bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological basis for the high salt tolerance of Debaryomyces hansenii
Authors: Prista C , Almagro A , Loureiro-Dias M C , Ramos J
Published: January 2020

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na+ and Li+ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb+ uptake in D. hansenii (Vmax, 4.2 nmol mg [dry weight]-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb+ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na+ than is that of S. cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+. In low K+ environments, D. hansenii behaved as a halophilic yeast.
Group-Specific 16S rRNA-Targeted Oligonucleotide Probes To Identify Thermophilic Bacteria in Marine Hydrothermal Vents
Authors: Harmsen H , Prieur D , Jeanthon C
Published: January 2020

Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems. We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the genus Thermus, the genera Thermotoga and Thermosipho, and the Aquificales order. The temperature of dissociation of each probe was determined. Probe specificities to the target groups were demonstrated by whole-cell and dot blot hybridization against a collection of target and nontarget rRNAs. Whole-cell hybridizations with the specific probes were performed on cells extracted from hydrothermal vent chimneys. One of the samples contained cells that hybridized to the probe specific to genera Thermotoga and Thermosipho. No positive signals could be detected in the samples tested with the probes whose specificities encompassed either the genus Thermus or the thermophilic members of the genus Bacillus. However, when simultaneous hybridizations with the probe specific to the order Aquificales and a probe specific to the domain Bacteria (R. I. Amann, B. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl, Appl. Environ. Microbiol. 56:1919-1925, 1990) were performed on cells extracted from the top and exterior subsamples of chimneys, positive signals were obtained from morphologically diverse bacteria representing about 40% of the bacterial population. Since specificity studies also revealed that the bacterial probe did not hybridize with the members of the order Aquificales, the detected cells may therefore correspond to a new type of bacteria. One of the observed morphotypes was similar to that of a strictly anaerobic autotrophic sulfur-reducing strain that we isolated from the chimney samples. This work demonstrates that application of whole-cell hybridization with probes specific for different phylogenetic levels is a useful tool for detailed studies of hydrothermal vent microbial ecology.
Survival of Escherichia coli cells exposed to iodoacetate and chlorodinitrobenzene is independent of the glutathione-gated K+ efflux systems KefB and KefC
Authors: Ness L S , Ferguson G P , Nikolaev Y , Booth I R
Published: January 2020

The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH. However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation. No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.
Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium
Authors: Fang A , Pierson D L , Koenig D W , Mishra S K , Demain A L
Published: January 2020

Production of the antibacterial polypeptide microcin B17 (MccB17) by Escherichia coli ZK650 was inhibited by simulated microgravity. The site of MccB17 accumulation was found to be different, depending on whether the organism was grown in shaking flasks or in rotating bioreactors designed to establish a simulated microgravity environment. In flasks, the accumulation was cellular, but in the reactors, virtually all the microcin was found in the medium. The change from a cellular site to an extracellular one was apparently not a function of gravity, since extracellular production occurred in these bioreactors, irrespective of whether they were operated in the simulated microgravity or normal gravity mode. More probably, excretion is due to the much lower degree of shear stress in the bioreactors. Addition of even a single glass bead to the 50-ml medium volume in the bioreactor created enough shear to change the site of MccB17 accumulation from the medium to the cells.
Chemotaxis of Pseudomonas spp. to the polyaromatic hydrocarbon naphthalene
Authors: Grimm A C , Harwood C S
Published: January 2020

Two naphthalene-degrading bacteria, Pseudomonas putida G7 and Pseudomonas sp. strain NCIB 9816-4, were chemotactically attracted to naphthalene in drop assays and modified capillary assays. Growth on naphthalene or salicylate induced the chemotactic response. P. putida G7 was also chemotactic to biphenyl; other polyaromatic hydrocarbons that were tested did not appear to be chemoattractants for either Pseudomonas strain. Strains that were cured of the naphthalene degradation plasmid were not attracted to naphthalene.
Purification and characterization of staphylococcin BacR1, a broad-spectrum bacteriocin
Authors: Crupper S S , Gies A J , Iandolo J J
Published: January 2020

