bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolated from bovine ruminal contents and liver abscesses
Authors: Okwumabua O , Tan Z , Staats J , Oberst R D , Chengappa M M , Nagaraja T G
Published: January 2020

Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.
Biosynthesis of Novel Aromatic Copolyesters from Insoluble 11-Phenoxyundecanoic Acid by Pseudomonas putida BM01
Authors: Song J J , Yoon S C
Published: January 2020

Two types of novel aromatic copolyesters were synthesized from 11-phenoxyundecanoic acid (11-POU) as the sole carbon source and the cosubstrates 11-POU and octanoate, respectively, by isolated Pseudomonas putida BM01 that is known to accumulate high concentrations of medium-chain-length polyesters. Insoluble 11-POU was recrystallized in situ in buffer by alkaline treatment and pH adjustment, followed by autoclaving. The resulting microcrystals, whose structure was different from that of the commercially available crystalline powder, suspended in media were rapidly consumed by the bacterium. Synthesized polymers were characterized by gas chromatography, nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. The aromatic copolyesters synthesized from 11-POU were composed of two monomer units consisting of 3-hydroxy-5-phenoxyvalerate (5POHV) as the major component (72 to 85 mol%) and 3-hydroxy-7-phenoxyheptanoate (7POHH) as the minor component (15 to 28 mol%). The aromatic copolyesters showed a crystalline melting transition at 70(deg)C. When the bacterium was grown on the cosubstrates 11-POU and octanoate, the bacterium synthesized the copolyesters composed of aromatic and aliphatic monomers poly(5POHV-co-7POHH-co-3-hydroxy-9-phenoxynonanoate-co-3-hydroxyalkanoates) . The addition of octanoate in the feed shifted the major monomer unit in the polymer from 5POHV to 7POHH. A further-fragmented metabolite, 3-phenoxypropionate, whose concentration reached a steady state at the time of greatest polyester accumulation, was detected in the medium. The metabolic pathway of 11-POU is suggested.
The Lactococcal Plasmid pNP40 Encodes a Third Bacteriophage Resistance Mechanism, One Which Affects Phage DNA Penetration
Authors: Garvey P , Hill C , Fitzgerald G F
Published: January 2020

The lactococcal plasmid pNP40 mediates insensitivity to (phi)c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system, AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to (phi)c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to (phi)c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for (phi)c2.
Selective enumeration of Bacteroides vulgatus and B. distasonis organisms in the predominant human fecal flora by using monoclonal antibodies
Authors: Corthier G , Muller M C , L'Haridon R
Published: January 2020

The genus Bacteroides represents about one-third of the isolates from human fecal samples. The proportions of the different species are difficult to estimate because there is no method for rapid identification of mixtures of anaerobes. Monoclonal antibodies against Bacteroides vulgatus and B. distasonis were prepared. They did not react with the other Bacteroides species of the B. fragilis group. These reagents allowed direct enumeration of B. vulgatus and B. diastasonis organisms in human fecal samples. Anaerobic bacteria resistant to 1-h contact with air were enumerated in fecal human samples, a filter was layered on the colonies, and then B. vulgatus colonies were identified by an immunoassay performed with the prepared monoclonal antibodies. Healthy human adult volunteers were tested. Most of them harbored B. vulgatus at high levels, while the B. distasonis levels were always lower. Kinetic studies suggested that time variations for each volunteer were small. The simplified quantification of Bacteroides strains at the species level described here will prove useful in complementing our knowledge of the factors which may influence the predominant human fecal flora.
Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius
Authors: Camarero S , Bockle B , Martinez M J , Martinez A T
Published: January 2020

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.
Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures
Authors: Bayles D O , Annous B A , Wilkinson B J
Published: January 2020

Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigerator temperatures. Twelve cold shock proteins (Csps) with apparent M(r)s of 48,600, 41,000, 21,800, 21,100, 19,700, 19,200, 18,800, 18,800, 17,200, 15,500, 14,500, and 14,400 were induced by cold shocking L. monocytogenes 10403S from 37 to 5 degrees C, as revealed by labeling with L-[35S]methionine followed by two-dimensional gel electrophoresis. Strain SLCC53 showed a similar response. Cold acclimation proteins were observed in cultures of strain 10403S growing at 5 degrees C, and four of these proteins, with apparent M(r)s 48,000, 21,100, 19,700, and 18,800, were also Csps. Two cold-sensitive transposon-induced mutants were labeled less efficiently than the parent strain, but the Csp response of the mutant examined was very similar to that of the parent strain.
Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes
Authors: Datz M , Janetzki-Mittmann C , Franke S , Gunzer F , Schmidt H , Karch H
Published: January 2020

