Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages.

Brucella is an intracellular bacterial pathogen that causes chronic systemic infection in domesticated livestock and poses a zoonotic infectious risk to humans. The virulence of Brucella is critically dependent on its ability to replicate and survive within host macrophages. Brucella modulates host physiological pathways and cell biology in order to establish a productive intracellular replicative niche. Conversely, the host cell presumably activates pathways that limit infection. To identify host pathways contributing to this yin and yang during host cell infection, we performed a high-throughput chemical genetics screen of known inhibitors and agonists of host cell targets to identify host factors that contribute to intracellular growth of the model pathogen Brucella neotomae Using this approach, we identified the p38 mitogen-activated protein (MAP) kinase pathway and autophagy machinery as both a linchpin and an Achilles' heel in B. neotomae's ability to coopt host cell machinery and replicate within macrophages. Specifically, B. neotomae induced p38 MAP kinase phosphorylation and autophagy in a type IV secretion system-dependent fashion. Both p38 MAP kinase stimulation and an intact autophagy machinery in turn were required for phagosome maturation and intracellular replication. These findings contrasted with those for Legionella pneumophila, where chemical inhibition of the p38 MAP kinase pathway and autophagy factor depletion failed to block intracellular replication. Therefore, results from a chemical genetics screen suggest that intersections of the MAP kinase pathways and autophagy machinery are critical components of Brucella's intracellular life cycle.

Brucella neotomae Recapitulates Attributes of Zoonotic Human Disease in a Murine Infection Model.

Members of the genus Brucella are Gram-negative pathogens that cause chronic systemic infection in farm animals and zoonotic infection in humans. Study of the genus Brucella has been hindered by the need for biosafety level 3 select agent containment. Brucella neotomae, originally isolated from the desert pack rat, presented an opportunity to develop an alternative, non-select agent experimental model. Our prior in vitro work indicated that the cell biology and type IV secretion system (T4SS) dependence of B. neotomae intracellular replication were similar to observations for human-pathogenic select agent Brucella species. Therefore, here, we investigated the pathobiology of B. neotomae infection in the BALB/c mouse. During a sustained infectious course, B. neotomae replicated and persisted in reticuloendothelial organs. Bioluminescent imaging and histopathological and PCR-based analysis demonstrated that the T4SS contributed to efficient early infection of the liver, spleen, and lymph nodes; granuloma formation and hepatosplenomegaly; and early induction of Th1-associated cytokine gene expression. The infectious course and pathologies in the murine model showed similarity to prior observations of primate and native host infection with zoonotic Brucella species. Therefore, the B. neotomae BALB/c infection model offers a promising system to accelerate and complement experimental work in the genus Brucella.

Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases PhoR and AioS.

In environments where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which directly affects bioavailability, toxicity and bioaccumulation. Speciation reactions influence arsenic behaviour in environmental systems, directly affecting human and agricultural exposures. Arsenite oxidation decreases arsenic toxicity and mobility in the environment, and therefore understanding its regulation and overall influence on cellular metabolism is of significant interest. The arsenite oxidase (AioBA) is regulated by a three-component signal transduction system AioXSR, which is in turn regulated by the phosphate stress response, with PhoR acting as the master regulator. Using RNA-sequencing, we characterized the global effects of arsenite on gene expression in Agrobacterium tumefaciens 5A. To further elucidate regulatory controls, mutant strains for histidine kinases PhoR and AioS were employed, and illustrate that in addition to arsenic metabolism, a host of other functional responses are regulated in parallel. Impacted functions include arsenic and phosphate metabolism, carbohydrate metabolism, solute transport systems and iron metabolism, in addition to others. These findings contribute significantly to the current understanding of the metabolic impact and genetic circuitry involved during arsenite exposure in bacteria. This informs how arsenic contamination will impact microbial activities involving several biogeochemical cycles in nature.

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate.

