (29), in some cases in concert with an MCO (34).Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium (9) whose genetic tractability and ease of growth under standard laboratory conditions make it an ideal model system for studying the physiology and mechanism of Mn(II) oxidation. Consequently, several random transposon mutagenesis screens have been undertaken with this organism to identify genes required for Mn(II) oxidation. These screens have identified several categories of genes as important for oxidation or the export of the oxidase to the cell surface: the ccm operon of c-type cytochrome synthesis genes (8, 13), genes encoding components of the trichloroacetic acid (TCA) cycle and the tryptophan biosynthesis pathway (8) and genes encoding a general secretory pathway (12). The Mn(II) oxidation-defective mutant GB-1-007 has a transposon insertion in the gene cumA that encodes a putative MCO (6). Therefore, P. putida GB-1 has been thought to use a similar mechanism as L. discophora SS-1, Pedomicrobium sp. strain ACM 3067, and Bacillus sp. to oxidize Mn(II).Because the available data suggested that CumA was an MCO essential for Mn(II) oxidation, we wanted to study its function in greater detail. We were hampered in this, however, by the fact that the transposon insertion in cumA resulted in a growth defect due to its polar effect on expression of the downstream cumB gene (6). In order to assess the role of CumA in Mn(II) oxidation without the complications arising from polarity, we generated an in-frame deletion of cumA and tested the ability of the resulting ΔcumA strain to form Mn(IV) oxides. Our results showed that cumA is dispensable for Mn(II) oxidation and have instead revealed a complex two-component regulatory pathway essential for Mn(II) oxidation in P. putida GB-1.     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes

Manganese (Mn²⁺) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn²⁺-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn²⁺ concentration. Interestingly, at site III, Mg²⁺ alone promoted weak, but the same specific cleavage as Mn²⁺. When added with Mn²⁺, Mg²⁺ had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn²⁺ cleavage at site III also exhibited lower values of K (Mn²⁺ requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn²⁺-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.     

Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii

The essential coenzyme nicotinamide adenine dinucleotide (NAD⁺) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD⁺. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD⁺ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD⁺ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD⁺. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD⁺ metabolism and cell longevity.     

AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+ transporter

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na⁺, K⁺, and Mn²⁺ transport. We also report high-capacity uptake of ⁸⁶Rb⁺ in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of ⁸⁶Rb⁺ uptake in AtCCX3 cells by excess Na⁺, K⁺, and Mn²⁺. Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl₂. Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na⁺ accumulation and increased ⁸⁶Rb⁺ transport. Uptake of ⁸⁶Rb⁺ increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H⁺-dependent K⁺ transporter with apparent Na⁺ and Mn²⁺ transport properties distinct from those of previously characterized plant transporters.     

Spatially Resolved Characterization of Biogenic Manganese Oxide Production within a Bacterial Biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn⁺² [Mn⁺²_(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L_2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn⁺²_(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.     

A Combination of Three Mutations, dcd, pyrH, and cdd, Establishes Thymidine (Deoxyuridine) Auxotrophy in thyA+ Strains of Salmonella typhimurium

The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305–316, 1977). In the genetic background used in their selection, the joint acquisition of a dcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase. The level of UMP kinase activity in the dum mutant was found to be only 30% of that observed for the dum⁺ strain. Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH⁺ donor or by complementation with either of two pyrH⁺-carrying plasmids. Thymidine auxotrophy could be reconstructed in the dum⁺ derivative (KP1389) by the introduction of a mutant pyrH allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA⁺ strains, a dcd::Km null mutation was constructed. In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement. A third mutation, cdd (cytidine-deoxycytidine deaminase), was required together with the dcd and pyrH mutations to impart thymidine auxotrophy.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Inactivation of an Iron Transporter in Lactococcus lactis Results in Resistance to Tellurite and Oxidative Stress

In Lactococcus lactis, the interactions between oxidative defense, metal metabolism, and respiratory metabolism are not fully understood. To provide an insight into these processes, we isolated and characterized mutants of L. lactis resistant to the oxidizing agent tellurite (TeO₃²⁻), which generates superoxide radicals intracellularly. A collection of tellurite-resistant mutants was obtained using random transposon mutagenesis of L. lactis. These contained insertions in genes encoding a proton-coupled Mn²⁺/Fe²⁺ transport homolog (mntH), the high-affinity phosphate transport system (pstABCDEF), a putative osmoprotectant uptake system (choQ), and a homolog of the oxidative defense regulator spx (trmA). The tellurite-resistant mutants all had better survival than the wild type following aerated growth. The mntH mutant was found to be impaired in Fe²⁺ uptake, suggesting that MntH is a Fe²⁺ transporter in L. lactis. This mutant is capable of carrying out respiration but does not generate as high a final pH and does not exhibit the long lag phase in the presence of hemin and oxygen that is characteristic of wild-type L. lactis. This study suggests that tellurite-resistant mutants also have increased resistance to oxidative stress and that intracellular Fe²⁺ can heighten tellurite and oxygen toxicity.     

Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu²⁺ has been thoroughly reported, but the mechanism by which Cu²⁺ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu²⁺ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu²⁺ concentration. This suggests that supplemental Cu²⁺ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.     

Insertional Inactivation of Determinants for Mg2+ and Co2+ Transport as a Tool for Screening Recombinant Lactococcus Species Clones

Insertional inactivation of the plasmid-encoded determinants for Mg²⁺ and Co²⁺ transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl₂. The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl₂ that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.     

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α_1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 μM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.