Whole-cell Suspensions Research Articles

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus and Lactobacillus acidophilus on clinical isolates- an in vitro study

Background: This study evaluated the in vitro antimicrobial and antagonistic activity of both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus towards S. aureus, as an alternative to control the spread of MRSA, which is endemic in India. Materials and Methods: The study was conducted at School of Medical Education, Kerala, India, between October 2022 and May 2023. Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus MTCC 1408 and Lactobacillus acidophilus MTCC 10307 grown on MRS agar, was evaluated on a total of 76 clinical isolates of S. aureus from Kerala, India, by the Agar overlay method. The study involved a comparison of probiotic activities by both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus. Results: The study results indicate that the mean value of inhibition zones produced by L. rhamnosus was greater than that of L. acidophilus, and statistically significant data was obtained in which the pH-adjusted whole cell suspensions of L. rhamnosus exhibited larger inhibition zones than that of neat suspensions of the same. Also, the mean value of activity by pH-adjusted L. acidophilus suspension was slightly greater than its neat suspension. Conclusions: L. rhamnosus and L. acidophilus were found to possess in vitro anti-Staphylococcus aureus activity. The antimicrobial and antagonistic activity of pH-adjusted live suspensions suggests the possible use of L. acidophilus and L. rhamnosus as probiotic treatment options in sites other than the gastrointestinal tract, supporting their use as local administration and topical applications, in particular for the treatment and decolonization of S. aureus.

Multi-species endophytic Bacillus for improved control of potato soilborne and tuber-borne diseases in Tunisia: from laboratory to field conditions

BackgroundDue to the soilborne and tuber-borne nature of their causative agents, Fusarium wilt, Rhizoctonia root canker and black dot diseases are still leading to serious problems in potato production in Tunisia and worldwide. Among endophytic bacteria, the genus Bacillus is one of the most exploited microbial groups known as potent biocontrol agents against several potato diseases. In the present study, six strains belonging to five Bacillus species were screened for their abilities to inhibit root and wilt potato pathogens in vitro, in vivo and under natural conditions over three cropping seasons and to promote plant growth.ResultsBased on the dual-culture assays, the whole-cell suspensions of SV39 and SV104 (Bacillus tequilensis), SV41 (B. subtilis), SV44 (B. methylotrophicus) and SV65 (B. amyloliquefaciens subsp. plantarum) strains exhibited potent antifungal activity against important potato soilborne phytopathogens with ~ 65 to 70% inhibition rates. Significant inhibition rates were also induced by the cell-free culture filtrates, the butanolic and the chloroformic extracts depending on the target pathogens, the concentration used and the Bacillus strain tested. In pot experiment, a decrease in Rhizoctonia root canker severity, ranging from 43 to 65% compared to the inoculated and untreated control, was induced by all Bacillus spp. strain-based treatments, while SV39-, S42- and SV65-based treatments were the most effective in suppressing by 50–53 and 65–52%, black dot severity and the relative vascular discoloration extent induced by F. oxysporum f. sp. tuberosi, respectively, relative to positive control. This biocontrol potential was associated with an enhancement of potato growth parameters. Field studies indicated that soil treatment with the most of the Bacillus spp. strains had significantly controlled all the target fungal soilborne diseases and improved at the least two growth and/or production parameters depending on the strain used and the cropping seasons. SV39-, SV41-, SV44- and SV104-based treatments resulted in a significant increase in tuber yield in one cropping season.ConclusionThese Bacillus spp. strains could be used in combinations and/or introduced with other existing practices in order to provide supplemental control of target diseases and yield promotion under organic or conventional potato production systems.

Photorhabdus and Xenorhabdus as Symbiotic Bacteria for Bio-Control Housefly (Musca domestica L.)

<b>Background and Objective:</b> The housefly poses a threat to the public health of humans and domestic animals since it can carry and transmit pathogens. Despite there are many attempts to control this insect, most of them depend on conventional pesticides. Thus, the current study aimed to evaluate the efficacy of whole-cell suspension, cell-free supernatant and crude cells of the symbiotic bacteria <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., as bio-control agents for housefly stages. <b>Materials and Methods:</b> The <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., were isolated from the entomopathogenic nematodes, <i>Heterorhabditis indica</i> and <i>Steinernema feltiae</i>, respectively. The phenotypic, as well as the enzymatic characterizations of both bacteria, were determined. In addition, histopathological changes of the alimentary canal of <i>M. domestica</i> adults treated with whole-cell suspensions (at 3×10<sup>8 </sup>cells mL<sup></sup><sup>1</sup>) of both bacteria were carefully examined using transmission electron microscopy. <b>Results:</b> The results showed that both symbiotic bacteria significantly suppressed larvae, pupae and adults of <i>M. domestica</i>, particularly when they were applied as whole-cell suspensions. For example, the highest concentration of whole-cell suspension, cell-free supernatant and crude cells of <i>Photorhabdus</i> sp., induced larval mortalities by 94.7, 64.0 and 45.3%, while those of <i>Xenorhabdus</i> sp., induced larval mortalities by 58.7, 46.7 and 30.7% at 96 hrs, respectively. The results also showed that whole-cell suspensions of both symbiotic bacteria caused severe histopathological changes in the ultrastructure of the treated adults' alimentary canal. <b>Conclusion:</b> Both symbiotic bacteria can be effectively used, particularly the whole-cell suspension, as bio-control agents against the housefly either in the larval or adult stage.