Environmental arsenic poisoning affects roughly 200 million people worldwide. The toxicity and mobility of arsenic in the environment is significantly influenced by microbial redox reactions, with arsenite (AsIII ) being more toxic than arsenate (AsV ). Microbial oxidation of AsIII to AsV is known to be regulated by the AioXSR signal transduction system and viewed to function for detoxification or energy generation. Here, we show that AsIII oxidation is ultimately regulated by the phosphate starvation response (PSR), requiring the sensor kinase PhoR for expression of the AsIII oxidase structural genes aioBA. The PhoRB and AioSR signal transduction systems are capable of transphosphorylation cross-talk, closely integrating AsIII oxidation with the PSR. Further, under PSR conditions, AsV significantly extends bacterial growth and accumulates in the lipid fraction to the apparent exclusion of phosphorus. This could spare phosphorus for nucleic acid synthesis or triphosphate metabolism wherein unstable arsenic esters are not tolerated, thereby enhancing cell survival potential. We conclude that AsIII oxidation is logically part of the bacterial PSR, enabling the synthesis of the phosphate analog AsV to replace phosphorus in specific biomolecules or to synthesize other molecules capable of a similar function, although not for total replacement of cellular phosphate.

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

We established a new Brucella neotomaein vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitroBrucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A.

UNLABELLED: ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE: Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor.

Involvement of the Acr3 and DctA anti-porters in arsenite oxidation in Agrobacterium tumefaciens 5A.

Microbial arsenite (AsIII) oxidation forms a critical piece of the arsenic cycle in nature, though our understanding of how and why microorganisms oxidize AsIII remains rudimentary. Our model organism Agrobacterium tumefaciens 5A contains two distinct ars operons (ars1 and ars2) that are similar in their coding region content. The ars1 operon is located nearby the aio operon that is essential for AsIII oxidation. The AsIII/H(+) anti-porters encoded by acr3-1 and acr3-2 are required for maximal AsIII and antimonite (SbIII) resistance, but acr3-1 (negatively regulated by ArsR-1) appears more active in this regard and also required for AsIII oxidation and expression of aioBA. A malate-phosphate anti-porter DctA is regulated by RpoN and AsIII, and is required for normal growth with malate as a sole carbon source. Qualitatively, a ΔdctA mutant was normal for AsIII oxidation and AsIII/SbIII resistance at metalloid concentrations inhibitory to the Δacr3-1 mutant; however, aioBA induction kinetics was significantly phase-shift delayed. Acr3 involvement in AsIII/SbIII resistance is reasonably well understood, but the role of Acr3 and DctA anti-porters in AsIII oxidation and its regulation is unexpected, and suggests that controlled AsIII trafficking across the cytoplasmic membrane is important to a process understood to occur in the periplasm.

Arsenite oxidase also functions as an antimonite oxidase.

Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreased Vmax compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium.

A Versatile Nanoluciferase Reporter Reveals Structural Properties Associated with a Highly Efficient, N-Terminal Legionella pneumophila Type IV Secretion Translocation Signal.

Many Gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most reporter systems make use of fusions to reporter proteins, in particular, ß-lactamase (TEM) and calmodulin-dependent adenylate cyclase (CYA), that allow detection of translocated enzymatic activity inside host cells. However, both systems require costly reagents and use complex, multistep procedures for loading host cells with substrate (TEM) or for analysis (CYA). Therefore, we have developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by Legionella pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive mutagenesis in the NLuc N terminus suggested the importance of an α-helical domain spanning D5 to V9, as mutations predicted to disrupt this structure, with one exception, were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages transfected with GFPopt, thereby resulting in in vivo assembly of superfolder green fluorescent protein (GFP). This provided a bifunctional assay in which translocation could be assayed by fluorescence microplate, confocal microscopy, and/or luciferase assays. We further identified an optimal NLuc substrate which allowed a robust, inexpensive, one-step, high-throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken together, these results indicate that NLuc provides both new insight into and also tools for studying T4SS biology. IMPORTANCE Type IV secretion systems (T4SS) are used by Gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet-to-be-defined contributions of secondary and tertiary structure. Here, we unexpectedly identified nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate, and we provide extensive mutagenesis data suggesting that the first N-terminal, alpha-helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we found that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high-throughput exploration of secretion system biology.