The bacteriocin BacR1 was purified from culture supernatant of Staphylococcus aureus UT0007 by sequential ammonium sulfate precipitation, cation-exchange chromatography, and C4 reverse-phase chromatography steps. Mass spectrographic analysis indicated that the purified peptide has a molecular mass of 3,338 Da. It is resistant to environmental conditions, retaining full biological activity after exposure to pH extremes (pHs 3 to 11), heating at 95 degrees C for 15 min, and exposure to strong chaotropic agents. BacR1 was destroyed with a complete loss of biological activity after digestion with trypsin and proteinase K. Amino acid sequence analysis revealed a high concentration of Asx, Gly, and Pro residues and a high proportion of hydrophobic amino acids. The peptide is bactericidal and kills in a dose-dependent manner, but it does not lyse log-phase cells of Corynebacterium renale, the routine indicator organism for bacteriocin assay. A specific receptor for binding was detected on sensitive cells but not on insensitive cells. Competition assays showed that UV-inactivated cells could protect susceptible cells from antibacterial action. A partial inhibitory spectrum revealed that organisms from the following genera are susceptible: Staphylococcus, Streptococcus, Corynebacterium, Haemophilus, Bordetella, Moraxella, Pasteurella, Neisseria, and Bacillus.
Conversion of beta-methylbutyric acid to beta-hydroxy-beta-methylbutyric acid by Galactomyces reessii
Authors: Lee I Y , Nissen S L , Rosazza J P
Published: January 2020

beta-Hydroxy-beta-methylbutyric acid (HMB) has been shown to increase strength and lean mass gains in humans undergoing resistance-exercise training. HMB is currently marketed as a calcium salt of HMB, and thus, environmentally sound and inexpensive methods of manufacture are being sought. This study investigates the microbial conversion of beta-methylbutyric acid (MBA) to HMB by cultures of Galactomyces reessii. Optimal concentrations of MBA were in the range of 5 to 20 g/liter for HMB production. Preliminary shake flask experiments indicated that HMB yields were sensitive to dissolved oxygen levels and that cell growth decreased significantly as MBA concentrations increased. Degradation of HMB was faster at acidic pH, and pH 7.0 was optimal for HMB production. Resting cells obtained from media without MBA could efficiently convert MBA to HMB. Thus, a two-step, fed-batch fermentation procedure in which biomass was first produced, followed by coaddition of MBA and glucose, while dissolved oxygen was maintained at 20% of saturation, was designed. A maximum HMB concentration of 38 g/liter was obtained after 136 h, and the molar conversion yield was more than 0.50 mol of HMB/mol of MBA during the fermentation.
Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness
Authors: Fedi S , Tola E , Moënne-Loccoz Y , Dowling D N , Smith L M , O'Gara F
Published: January 2020

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.
Effects of dissolved organic carbon and salinity on bioavailability of mercury
Authors: Barkay T , Gillman M , Turner R R
Published: January 2020

Hypotheses that dissolved organic carbon (DOC) and electrochemical charge affect the rate of methylmercury [CH3Hg(I)] synthesis by modulating the availability of ionic mercury [Hg(II)] to bacteria were tested by using a mer-lux bioindicator (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993). A decline in Hg(II)-dependent light production was observed in the presence of increasing concentrations of DOC, and this decline was more pronounced at pH 7 than at pH 5, suggesting that DOC is a factor controlling the bioavailability of Hg(II). A thermodynamic model (MINTEQA2) was used to select assay conditions that clearly distinguished among various Hg(II) species. By using this approach, it was shown that negatively charged forms of mercuric chloride (HgCl3-/HgCl(4)2-) induced less light production than the electrochemically neutral form (HgCl2), and no difference was observed between the two neutral forms, HgCl2 and Hg(OH)2. These results suggest that the negative charge of Hg(II) species reduces their availability to bacteria and may be one reason why accumulation of CH3Hg(I) is more often reported to occur in freshwater than in estuarine and marine biota.
Biodegradation of [(sup14)C]Benzo[a]pyrene Added in Crude Oil to Uncontaminated Soil
Authors: Kanaly R , Bartha R , Fogel S , Findlay M
Published: January 2020