KS01.6.A Comparative analysis of the immune compartment in human glioblastoma and IDH-mutant WHO grade 4 astrocytoma reveals profound differences in microglia phenotypes

Abstract BACKGROUND Glioblastoma (GBM) are the most common primary brain tumors. If untreated the average survival is around 12–18 months. Unfortunately, despite extensive research efforts, the therapeutic options remain limited. One major aspect complicating therapeutic development is an immunosuppressive tumor microenvironment. Isocitrate dehydrogenase (IDH)-mutant, WHO grade 4 astrocytomas appear histologically indistinguishable from GBM, but show significantly longer survival times. IDH mutations lead to changes in the tumor microenvironment with accrual of the neometabolite R-2-hydroxyglutarate. Previous studies on bulk transcriptomes have shown differences in the immune compartment of both tumor entities that were linked to the differences in clinical behavior. MATERIAL AND METHODS We have conducted high-dimensional comparative analyses of the myeloid compartment in surgically resected human GBM and IDH-mutant WHO grade 4 astrocytomas using single-cell RNA-Sequencing and immunohistochemistry. Histologically normal brain regions from epilepsy patients were used as controls. For analysis, whole-cell suspensions were prepared from freshly resected tumors or controls. Fluorescence activated cell sorting was used for myeloid cell enrichment. Samples were processed using the high-sensitivity single-cell RNA sequencing protocol CEL-Seq2. Seurat and StemID2 algorithms were used for clustering, differential gene expression and pseudotime analysis. Protein validation was achieved using immunohistochemistry. RESULTS We identified profound transcriptional changes of glioma-associated microglia in GBM with respect to control brain samples. Namely, we observed a global upregulation of major histocompatibility complex associated genes in GBM across all clusters. Additionally, we identified distinct myeloid subsets with phagocytic, hypoxia-associated and chemotactic transcriptomic signatures. Pseudotime analysis finely resolved transitional cell states. These changes were dramatically attenuated in IDH-mutant WHO grade 4 astrocytomas. The myeloid cells in these tumors resembled homeostatic microglia and showed an increased expression of cytokine and chemokine genes. CONCLUSION Here, we present a high-dimensional transcriptomic atlas of the myeloid compartment in human GBM and IDH-mutant WHO grade 4 astrocytomas. The identified differences point towards targeted therapeutic options via the modulation of the tumor microenvironment.

Production of a Recombinant Catechol 2,3-Dioxygenase for the Degradation of Micropollutants.

Phenolic compounds such as catechol represent a particular type of micropollutant whose high stability prevents rapid decay and metabolization in the environment. We successfully cloned a catechol 2,3-dioxygenase (C2,3O) from Pseudomonas putida mt-2 and expressed it in Escherichia coli BER2566. The biomass isolated from shake-flask fermentations was used to partially purify the enzyme. The enzyme proved unstable in clarified liquid fractions (50 mM Tris buffer, pH 7.6) and lost more than 90% of its activity over 7 h at 25 °C. In the presence of 10% acetone, the process was slowed down and 30% residual activity was still present after 7 h incubation. Storage of the enzyme in clear liquid fractions also proved difficult and total inactivation was achieved after 2 weeks even when kept frozen at -20 °C. Lowering the storage temperature to -80 °C preserved 30% activity over the same period. Only minor reactivation of the affected enzyme could be achieved after incubation at 20 °C in the presence of FeSO4 and/or ascorbic acid. Activity loss seems to be due mostly to Fe2+ oxidation as well as to subunit dissociation in the tetrameric structure. However, complete degradation of 1.0 mM catechol could be achieved at 20 °C and pH 7.6 over a 3 h period when using a suspension of whole cells or alginate-encapsulated cells for the biotransformation. Contrary to the clear liquid fractions, these forms of biocatalyst showed no significant sign of inactivation under the working conditions.