Integrated co-regulation of bacterial arsenic and phosphorus metabolisms.

Arsenic ranks first on the US Environmental Protection Agency Superfund List of Hazardous Substances. Its mobility and toxicity depend upon chemical speciation, which is significantly driven by microbial redox transformations. Genome sequence-enabled surveys reveal that in many microorganisms genes essential to arsenite (AsIII) oxidation are located immediately adjacent to genes coding for functions associated with phosphorus (Pi) acquisition, implying some type of functional importance to the metabolism of As, Pi or both. We extensively document how expression of genes key to AsIII oxidation and the Pi stress response are intricately co-regulated in the soil bacterium Agrobacterium tumefaciens. These observations significantly expand our understanding of how environmental factors influence microbial AsIII metabolism and contribute to the current discussion of As and P metabolism in the microbial cell.

Involvement of RpoN in regulating bacterial arsenite oxidation.

In this study with the model organism Agrobacterium tumefaciens, we used a combination of lacZ gene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of As(III) oxidation. A deletion mutation that removed the RpoN binding site from the aioBA promoter and an aacC3 (gentamicin resistance) cassette insertional inactivation of the rpoN coding region eliminated aioBA expression and As(III) oxidation, although rpoN expression was not related to cell exposure to As(III). Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters for aioB, phoB1, pstS1, dctA, glnA, glnB, and flgB that were examined by using qualitative RT-PCR and lacZ reporter fusions to assess the relative contribution of RpoN to their transcription. The expressions of aioB and dctA in the wild-type strain were considerably enhanced in cells exposed to As(III), and both genes were silent in the rpoN::aacC3 mutant regardless of As(III). The expression level of glnA was not influenced by As(III) but was reduced (but not silent) in the rpoN::aacC3 mutant and further reduced in the mutant under N starvation conditions. The rpoN::aacC3 mutation had no obvious effect on the expression of glnB, pstS1, phoB1, or flgB. These experiments provide definitive evidence to document the requirement of RpoN for As(III) oxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by As(III) or by this sigma factor.

Introducing the ArsR-Regulated Arsenic Stimulon.

The microbial ars operon encodes the primary bacterial defense response to the environmental toxicant, arsenic. An important component of this operon is the arsR gene, which encodes ArsR, a member of the family of proteins categorized as DNA-binding transcriptional repressors. As currently documented, ArsR regulates its own expression as well as other genes in the same ars operon. This study examined the roles of four ArsR proteins in the well-developed model Gram-negative bacterium Agrobacterium tumefaciens 5A. RNASeq was used to compare and characterize gene expression profiles in ± arsenite-treated cells of the wild-type strain and in four different arsR mutants. We report that ArsR-controlled transcription regulation is truly global, extending well beyond the current ars operon model, and includes both repression as well as apparent activation effects. Many cellular functions are significantly influenced, including arsenic resistance, phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, iron homeostasis, and many others. While there is evidence of some regulatory overlap, each ArsR exhibits its own regulatory profile. Furthermore, evidence of a regulatory hierarchy was observed; i.e. ArsR1 represses arsR4, ArsR4 activates arsR2, and ArsR2 represses arsR3. Additionally and unexpectedly, aioB (arsenite oxidase small subunit) expression was shown to be under partial positive control by ArsR2 and ArsR4. Summarizing, this study demonstrates the regulatory portfolio of arsenite-activated ArsR proteins and includes essentially all major cellular functions. The broad bandwidth of arsenic effects on microbial metabolism assists in explaining and understanding the full impact of arsenic in natural ecosystems, including the mammalian gut.

Trade-off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1.