To investigate the possible cometabolic biodegradation of benzo[a]pyrene (BaP), crude oil spiked with [7-(sup14)C]BaP and unlabeled BaP was added to soil with no known pollution history, to give 34 g of oil and 67 mg of BaP/kg of dry soil. The oil-soil mixture was amended with mineral nutrients and incubated in an airtight container with continuous forced aeration. Total CO(inf2) and (sup14)CO(inf2) in the off-gas were trapped and quantified. Soil samples were Soxhlet extracted with dichloromethane at seven time points during the 150-day incubation period, and the extracted soil was subjected to further fractionation in order to recover reversibly and irreversibly bound radiocarbon. Radiocarbon recovery was 100% (plusmn) 3% for each time point. During the first 50 days of incubation, no (sup14)CO(inf2) was evolved, but over the next 100 days, 50% of the BaP radiocarbon was evolved as (sup14)CO(inf2). At 150 days, only 5% of the intact BaP and 23% of the crude oil remained. Of the remaining radiolabel, 20% was found in solvent-extractable metabolites and 25% was incorporated into soil organic matter. Only 1/10 of this could be solubilized by chemical hydrolysis. An abiotic control experiment exhibited binding of only 2% of the BaP, indicating the microbial nature of the BaP transformations. We report that in soil containing suitable cosubstrates, BaP can be completely degraded.
Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria
Authors: Yoch D C , Ansede J H , Rabinowitz K S
Published: January 2020

Volume 63, no. 8, p. 3183, column 1, line 3 from the bottom: "0.012 versus 0.730" should read "0.022 versus 0.065." [This corrects the article on p. 3182 in vol. 63.].
Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon
Authors: Moniruzzaman M , Lai X , York S W , Ingram L O
Published: January 2020

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.
Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes
Authors: Wang G C , Wang Y
Published: January 2020

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.
Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis
Authors: Lee H K , Gill S S
Published: January 2020

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.
Ribotyping to compare Fusobacterium necrophorum isolates from bovine liver abscesses, ruminal walls, and ruminal contents
Authors: Narayanan S , Nagaraja T G , Okwumabua O , Staats J , Chengappa M M , Oberst R D
Published: January 2020

Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal. Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter. F. necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples. Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli. EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison. Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI. The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples. None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates. The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F. necrophorum isolates of liver abscesses originate from the rumen.
Spatial distribution and inhibition by ammonium of methane oxidation in intertidal freshwater marshes
Authors: Van Der Nat F , De Brouwer J , Middelburg J J , Laanbroek H J
Published: January 2020

In two intertidal marshes, the vertical distribution in the sediment and inhibition by ammonium of methane oxidation were investigated by slurry incubation experiments. The two sites differ in their dominant vegetation type, i.e., reed and bulrush, and in their heights above sea level. The reed site was elevated with respect to the bulrush site, resulting in a lower frequency and duration of flooding and, consequently, a higher potential for methane oxidation. Methane oxidation decreased with depth in the bulrush and reed slurries, although methane oxidation associated with root material from the bulrush plants increased with depth. Reed root material had a limited capacity for methane oxidation and showed no significant increase with depth. Inhibition of methane oxidation by ammonium was observed in all samples and depended on methane and ammonium concentrations. Increasing ammonium concentrations resulted in greater inhibition, and increasing methane concentrations resulted in less. Ammonium concentrations had to exceed methane concentrations by at least 30-fold to become effective for inhibition. This ratio was found only in the surface layer of the sediment. Hence, the ecological relevance for ammonium inhibition of methane oxidation in intertidal marshes is rather limited and is restricted to the surface layer. Nitrate production was restricted to the 0- to 5-cm-depth slurries.
Purification and characterization of a cephalosporin esterase from Rhodosporidium toruloides
Authors: Politino M , Tonzi S M , Burnett W V , Romancik G , Usher J J
Published: January 2020