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.
Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans
Authors: Zhang D , Freeman J P , Sutherland J B , Walker A E , Yang Y , Cerniglia C E
Published: January 2020

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.
Involvement of Pyochelin and Pyoverdin in Suppression of Pythium-Induced Damping-Off of Tomato by Pseudomonas aeruginosa 7NSK2
Authors: Buysens S , Heungens K , Poppe J , Hofte M
Published: January 2020

The plant growth-promoting rhizobacterium Pseudomonas aeruginosa 7NSK2 produces three siderophores when iron is limited: the yellow-green fluorescent pyoverdin, the salicylate derivative pyochelin, and salicylic acid. This Pseudomonas strain was shown to be an efficient antagonist of Pythium-induced damping-off. The role of pyoverdin and pyochelin in the suppression of Pythium splendens was investigated by using various siderophore-deficient mutants derived from P. aeruginosa 7NSK2 in a bioassay with tomato (Lycopersicon esculentum). To provide more insight into the role of pyochelin in antagonism, mutant KMPCH, deficient in the production of pyoverdin and pyochelin, was complemented for pyochelin production. The complementing clone was further characterized by subcloning and transposon mutagenesis and used to generate a pyochelin-negative, pyoverdin-positive mutant by marker exchange. All mutants were able to reduce Pythium-induced preemergence damping-off to some extent. Production of either pyoverdin or pyochelin proved to be necessary to achieve wild-type levels of protection against Pythium-induced postemergence damping-off. Mutant KMPCH inhibited P. splendens but was less active than the parental strain. This residual protection could be due to the production of salicylic acid. Since pyoverdin and pyochelin are both siderophores, siderophore-mediated iron competition could explain the observed antagonism and the apparent interchangeability of the two compounds. We cannot, however, exclude the possibility that both siderophores act in an indirect way.
Phylogenetic position of the spirochetal genus Cristispira
Authors: Paster B J , Pelletier D A , Dewhirst F E , Weisburg W G , Fussing V , Poulsen L K , Dannenberg S , Schroeder I
Published: January 2020

Comparative sequence analysis of 16S rRNA genes was used to determine the phylogenetic relationship of the genus Cristispira to other spirochetes. Since Cristispira organisms cannot presently be grown in vitro, 16S rRNA genes were amplified directly from bacterial DNA isolated from Cristispira cell-laden crystalline styles of the oyster Crassostrea virginica. The amplified products were then cloned into Escherichia coli plasmids. Sequence comparisons of the gene coding for 16S rRNA (rDNA) insert of one clone, designated CP1, indicated that it was spirochetal. The sequence of the 16S rDNA insert of another clone was mycoplasmal. The CP1 sequence possessed most of the individual base signatures that are unique to 16S rRNA (or rDNA) sequences of known spirochetes. CP1 branched deeply among other spirochetal genera within the family Spirochaetaceae, and accordingly, it represents a separate genus within this family. A fluorescently labeled DNA probe designed from the CP1 sequence was used for in situ hybridization experiments to verify that the sequence obtained was derived from the observed Cristispira cells.
Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation
Authors: Migaud M E , Chee-Sanford J C , Tiedje J M , Frost J W
Published: January 2020

E-Phenylitaconic acid has been isolated as a metabolite generated by Azoarcus tolulyticus Tol-4 along with benzylsuccinic acid during anaerobic degradation of toluene. Strain Tol-4 converted 1 to 2% of toluene carbon to E-phenylitaconate and benzylsuccinate (10:1). The identification of E-phenylitaconic acid was based on 1H nuclear magnetic resonance (NMR) characterization of degradation products derived from 13C-labeled toluene followed by comparison of spectroscopic and chromatographic data for the isolated, unlabeled metabolite with those for chemically synthesized benzylfumaric acid, benzylmaleic acid, E-phenylitaconic acid, and Z-phenylitaconic acid. Spectroscopic comparisons included 1H NMR, 13C NMR, and nuclear overhauser effect correlations. High-pressure liquid chromatography (HPLC) retention times and HPLC coinjections with synthetic dioic acids provided another reliable line of evidence for structure assignment. The formation of E-phenylitaconic acid differs from previous reports of benzylfumaric acid generation along with benzylsuccinic acid during anaerobic microbial degradation of toluene. This has important implications relevant to elaboration of the metabolic route for anaerobic toluene degradation by strain Tol-4 and related organisms. Similar amounts of E-phenylitaconic acid were also produced by seven other strains of A. tolulyticus.
The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase
Authors: Eggert C , Temp U , Eriksson K E
Published: January 2020