Involvement of lipopeptide antibiotics and chitinase genes and induction of host defense in suppression of Fusarium wilt by endophytic Bacillus spp. in tomato

Five endophytic Bacillus spp. isolated from surface-sterilized stem tissues of wild Solanaceous species, i.e. Datura metel, Solanum nigrum and S. elaeagnifolium, were screened for their in vitro and in vivo antagonistic potential against Fusarium oxysporum f. sp. lycopersici (FOL). Pathogen sporulation was totally suppressed by these five strains and its mycelial growth was significantly decreased using bacterial whole-cell suspensions and cell-free culture filtrates. Extracellular metabolites remained effective after heating at 50–100 °C with a decline in activity with treatments involving temperatures at 100 °C, proteinase K addition, and pH adjustments to 2 or 12. Chitinase and lipopeptide antibiotic genes were detected using PCR amplification. The five strains produced salicylic acid when grown in succinate medium, with the greatest production exhibited by B. subtilis str. SV41. In planta application of Bacillus spp. cell-free culture filtrates and whole-cell suspensions significantly decreased Fusarium wilt severity by 87–100% and enhanced tomato growth by 38–80%, compared to FOL-inoculated and untreated controls. The two Bacillus strains exhibiting the strongest decrease in Fusarium wilt severity (B. subtilis str. SV41 and B. amyloliquefaciens subsp. plantarum str. SV65) were further evaluated for their ability to induce systemic resistance (ISR) in FOL-infected and uninfected tomato plants. Using quantitative RT-PCR, the expression of the acidic PR-1 and PR-3 genes as well as the lipoxygenase (LOXD) gene were significantly induced in plants treated with B. amyloliquefaciens subsp. plantarum str. SV65. This effect was observed in both FOL-inoculated and uninoculated plants.

A putative endophytic Bacillus cereus str. S42 from Nicotiana glauca for biocontrol of Fusarium wilt disease in tomato and gas chromatography-mass spectrometry analysis of its chloroform extract

A putative endophytic Bacillus cereus str. S42 (KP993206), recovered from surface-sterilised stems of Nicotiana glauca was assessed in vitro and in vivo for its antifungal potential towards Fusarium oxysporum f. sp. lycopersici (FOL). Pathogen sporulation was significantly inhibited by B. cereus str. S42. FOL mycelial growth was reduced using its whole-cell suspensions, cell-free culture supernatant and chloroform extract. Its extracellular metabolites remained effective after heating at 50–100 °C with a decline in their activity was observed beyond 100 °C, when added with proteinase K and/or after pH adjustment to 2 and 12. Chitinase gene was detected using PCR amplification. Gas chromatography–mass spectrometry analysis of its chloroform extract matched phthalic acid, dibutyl ester with high level of similarity. B. cereus str. S42 cell-free culture supernatant and whole-cell suspensions had significantly suppressed Fusarium wilt severity by 87–96% and enhanced tomato growth by 39–79% compared to FOL-inoculated and untreated control.

The obligate alkaliphile Bacillus clarkii K24-1U retains extruded protons at the beginning of respiration

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (Delta pH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Delta psi), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the Delta pH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na(+) channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Delta psi-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient Delta pH under alkaline conditions.

Phase I Study with SV-BR-1 Breast Cancer Cell Line Vaccine and GMCSF: Clinical Experience in 14 Patients

We evaluated the safety and feasibility of breast cancer vaccine therapy using a new cell line, SV-BR-1, in conjunction with repeated injections of GM-CSF, as a prototype for a subsequent genetically engineered vaccine. Also addressed with this preliminary trial were monitoring effects on quality of life, screening for potential immune responses, and evaluating any clinical effects on tumor progression and patient survival. Fourteen patients with metastatic breast cancer were treated with SV-BR-1 tumor cells. The cell line strongly overexpresses HER2/neu, and has an unusual variety of cytogenetic abnormalities. Irradiated whole-cell suspensions (median dose 14 x 10 6 viable cells) were inoculated via intradermal injection or intralymphatic cannulation, the former usually including an equal amount of irradiated autologous peripheral blood lymphocytes (PBL). Vaccine was initially given at 2-week intervals x3, then monthly. Low-dose cyclophosphamide 300mg/m 2 was given 48-72 hrs prior to vaccine, and GM-CSF (125 mcg) was given subcutaneously immediately prior and for 8 days subsequently. The Kaplan-Meier mean survival for all 14 patients was 17.2 months (CI 5.8-28.5 months) and was 21.1 months (95% CI 5.0-37.2 months) for nine patients treated with the tumor cell/PBL admixture, although there were no clinical regressions. SV-BR-1 as a whole-cell vaccine appears feasible and without obvious major toxicity. The overall survival compares favorably with current Phase I studies. Based on these observations, we have initiated a clinical trial using the SV-BR1 cell line transfected to release GM-CSF in situ with four patients to date. One case responded with clear evidence of tumor regressions in multiple sites and the median survival of 35.0 months is three times longer than usual expectations for salvage programs.