A novel cephalosporin esterase (EC 3.1.1.41) from Rhodosporidium toruloides was purified to gel electrophoretic homogeneity. The enzyme is a glycoprotein with a molecular mass of 80 kDa. Upon deglycosylation, several forms of the enzyme were observed with a molecular mass range between 60 and 66 kDa. The isoelectric point of the enzyme is approximately 5.6, with the pH optimum for activity occurring at 6.0. The optimal activity of the enzyme occurred at 25 degrees C, with the enzyme rapidly losing activity at temperatures above 25 degrees C. The enzyme deacetylated a variety of cephalosporin derivatives, including cephalosporin C; the Km for this substrate is 51.8 mM, and the Vmax is 7.9 mumol/min/mg. In addition to cephalosporins, the enzyme hydrolyzed short-chain p-nitrophenyl esters, with the activity decreasing with increasing ester chain length. The enzyme also has the ability to acetylate desacetyl cephalosporins in high yields under mild conditions in the presence of various acetyl donors. A comparison of the physical properties of the esterase with those of other well-characterized cephalosporin esterases indicates that the enzyme is unique in this class.
Factors affecting lactate and malate utilization by Selenomonas ruminantium
Authors: Evans J D , Martin S A
Published: January 2020

Lactate utilization by Selenomonas ruminantium is stimulated in the presence of malate. Because little information is available describing lactate-plus-malate utilization by this organism, the objective of this study was to evaluate factors affecting utilization of these two organic acids by two strains of S. ruminantium. When S. ruminantium HD4 and H18 were grown in batch culture on DL-lactate and DL-malate, both strains coutilized both organic acids for the initial 20 to 24 h of incubation and acetate, propionate, and succinate accumulated. However, when malate and succinate concentrations reached 7 mM, malate utilization ceased, and with strain H18, there was a complete cessation of DL-lactate utilization. Malate utilization by both strains was also inhibited in the presence of glucose. S. ruminantium HD4 was unable to grow on 6 mM DL-lactate at extracellular pH 5.5 in continuous culture (dilution rate, 0.05 h-1) and washed out of the culture vessel. Addition of 8 mM DL-malate to the medium prevented washout on 6 mM DL-lactate at pH 5.5 and resulted in succinate accumulation. Addition of malate also increased bacterial protein, acetate, and propionate concentrations in continuous culture. These results suggest that 8 mM DL-malate enhances the ability of strain HD4 to grow on 6 mM DL-lactate at extracellular pH 5.5.
Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei
Authors: Nykanen M , Saarelainen R , Raudaskoski M , Nevalainen K , Mikkonen A
Published: January 2020

Localization of expression and secretion of a heterologous barley cysteine endopeptidase (EPB) and the homologous main cellobiohydrolase I (CBHI) in a Trichoderma reesei transformant expressing both proteins were studied. The transformant was grown on solid medium with Avicel cellulose and lactose to induce the cbh1 promoter for the synthesis of the native CBHI and the recombinant barley protein linked to a cbh1 expression cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirect immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher molecular weight than the native barley protein, probably due to glycosylation and differential processing in the fungal host. As was found with its transcripts, recombinant EPB was localized in apical and subapical compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI was also secreted from these. In immunoelectron microscopy, the endoplasmic reticulum and spherical vesicles assumed to contribute to secretion were labeled by both CBHI and EPB antibodies while only CBHI was localized in elongated vesicles close to the plasma membrane and in hyphal walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins.
A mutant of Listeria monocytogenes LO28 unable to induce an acid tolerance response displays diminished virulence in a murine model
Authors: Marron L , Emerson N , Gahan C G , Hill C
Published: January 2020