Rifampicin, a bactericidal antibiotic drug, is routinely used to make an environmental recipient selective in laboratory-conjugation experiments. We noticed, inadvertently, that the rifampicin-resistant Acinetobacter sp. strain DR1, a recently discovered hexadecane-degrading environmental isolate, exhibited a substantial loss of quorum sensing signalling. The domesticated ampicillin-resistant strain, DR1, evidenced more dramatic phenotypic changes than were observed in the rifampicin-resistant cells: a complete loss of quorum sensing, a loss in swimming and swarming motilities, poor fimbrial expression, increased rigidity in membrane fatty acid composition and reduced hexadecane degradation capability. Interestingly, the motility of strain DR1 grown adjacent to a streptomycin-producing Streptomyces griceus was permanently abrogated, where this change was heritable and other phenotypic changes could not be detected. In this study, we have reported for the first time that the in situ acquisition of antibiotic resistance may reduce biological fitness, including losses in the production of quorum sensing signals, motility and substrate utilization, and each antibiotic is associated with different degrees of phenotypic and genetic alterations. Our data also suggested that the domestication of environmental isolates should be approached with caution, as there are phenotypic variations in antibiotic-resistant cells that might not be noticeable unless all possible phenotypic assays are conducted.

Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in Agrobacterium tumefaciens 5A.

Arsenite (AsIII) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for AsIII oxidation (aio) encode a periplasmic AsIII sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the AsIII oxidase AioBA. The aio genes are under ultimate control of the phosphate stress response via histidine kinase PhoR. To better understand the cell-wide impacts exerted by these key histidine kinases, we employed 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-coupled mass spectrometry (LC-MS) metabolomics to characterize the metabolic profiles of ΔphoR and ΔaioS mutants of Agrobacterium tumefaciens 5A during AsIII oxidation. The data reveals a smaller group of metabolites impacted by the ΔaioS mutation, including hypoxanthine and various maltose derivatives, while a larger impact is observed for the ΔphoR mutation, influencing betaine, glutamate, and different sugars. The metabolomics data were integrated with previously published transcriptomics analyses to detail pathways perturbed during AsIII oxidation and those modulated by PhoR and/or AioS. The results highlight considerable disruptions in central carbon metabolism in the ΔphoR mutant. These data provide a detailed map of the metabolic impacts of AsIII, PhoR, and/or AioS, and inform current paradigms concerning arsenic-microbe interactions and nutrient cycling in contaminated environments.

Inhibitory effect of aged-petroleum hydrocarbons on the survival of inoculated microorganism in a crude-oil contaminated site.

We studied the effects of aged total petroleum hydrocarbons (aged TPH) on the survival of allochthonous diesel-degrading Rhodococcus sp. strain YS-7 in both laboratory and field investigations. The aged TPH extracted from a crude oil-contaminated site were fractionized by thin-layer chromatography/flame ionization detection (TLC/FID). The three fractions identified were saturated aliphatic (SA), aromatic hydrocarbon (AH), and asphaltene-resin (AR). The ratio and composition of the separated fractions in the aged TPH were quite different from the crude-oil fractions. In the aged TPH, the SA and AH fractions were reduced and the AR fraction was dramatically increased compared to crude oil. The SA and AH fractions (2 mg/L each) of the aged TPH inhibited the growth of strain YS-7. Unexpectedly, the AR fraction had no effect on the survival of strain YS-7. However, crude oil (1,000 mg/L) did not inhibit the growth of strain YS-7. When strain YS-7 was inoculated into an aged crude oil-contaminated field and its presence was monitored by fluorescent in situ hybridization (FISH), we discovered that it had disappeared on 36 days after the inoculation. For the first time, this study has demonstrated that the SA and AH fractions in aged TPH are more toxic to an allochthonous diesel-degrading strain than the AR fraction.

Molecular characterization of fprB (ferredoxin-NADP+ reductase) in Pseudomonas putida KT2440.