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.
Influence of environmental parameters on phosphatidylcholine phospholipase C production in Listeria monocytogenes: a convenient method to differentiate L. monocytogenes from other Listeria species
Authors: Coffey A , Rombouts F M , Abee T
Published: January 2020

The ability to produce phosphatidylcholine phospholipase C (lecithinase) is associated with virulence in pathogenic species of Listeria. Levels of production vary greatly among members of the genus, and this virulence factor is not readily detectable in many members of the pathogenic species on conventional agar media containing egg yolk, a common substrate for the enzyme. In this study, the influence of a variety of environmental parameters, including temperature, pH, and salt concentration, on the production of lecithinase by a number of strains was evaluated. Lecithinase production by Listeria monocytogenes LO28 in brain heart infusion medium was optimal at 1.75 to 2.0% NaCl; pH 7.0 to 7.3, and 37 to 40 degrees C, and the presence of oxygen had no effect. In a chemically defined medium, the optimal NaCl concentration and temperature were lower at 0.75 to 1.0% NaCl and 33.5 degrees C. As detection of virulence factors is useful to assist in the identification and differentiation of Listeria species, this report shows that lecithinase activity can conveniently be detected within 36 h on a relatively inexpensive medium. Under the conditions described, L. monocytogenes could be distinguished from other members of the genus as a result of distinct lecithin degradation which was not evident in L. innocua, L. seeligeri, L. ivanovii, L. welshimeri, or L. murrayi/grayi.
Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos
Authors: Labarre C , Guzzo J , Cavin J F , Diviès C
Published: January 2020

Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank. Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments. Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure. The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present. The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells. The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species. The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core. Energy-dependent L-[14C]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector. Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.
Nucleotide sequence and taxonomical distribution of the bacteriocin gene lin cloned from Brevibacterium linens M18
Authors: Valdes-Stauber N , Scherer S
Published: January 2020

Linocin M18 is an antilisterial bacteriocin produced by the red smear cheese bacterium Brevibacterium linens M18. Oligonucleotide probes based on the N-terminal amino acid sequence were used to locate its single copy gene, lin, on the chromosomal DNA. The amino acid composition, N-terminal sequence, and molecular mass derived from the nucleotide sequence of an open reading frame of 798 nucleotides coding for 266 amino acids found on a 3-kb BamHI restriction fragment correspond closely to those obtained from the purified protein (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994). No sequence homology to any protein or nucleotide sequences deposited in databases was found. Comparison of the nucleotide sequence and the N-terminal amino acid sequence derived from the protein suggests that B. linens M18 produces an N-formyl-methionyl-CAC tRNA. A wide taxonomical distribution of the gene within coryneform bacteria has been demonstrated by PCR amplification. The structural gene from linocin M18 is present at least in three Brevibacterium species, five Arthrobacter species, and five Corynebacterium species.
Inactivation of tryptic activity by a human-derived strain of Bacteroides distasonis in the large intestines of gnotobiotic rats and mice
Authors: Ramare F , Hautefort I , Verhe F , Raibaud P , Iovanna J
Published: January 2020

Tryptic activity disappeared and trypsin was no longer detected with an antitrypsin antiserum in the large intestines of gnotobiotic rats and mice monoassociated with a human-derived strain of Bacteroides distasonis, whereas tryptic activity was not modified in the small intestines. This function was shown to be strain specific.
Legionella contamination of dental-unit waters
Authors: Atlas R M , Williams J F , Huntington M K
Published: January 2020

Volume 61, no. 4, p. 1211, column 2, line 40: this sentence should read as follows. "The viable-culture methods for Legionella sp. detection, however, often fail, and the Centers for Disease Control and Prevention has turned, when necessary, to PCR for epidemiological investigations of Legionnaires' disease and Pontiac fever (19)." Line 46: "(6)" should read "(11)." [This corrects the article on p. 1208 in vol. 61.].
Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture
Authors: Lendenmann U , Snozzi M , Egli T
Published: January 2020

In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle.
Anaerobic Desulfonation of 4-Tolylsulfonate and 2-(4-Sulfophenyl) Butyrate by a Clostridium sp
Authors: Denger K , Kertesz M A , Vock E H , Schon R , Magli A , Cook A M
Published: January 2020