Exposing Listeria monocytogenes LO28 to sublethal pH induces protection against normally lethal pH conditions, a phenomenon known as the acid tolerance response. We identified a mutant, L. monocytogenes ATR1, which is incapable of inducing such tolerance, either against low pH or against any other stress tested. The virulence of this mutant was considerably decreased, suggesting that the acid tolerance response contributes to in vivo survival of L. monocytogenes.
Mosquito larvicidal activity of transgenic Anabaena strain PCC 7120 expressing combinations of genes from Bacillus thuringiensis subsp. israelensis
Authors: Xiaoqiang W , Vennison S J , Huirong L , Ben-Dov E , Zaritsky A , Boussiba S
Published: January 2020

Various combinations of the genes cryIVA (cry4A), cryIVD (cry11A), and p20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 by means of Escherichia coli-Anabaena shuttle vector pRL488p and were expressed under control of two tandem strong promoters, a cyanobacterial promoter (PpsbA) and an E. coli T7 promoter (PA1). Two of the clones carrying cryIVA plus cryIVD, one with p20 and one without p20, displayed toxicity against third-instar larvae of Aedes aegypti at levels greater than any level previously reported for transgenic cyanobacteria.
Vibrio sp. strain NM 10, isolated from the intestine of a Japanese coastal fish, has an inhibitory effect against Pasteurella piscicida
Authors: Sugita H , Matsuo N , Hirose Y , Iwato M , Deguchi Y
Published: January 2020

Vibrio sp. strain NM 10 with an inhibitory activity against Pasteurella piscicida K-III was isolated from the intestine of a spotnape ponyfish (Leiognathus nuchalis). This bacterium efficiently produced an antibacterial substance after growth at 20 degrees C for 24 h on 1/5 PYBG agar prepared with 50% seawater at pHs of 7.5 to 9.0. The antibacterial substance was heat labile and proteinaceous, with a molecular mass of less than 5 kDa, possibly a bacteriocin or a bacteriocin-like substance.
Anoxygenic photosynthesis and nitrogen fixation by a microbial mat community in a bahamian hypersaline lagoon
Authors: Pinckney J L , Paerl H W
Published: January 2020

Simultaneous measurements of photosynthesis (both oxygenic and anoxygenic) and N(inf2) fixation were conducted to discern the relationships between photosynthesis, N(inf2) fixation, and environmental factors potentially regulating these processes in microbial mats in a tropical hypersaline lagoon (Salt Pond, San Salvador Island, Bahamas). Major photoautotrophs included cyanobacteria, purple phototrophic bacteria, and diatoms. Chemosystematic photopigments were used as indicators of the relative abundance of mat phototrophs. Experimental manipulations consisted of light and dark incubations of intact mat samples exposed to the photosystem II inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a dissolved organic carbon source (D-glucose), and normal seawater (37(permil)). Photosynthetic rates were measured by both O(inf2) and (sup14)C methods, and nitrogenase activity (NA) was estimated by the acetylene reduction assay. Moderate reductions in salinity (from 74 to 37(permil)) had no measurable effect on photosynthesis, O(inf2) consumption, or NA. CO(inf2) fixation in DCMU-amended samples was (symbl)25% of that in the control (nonamended) samples and demonstrated photosynthetic activity by anoxygenic phototrophs. NA in DCMU-amended samples, which was consistently higher (by a factor of 2 to 3) than the other (light and dark) treatments, was also attributed to purple phototrophic bacteria. The ecological implication is that N(inf2) fixation by anoxygenic phototrophs (purple phototrophic bacteria and possibly cyanobacteria) may be regulated by the activity of oxygenic phototrophs (cyanobacteria and diatoms). Consortial interactions that enhance the physiological plasticity of the mat community may be a key for optimizing production, N(inf2) fixation, and persistence in these extreme environments.
Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain
Authors: Saito Y , Ishii Y , Hayashi H , Imao Y , Akashi T , Yoshikawa K , Noguchi Y , Soeda S , Yoshida M , Niwa M , Hosoda J , Shimomura K
Published: January 2020