The fpr gene, which encodes a ferredoxin-NADP+ reductase, is known to participate in the reversible redox reactions between NADP+/NADPH and electron carriers, such as ferredoxin or flavodoxin. The role of Fpr and its regulatory protein, FinR, in Pseudomonas putida KT2440 on the oxidative and osmotic stress responses has already been characterized [Lee at al. (2006). Biochem. Biophys. Res. Commun. 339, 1246-1254]. In the genome of P. putida KT2440, another Fpr homolog (FprB) has a 35.3% amino acid identity with Fpr. The fprB gene was cloned and expressed in Escherichia coli. The diaphorase activity assay was conducted using purified FprB to identify the function of FprB. In contrast to the fpr gene, the induction of fprB was not affected by oxidative stress agents, such as paraquat, menadione, H2O2 and t-butyl hydroperoxide. However, a higher level of fprB induction was observed under osmotic stress. Targeted disruption of fprB by homologous recombination resulted in a growth defect under high osmotic conditions. Recovery of oxidatively damaged aconitase activity was faster for the fprB mutant than for the fpr mutant, yet still slower than that for the wild type. Therefore, these data suggest that the catalytic function of FprB may have evolved to augment the function of Fpr in P. putida KT2440.

Analysis of EST and lectin expressions in hemocytes of Manila clams (Ruditapes philippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni.

The hemocytes of invertebrates play key roles in both cellular and humoral immune reactions by phagocytosis or delivering immune factors such as lectin and anti-microbial peptides. Bacterial infection causes changes in components such as lectins, anti-bacterial peptides, and lysosomal enzymes of plasma or hemolymph in molluscs. Previously, we found that infection with the protozoan parasite, Perkinsus, increases lectin synthesis in hemocytes. In order to investigate the patterns of genes expressed in Manila clams (Ruditapes philippinarum) infected with the protozoan parasite Perkinsus olseni, we constructed a cDNA library and sequenced 1850 clones (expressed sequence tags). A total of 79 ESTs, were related to 29 functional immune genes such as C-type lectin, lysozyme, and cystatin B, in Manila clams. Lectins were the largest group of immune-function ESTs found in our Manila clams library. Among 7 lectin clones, two full length cDNAs of lectins were cloned. MCL-3, which is a simple C-type lectin composed of 151 amino acids, has a relatively short signal sequence of 17aa and single carbohydrate-recognition domain (CRD) of approximately 130 residues. It is highly homologous to eel C-type lectin. The sequence of mc-sialic acid-binding lectin consists of 168 amino acid residues with molecular weight of 19.2 and shows high homology to sialic acid-binding lectin from the snail, Cepaea hortensis. The expression of 7 different lectins in hemocytes was analyzed by RT-PCR using gene-specific primers. Hemocytes from Perkinsus-infected clam expressed different sets of lectins than with Vibrio infection. These results demonstrate that several lectins are involved in Manila clam innate immunity and different challenges induce expression of different lectins.

Purification and characterization of a catalase from photosynthetic bacterium Rhodospirillum rubrum S1 grown under anaerobic conditions.

The photosynthetic bacterium, Rhodospirillum rubrum S1, when grown under anaerobic conditions, generated three different types of catalases. In this study, we purified and characterized the highest molecular weight catalase from the three catalases. The total specific catalase activity of the crude cell extracts was 88 U/mg. After the completion of the final purification step, the specific activity of the purified catalase was 1,256 U/mg. The purified catalase evidenced an estimated molecular mass of 318 kDa, consisting of four identical subunits, each of 79 kDa. The purified enzyme exhibited an apparent Km value of 30.4 mM and a Vmax of 2,564 U against hydrogen peroxide. The enzyme also exhibited a broad optimal pH (5.0-9.0), and remained stable over a broad temperature range (20 degrees C-60 degrees C). It maintained 90% activity against organic solvents (ethanol/chloroform) known hydroperoxidase inhibitors, and exhibited no detectable peroxidase activity. The catalase activity of the purified enzyme was reduced to 19% of full activity as the result of the administration of 10 mM 3-amino-1,2,4-triazole, a heme-containing catalase inhibitor. Sodium cyanide, sodium azide, and hydroxylamine, all of which are known heme protein inhibitors, inhibited catalase activity by 50% at concentrations of 11.5 microM, 0.52 microM, and 0.11 microM, respectively. In accordance with these findings, the enzyme was identified as a type of monofunctional catalase.