Alkyl- and arylsulfonates were tested as sole added sources of sulfur for the growth of enrichment cultures under strictly anaerobic denitrifying or fermentative conditions. Cultures that utilized taurine, ethylsulfonate, the dyestuffs orange II and acid red I, tolylsulfonate, 2-(4-sulfophenyl)butyrate (SPB), a dialkyltetralinesulfonate, and 1-(4-sulfophenyl)octane were readily obtained. We chose to work with the simple aromatic compounds and isolated a fermentative bacterium, strain EV4, which utilized SPB as the sole added source of sulfur in glucose-mineral medium. The organism was identified as a Clostridium sp. related to Clostridium beijerinckii. Clostridium sp. strain EV4 utilized seven of seven tested arylsulfonates quantitatively. The growth yield was about 3 kg of protein per mol of sulfur, whether sulfonate or sulfate was utilized. A major product specific to each sulfonate could be observed. Although no product was identified, the existence of anaerobic desulfonation has been established.
Model for the combined effects of temperature, pH, and sodium lactate on growth rates of Listeria innocua in broth and Bologna-type sausages
Authors: Houtsma P C , Kant-Muermans M L , Rombouts F M , Zwietering M H
Published: January 2020

A modified Monod equation was successfully applied to describe the maximum specific growth rate of Listeria innocua in a broth model in the presence of various concentrations of sodium lactate or NaCl. The combined effects of temperature and pH were assessed by translating the parameters of the modified Monod equation mu(m), alpha, and p') as functions of pH and/or temperature. As a result, the area in which the growth rate could be predicted was extended to include as a variable not only the salt concentration but also pH and temperature. The number of parameters needed to describe the experimental data was thereby reduced from 48 to 4 (NaCl) and from 42 to 5 (sodium lactate). The decline in the goodness of fit that accompanied the reduction in the number of parameters was within statistically acceptable ranges. The resulting model was compared with a polynomial fit, and it was proposed that the former was more suitable for the purpose of this study. The broth model for sodium lactate was evaluated with Bologna-type sausages. Because of the "worst-case" design of the broth model, it was necessary to reestimate one or all parameters to obtain a good description of the growth rate of L. innocua in the meat product. However, the simplicity of the model and the practical usefulness of its parameters offer considerable prospects for its use in predictive microbiology.
Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence
Authors: O'Driscoll B , Gahan C G , Hill C
Published: January 2020

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.
Xylulose and glucose fermentation by Saccharomyces cerevisiae in chemostat culture
Authors: Jeppsson H , Yu S , Hahn-Hägerdal B
Published: January 2020

Saccharomyces cerevisiae ATCC 24860 was cultivated in chemostat culture under anoxic conditions with 111.1 mmol of glucose liter-1 alone or with a mixture of 66.7 mmol of xylulose liter-1 and 111.1 mmol of glucose liter-1. The substrate consumption rate was 5.4 mmol g of cells-1 h-1 for glucose, whereas for xylulose it was 1.0 mmol g of cells-1 h-1. The ethanol yield decreased from 0.52 carbon mole of ethanol produced per carbon mole of sugar consumed during the utilization of glucose alone to 0.49 carbon mole produced per carbon mole consumed during the simultaneous utilization of xylulose and glucose, while cell biomass was maintained at 2.04 to 2.10 g liter-1. Xylulose coutilization was accompanied by a shift in product formation from ethanol to acetate and arabinitol. Xylulokinase activity was absent during glucose metabolism but detectable during simultaneous utilization of xylulose and glucose. Xylulose cometabolism resulted in increased in vitro activity of pyruvate decarboxylase and an increased concentration of the intracellular metabolite fructose 1,6-diphosphate without significant changes in the concentrations of 6-phosphogluconate and pyruvate. The results are discussed in relation to (i) altered enzyme activities and (ii) the redox flux of the cell.
Influence of Bovine Slurry Deposition on the Structure of Nodulating Rhizobium leguminosarum bv. viciae Soil Populations in a Natural Habitat
Authors: Labes G , Ulrich A , Lentzsch P
Published: January 2020

The population of nodulating R. leguminosarum bv. viciae in soil from a grass-covered valley area which had been used for bovine slurry deposition over a period of 5 years was analyzed. For these studies, a rapid and reproducible method based on enterobacterial repetitive intergenic consensus (ERIC)-PCR was applied to identify Rhizobium strains which had infected pea nodules. Soil samples were taken from different areas and further analyzed in plant tests to determine the impact of the application of slurry (polluted or nonpolluted), the slope position (summit or toe), and exposure (north or south). After comparison of all PCR fingerprint patterns, 24 strain groups were defined. Some strain groups from the nonpolluted soil were suppressed in the polluted samples, and new strain groups were detected in the slurry-polluted soil. After analyzing relationships between the strain groups, we determined the influences of local factors on the nodulating R. leguminosarum bv. viciae population. We show that one of those local parameters, slope position, had significantly greater impact on the composition of the Rhizobium population than the presence of slurry.
Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments
Authors: Kukor J J , Olsen R H
Published: January 2020