We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.
Stable-Isotope Analysis of a Combined Nitrification-Denitrification Sustained by Thermophilic Methanotrophs under Low-Oxygen Conditions
Authors: Pel R , Oldenhuis R , Brand W , Vos A , Gottschal J C , Zwart K B
Published: January 2020

To simulate growth conditions experienced by microbiota at O(inf2)-limited interfaces of organic matter in compost, an experimental system capable of maintaining dual limitations of oxygen and carbon for extended periods, i.e., a pO(inf2)-auxostat, has been used. (sup15)N tracer studies on thermophilic (53(deg)C) decomposition processes occurring in manure-straw aggregates showed the emission of dinitrogen gas from the reactor as a result of simultaneous nitrification and denitrification at low pO(inf2) values (0.1 to 2.0%, vol/vol). The N loss was confirmed by nitrogen budget studies of the system. Depending on the imposed pO(inf2), 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N(inf2). When the pO(inf2) was raised, the rates of both nitrification and denitrification increased instantaneously, indicating that ammonia oxidation was limited by oxygen. In auxostats permanently running at pO(inf2) >= 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N(inf2) gas. Labelling of the auxostat with [(sup13)C]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin. Incorporation of (sup13)CO(inf2) into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fatty acids (FAs) by using capillary gas chromatography and subsequently analyzing the (sup13)C/(sup12)C ratios of the individual FAs, after their combustion to CO(inf2), by isotope ratio mass spectrometry. Apart from the observed label incorporation into FAs originating from a microflora belonging to the genus Methylococcus (type X group), supporting nitrification of a methylotrophic nature, this analysis also corroborated the absence of truly autotrophic nitrifying populations. Nevertheless, the extent to which ammonia oxidation continued to exist in this thermophilic community suggested that a major energy gain could be associated with it.
Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels
Authors: Gouka R J , Punt P J , van den Hondel C A
Published: January 2020

In this study we have analyzed the effects of a glucoamylase gene fusion on the mRNA levels and protein levels for the human interleukin-6 gene (hil6) and the guar alpha-galactosidase gene (aglA). Previously it was shown that production of nonfused alpha-galactosidase and hIL-6 in Aspergillus awamori was limited at transcriptional and (post)translational levels, respectively (R. J. Gouka, P. J. Punt, J. G. M. Hessing, and C. A. M. J. J. van den Hondel, Appl. Environ. Microbiol. 62:1951-1957, 1996). Vectors were constructed which contained either the hil6 or aglA gene fused to the Aspergillus niger glucoamylase gene (glaA) under control of the efficient 1,4-beta-endoxylanase A promoter and transcription terminator. For comparison, the vectors were integrated in a single copy at the pyrG locus of A. awamori. A glaA fusion to the 5' end of the hil6 gene resulted in a large increase in hIL-6 yield, whereas with a glaA fusion to the 3' end of the hil6 gene, almost no protein was produced. Nevertheless, the steady-state mRNA levels of both fusions were very similar and not clearly increased compared to those of a strain expressing nonfused hIL-6. Fusions of glaA to the 5' end of the wild-type guar aglA gene resulted in truncated mRNA lacking almost 900 bases (> 80%) of the aglA sequence. When the coding sequence of the wild-type aglA gene was replaced by a synthetic aglA gene with optimized Saccharomyces cerevisiae codon usage, full-length mRNA was obtained. Compared to a nonfused synthetic aglA gene, a glaA fusion with the synthetic aglA gene resulted in a 25-fold increase in the mRNA level and, as a consequence, a similar increase in the alpha-galactosidase protein level. The truncated transcripts derived from the wild-type aglA gene were further analyzed by nuclear run-on transcription assays. These experiments indicated that transcription elongation in the nucleus proceeded at least 400 bases downstream of the site where the truncation was determined, indicating that transcription elongation or premature termination was not the reason for the generation of truncated mRNAs. As the truncated mRNA also contained a poly(A) tail, truncation most likely occurs by incorrect processing of the aglA mRNA in the nucleus.
Purification and Characterization of Cystathionine (gamma)-Lyase from Lactococcus lactis subsp. cremoris SK11: Possible Role in Flavor Compound Formation during Cheese Maturation
Authors: Bruinenberg P G , De Roo G , Limsowtin G
Published: January 2020