Recent Advances in Antigen-Loaded Dendritic Cell-Based Strategies for Treatment of Minimal Residual Disease in Acute Myeloid Leukemia

Therapeutic vaccination with dendritic cells (DCs) is recognized as an important experimental therapy for the treatment of minimal residual disease in acute myeloid leukemia. Many sources of leukemia-associated antigens and different methods for antigen loading of DCs have been used in an attempt to optimize anti-tumor responses. For instance, monocyte-derived DCs have been loaded with apoptotic whole-cell suspensions, necrotic cell lysates, tumor-associated peptides, eluted peptides and cellular DNA or RNA. Furthermore, monocyte-derived DCs can be chemically or electrically fused with leukemic blasts, and DCs have been cultured out of leukemic blasts. However, it remains a challenge in cancer immunotherapy to identify which of these methods is the most optimal for antigen loading and activation of DCs. This review discusses recent advances in DC research and the application of this knowledge towards new strategies for antigen loading of DCs in the treatment of minimal residual disease in acute myeloid leukemia.

Growth of Pseudomonas chloritidismutans AW-1T on n-alkanes with chlorate as electron acceptor

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day−1, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.

Enzymatic hydrolysis of soybean protein using lactic acid bacteria

The proteolytic activity of 12 lactic acid bacteria (LAB) strains, assayed on soy protein extract at a temperature of 37 °C for 6 h, was evaluated by SDS–PAGE, reverse-phase HPLC and free-amino acid analyses. The results indicated that α- and α′-subunits of β-conglycinin were the preferred substrates for the majority of the LAB. Only a few strains exerted some action against the basic polypeptides of glycinin, this fraction was the least degraded of all soy protein fractions. Whole-cell suspensions of LAB used in this study generated hydrophilic and hydrophobic peptides from mainly soy protein fractions. RP-HPLC analyses indicated differences in the profiles of the hydrolysates, with several peaks decreasing in size and new peaks being formed. Three of the selected strains assayed increased the level of total free amino acids in the soy protein extract (SPE) and hydrolyzed principally essential amino acids and flavour precursor amino acids.

Identification of a Novel Laser Dye Substrate of Mammalian Cytochromes P450: Application in Rapid Kinetic Analysis, Inhibitor Screening, and Directed Evolution

The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)-supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) k(cat) = 3 to 6 min(-1), 2) K(m, CuOOH) = 100 to 200 microM, and 3) S(50, C152) = 10 to 20 microM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high k(ca)t (27 and 38 min(-1), respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)-supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution.

Characterization of membrane-bound Fe(III)-EDTA reductase activities of the thermophilic gram-positive dissimilatory iron-reducing bacterium Thermoterrabacterium ferrireducens

Whole-cell suspensions of T. ferrireducens reduced Fe(III) citrate, Fe(III)-EDTA, and ferrihydrite with glycerol as an electron donor. After cell disruption, the highest activity was registered with Fe(III)-EDTA as the electron acceptor and NADH or NAD(P)H as electron donors. About 80% of the NAD(P)H-dependent Fe(III)-EDTA reductase activities were associated with the membrane fraction of the cells. Treatment of the membranes with lauryl maltoside led to complete solubilization of the NADH-dependent and 70% solubilization of the NADPH-dependent Fe(III)-EDTA reductase activities. After purification by ion-exchange chromatography, the NADH-dependent activity was concentrated 8-fold, and the NADPH-dependent activity was concentrated 11-fold, with a yield of about 10% for both activities. The Fe(III)-EDTA-reducing enzyme complex included c-type cytochromes and a protein with a molecular mass of ca. 115 k Da, consisting of two polypeptides. This is the first description of membrane-bound Fe(III)-reducing oxidoreductase activities from a gram-positive dissimilatory Fe(III)-reducing bacterium.

Isolation of Methylophaga spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent K(m)s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 microM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.

Protease and esterase activity of staphylococci

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing l-methionine, l-leucine and l-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were ρ-nitrophenyl (ρ-NP) butyrate and ρ-NP caprylate and, secondly, ρ-NP palmitate. The extracellular extracts of most of the strains were only active against ρ-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed ρ-NP caprylate and ρ-NP palmitate. The cell-free extracts and whole cells were mainly active against ρ-NP butyrate and ρ-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.

Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the DeltanarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the DeltanarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

Facultative Methanotrophs Revisited

Facultative Methanotrophs Revisited

Cytochrome bd Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium Moorella thermoacetica

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.