We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.
A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus
Authors: Brahamsha B
Published: January 2020

Unicellular cyanobacteria of the genus Synechococcus are among the most abundant members of the picoplankton in the open ocean, and their contribution to primary production is considerable. While several isolates have been used for physiological, biochemical, and molecular studies of their unique adaptations to the marine environment, it has become necessary to develop molecular genetic methods for one or more model open-ocean cyanobacteria in order for studies of these organisms and their unique properties to progress. A number of molecular tools for the genetic manipulation of Synechococcus sp. strains WH7803, WH8102, and WH8103 have been developed. These include a plating technique for obtaining isolated colonies at high efficiencies and a conjugation method for introducing both a replicative vector and a suicide vector. In addition, a method for the generation of random, tagged chromosomal insertions (N. Dolganov and A. R. Grossman, J. Bacteriol. 175:7644-7651, 1993; N. F. Tsinoremas, A. K. Kutach, C. A. Strayer, and S. S. Golden, J. Bacteriol. 176:6764-6768, 1994) has been applied to these organisms.
Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola
Authors: Lou J , Dawson K A , Strobel H J
Published: January 2020

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.
Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin
Authors: Vionis A P , Niemeyer F , Karagouni A D , Schrempf H
Published: January 2020

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.
Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media
Authors: Ryser E T , Arimi S M , Bunduki M M , Donnelly C W
Published: January 2020

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.
Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation
Authors: Bogan B W , Lamar R T , Hammel K E
Published: January 2020

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.
Use of colicin e3 for biological containment of microorganisms
Authors: Munthali M T , Timmis K N , Diaz E
Published: January 2020

The genetic determinant of the lethal antibiotic colicin E3 was cloned under the control of a tightly regulated promoter in the absence of the gene for its cognate inhibitor. Combination of this killing cassette with a stringent regulatory element provided a substrate-dependent conditional suicide system that was exploited for the biological containment of a Pseudomonas putida strain. The lethality of a single gene copy and the distinct and universal cellular target of the antibiotic suggest colicin E3 as an ideal candidate for combination with other lethal functions to design highly efficient containment systems for microorganisms.
Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates
Authors: Velraeds M M , van der Mei H C , Reid G , Busscher H J
Published: January 2020

In this study, 15 Lactobacillus isolates were found to produce biosurfactants in the mid-exponential and stationary growth phases. The stationary-phase biosurfactants from lactobacillus casei subsp. rhamnosus 36 and ATCC 7469, Lactobacillus fermentum B54, and Lactobacillus acidophilus RC14 were investigated further to determine their capacity to inhibit the initial adhesion of uropathogenic Enterococcus faecalis 1131 to glass in a parallel-plate flow chamber. The initial deposition rate of E. faecalis to glass with an adsorbed biosurfactant layer from L. acidophilus RC14 or L. fermentum B54 was significantly decreased by approximately 70%, while the number of adhering enterococci after 4 h of adhesion was reduced by an average of 77%. The surface activity of the biosurfactants and their activity inhibiting the initial adhesion of E. faecalis 1131 were retained after dialysis (molecular weight cutoff, 6,000 to 8,000) and freeze-drying. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy revealed that the freeze-dried biosurfactants from L. acidophilus RC14 and L. fermentum B54 were richest in protein, while those from L. casei subsp. rhamnosus 36 and ATCC 7469 had relatively high polysaccharide and phosphate contents.
An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402
Authors: Brass S , Ernst A , Böger P
Published: January 2020

The unicellular diazotrophic cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, contains a novel insertion sequence, IS8402, in the apcA gene encoding a pigmented protein of phycobilisomes. IS8402 comprises 1,322 bp, flanked by two inverted repeats of 15 bp. Upon insertion in the target DNA, direct duplications of 8 nucleotides were generated. One open reading frame, potentially coding for a protein of 399 amino acids, was found. The deduced amino acid sequence shows homology to putative transposases of the IS4 family. Precise excision of the insertion element resulted in a spontaneous revertant, Synechocystis sp. strain BO 9201, that had regained the ability to form hemidiscoidal phycobilisomes. Apart from the unique insertion of IS8402 into apcA in strain BO 8402 both strains contain at least 12 further homologous insertion elements at corresponding sites in the genomes. The unique insertion in strain BO 8402 prevents the expression of apcABC operon and hence abolishes the formation of intact phycobilisomes. This decreases the quantum efficiency of photosystem II and promotes anaerobic N2 fixation in a unicellular cyanobacterium with a highly oxygen-sensitive nitrogenase.