A cystathionine (gamma)-lyase (EC 4.4.1.1) ((gamma)-CTL) was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris SK11 by a procedure including anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The activity of SK11 (gamma)-CTL is pyridoxal-5(prm1)-phosphate dependent, and the enzyme catalyzes the (alpha),(gamma)-elimination reaction of L-cystathionine to produce L-cysteine, (alpha)-ketobutyrate, and ammonia. The native enzyme has a molecular mass of approximately 120 to 200 kDa and apparently consists of at least six identical subunits of 20 kDa. In this respect, the SK11 enzyme clearly differs from other bacterial cystathionine lyases, which are all tetrameric proteins with identical subunits of approximately 40 kDa. In addition, the specific activity of purified SK11 (gamma)-CTL toward L-cystathionine is relatively low compared with those reported for other bacterial cystathionine lyases. The SK11 enzyme shows a broad substrate specificity. In the case of L-methionine, the action of SK11 (gamma)-CTL results in the formation of methanethiol, a volatile sulfur compound known to be required in flavor development in cheddar cheese. The (alpha),(beta)-elimination reaction of L-cysteine is also efficiently catalyzed by the enzyme, resulting in the formation of hydrogen sulfide. Although the conditions are far from optimal, cystathionine (gamma)-lyase is still active under cheddar cheese-ripening conditions, namely, pH 5.0 to 5.4 and 5% (wt/vol) NaCl. The possible role of the enzyme in cheese flavor development is discussed.
Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins
Authors: Van der Vaart J M , te Biesebeke R , Chapman J W , Toschka H Y , Klis F M , Verrips C T
Published: January 2020

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.
Competitive Interactions among Symbiotic Fungi of the Southern Pine Beetle
Authors: Klepzig K D , Wilkens R T
Published: January 2020

The southern pine beetle, a damaging pest of conifers, is intimately linked to three symbiotic fungi. Two fungi, Ceratocystiopsis ranaculosus and Entomocorticium sp. A, are transported within specialized structures (mycangia) in the beetle exoskeleton and are mutualists of the beetle. A third fungus, Ophiostoma minus, is transported externally on the beetle exoskeleton (phoretically) and is an antagonist of the beetle. This study examined competitive interactions among these three fungi. The results of de Wit replacement series and primary and secondary resource capture assays with these fungi provide strong evidence for differential competition between the phoretic and mycangial fungi. O. minus was the most able to capture both uncolonized and colonized resources. Entomocorticium sp. A and C. ranaculosus, although equal to one another in competitive abilities, differed in their ability to compete with O. minus. Entomocorticium sp. A was able to maintain space free of O. minus to a much greater degree than was C. ranaculosus. The outcome of such competitive interactions may have significant impacts on the biology of this ecologically and economically important beetle.
Limitations of highly sensitive enzymatic presence-absence tests for detection of waterborne coliforms and Escherichia coli
Authors: Van Poucke S O , Nelis H J
Published: January 2020

This study presents evidence for the unfeasibility of enzymatic presence-absence tests to detect one total coliform or one Escherichia coli organism in 100 ml of drinking water within a working day. The results of field trials with prototype chemiluminometric procedures indicated that the sensitivity-boosting measures that are essential to achieve the required speed compromise the specificity of the tests.
Purification and Some Properties of a Xylanase from Aspergillus sydowii MG49
Authors: Ghosh M , Nanda G
Published: January 2020

Volume 62, no. 10, p. 3915, Author's Correction (Ghosh and Nanda). Errors were made in the original identification of the microorganism used in this study and in the reporting of the corrected species identification. The culture resubmitted to the American Type Culture Collection was identified by Dr. Frank Dugan as Aspergillus flavus, not Aspergillus citrinum, and is available as ATCC 96916. [This corrects the article on p. 3915b in vol. 62.].
Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State
Authors: Whitesides M D , Oliver J D
Published: January 2020

Like many other gram-negative bacteria, the human pathogen Vibrio vulnificus is induced into a viable but nonculturable (VBNC) state by incubation at low temperatures. The ability of any bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state is truly a survival strategy. The question as to whether the culturable cells which appear following removal of the inducing stress are a result of true resuscitation or of regrowth of a few residual culturable cells has long been debated. V. vulnificus was examined for its ability to resuscitate from this state following a temperature upshift. Several lines of investigation, including dilution studies, determination of the time necessary for appearance of a culturable population, and the effects of nutrient on recovery, all indicated that, at least for V. vulnificus, true resuscitation does occur. Our studies further suggest that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC state and that studies which add nutrient in an attempt to detect resuscitation are able to detect only residual culturable cells which might be present and which were not inhibited by the added nutrient.
Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria
Authors: Schofield P , Pell A N , Krause D O
Published: January 2020

Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus 8 and F. succinogenes S85.
Oxidation and assimilation of atmospheric methane by soil methane oxidizers
Authors: Roslev P , Iversen N , Henriksen K
Published: January 2020

The metabolism of atmospheric methane in a forest soil was studied by radiotracer techniques. Maximum (sup14)CH(inf4) oxidation (163.5 pmol of C cm(sup-3) h(sup-1)) and (sup14)C assimilation (50.3 pmol of C cm(sup-3) h(sup-1)) occurred at the A(inf2) horizon located 15 to 18 cm below the soil surface. At this depth, 31 to 43% of the atmospheric methane oxidized was assimilated into microbial biomass; the remaining methane was recovered as (sup14)CO(inf2). Methane-derived carbon was incorporated into all major cell macromolecules by the soil microorganisms (50% as proteins, 19% as nucleic acids and polysaccharides, and 5% as lipids). The percentage of methane assimilated (carbon conversion efficiency) remained constant at temperatures between 5 and 20(deg)C, followed by a decrease at 30(deg)C. The carbon conversion efficiency did not increase at methane concentrations between 1.7 and 1,000 ppm. In contrast, the overall methane oxidation activity increased at elevated methane concentrations, with an apparent K(infm) of 21 ppm (31 nM CH(inf4)) and a V(infmax) of 188 pmol of CH(inf4) cm(sup-3) h(sup-1). Methane oxidizers from soil depths with maximum methanotrophic activity respired approximately 1 to 3% of the assimilated methane-derived carbon per day. This apparent endogenous respiration did not change significantly in the absence of methane. Similarly, the potential for oxidation of atmospheric methane was relatively insensitive to methane starvation. Soil samples from depths above and below the zone with maximum atmospheric methane oxidation activity showed a dramatic increase in the turnover of the methane assimilated (>20 times increase). Physical disturbance such as sieving or mixing of soil samples decreased methane oxidation and assimilation by 50 to 58% but did not alter the carbon conversion efficiency. Ammonia addition (0.1 or 1.0 (mu)mol g [fresh weight](sup-1)) decreased both methane oxidation and carbon conversion efficiency. This resulted in a dramatic decrease in methane assimilation (85 to 99%). In addition, ammonia-treated soil showed up to 10 times greater turnover of the assimilated methane-derived carbon (relative to untreated soil). The results suggest a potential for microbial growth on atmospheric methane. However, growth was regulated strongly by soil parameters other than the methane concentration. The pattern observed for metabolism of atmospheric methane in soils was not consistent with the physiology of known methanotrophic bacteria.